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The long non-coding RNA TUG1 indicates a poor prognosis for colorectal cancer and promotes metastasis by affecting epithelial-mesenchymal transition
Junfeng Sun1,
Chaohui Ding1,
Zhen Yang1,
Tao Liu1,
Xiefu Zhang1,
Chunlin Zhao1 &
Jiaxiang Wang2
A Correction to this article was published on 22 December 2020
This article has been updated
Long intergenic non-coding RNAs (lncRNAs) are a class of non-coding RNAs that are involved in gene expression regulation. Taurine up-regulated gene 1 (TUG1) is a cancer progression related lncRNA in some tumor oncogenesis; however, its role in colorectal cancer (CRC) remains unclear. In this study, we determined the expression patterns of TUG1 in CRC patients and explored its effect on CRC cell metastasis using cultured representative CRC cell lines.
The expression levels of TUG1 in 120 CRC patients and CRC cells were determined using quantitative real-time PCR. HDACs and epithelial-mesenchymal transition (EMT)-related gene expression were determined using western blot. CRC cell metastasis was assessed by colony formation, migration assay and invasion assay.
Our data showed that the levels of TUG1 were upregulated in both CRC cell lines and primary CRC clinical samples. TUG1 upregulation was closely correlated with the survival time of CRC patients. Overexpression of TUG1 in CRC cells increased their colony formation, migration, and invasion in vitro and promoted their metastatic potential in vivo, whereas knockdown of TUG1 inhibited the colony formation, migration, and invasion of CRC cells in vitro. It is also worth pointing out that TUG1 activated EMT-related gene expression.
Our data suggest that tumor expression of lncRNA TUG1 plays a critical role in CRC metastasis. TUG1 may have potential roles as a biomarker and/or a therapeutic target in colorectal cancer.
Colorectal cancer (CRC) remains a primarily world-wide health concern in spite of significant improvements in its diagnosis and therapy modalities. Over 1.2 million new CRC cancer cases and 608,700 deaths are recorded annually [1]. Historically, comprehensive cancer treatment in cases of cancer metastases has always been very challenging [2]. Currently, the need to elaborate knowledge on the underlying molecular mechanisms for cancer metastasis in CRC is urgently needed.
Mammalian genomes encode a wide variety of conserved non-coding RNA transcripts [3, 4]. In addition to classical 'housekeeping' RNAs (such as ribosome RNAs, transfer RNAs, and others) and widely-defined microRNAs, long non-coding RNAs (lncRNAs) have recently been identified as one of fraction of untranslated RNA molecules. lncRNAs, transcribed by RNA polymerase II (RNA pol II), are characterized by lengths of 200 nucleotides to ~100 kilobases (kb) and by their lack of a significant open reading frame [3]. These mRNA-like molecules are pervasively transcribed and roughly classified as antisense, based on their position relative to the protein-coding genes [5] and exhibit cis- or trans- regulatory capabilities for gene expression. Gene expression patterns indicate that these lncRNAs are implicated in diverse biological processes, including nuclear architecture, regulation of gene expression, immune surveillance, or embryonic stem cell pluripotency. Recently, evidence revealing the molecular mechanisms by which these RNA species function has provided some insight into the functional roles they may play in tumorigenesis [6]. Aberrant lncRNA expression participates in carcinogenesis by disrupting major biological processes, such as redirecting chromatin remodeling complexes or inactivating major tumor suppressor genes [6, 7].
Among these, LincRNA taurine up-regulated gene 1 (TUG1; also known as TI-227H; Linc00080; ncRNA00080) was originally identified as a transcript up-regulated by taurine and is found to be expressed in various human cancer cell lines and tumors [8]. Upregulation of the long noncoding RNA TUG1 promotes proliferation and migration of esophageal squamous cell carcinoma [9]. Downregulation of long non-coding RNA TUG1 inhibits osteosarcoma cell proliferation and promotes apoptosis [10]. However, the role of TUG1 in colorectal cancer remains unclear. This study was established due to our speculation that TUG1 may have roles to play in the CRC pathological process.
Previously, research has found that the metastasis and invasion of cancer cells are common events that mediate changes in cellular behavior, and induce different steps in the metastatic cascade [11, 12]. One of the most crucial steps in the tumor cell metastatic cascade is the acquisition of invasive capabilities, including destroying cell–cell junctions, degrading the cell matrix, and activating pathways that control the cytoskeletal dynamics of cancer cells. Over the past decade, cell and tumor biologists have identified the key role of epithelial-mesenchymal transition (EMT)-a biological process in which epithelial cells lose their polarity and transition into a mesenchymal phenotype-in cancer cell metastasis [13]. Evidence suggests that EMT enhances tumor cell invasion in response to environmental triggers, augments invasive functions, and also contributes to cell growth and survival [14, 15].
In preliminary experiments, we found that upregulated UTR1 expression in metastatic CRC cell lines predicted poor survival for CRC patients. This current study was designed to search the evidence supporting a role for TUG1 in the metastasis of CRC in representative CRC cell lines. We further attempted the preliminary exploration of the possible association of EMT-related gene expression by TUG1.
Clinical samples
We obtained paired CRC tumor samples (bulk samples) and adjacent non-tumor colorectal tissues from 120 patients who had undergone gastrointestinal surgery between 2008 and 2013 at the First Affiliated Hospital of Zhengzhou University, China. All specimens were immediately frozen in liquid nitrogen and stored at 80 °C until total RNA extraction. Written informed consent was obtained from all patients. No patient received chemotherapy or radiotherapy before surgery. The follow-up periods ranged from 2 months to 5 years, with a mean of 3 years. Our study was approved by the Research Ethics Committee of Zhengzhou University.
Cell lines and cultivation
Human colorectal cancer cell lines including SW480, HCT116, HT29, SW620, and LOVO, were obtained from the Key Laboratory of Cancer Prevention and Intervention, Cancer Institute, The Affiliated Hospital, Zhengzhou University School of Medicine, Zhengzhou, China. The SW480, SW620, and LOVO cell lines were cultured in L-15 (with 10 % FBS and 1 % streptomycin/penicillin); HCT-116 cell lines were cultured in McCoy's 5A (with 10 % FBS and 1 % streptomycin/penicillin); and HT-29 cell lines were cultured in RPMI-1640 (with 10 % FBS and 1 % streptomycin/penicillin). Normal human colorectal cells were purchased from the American Type Culture Collection and cultured in RPMI1640 supplemented with 10 % FBS and 2 mM l-Glutamine (Gibco). All cell lines were maintained at 37 °C and 5 % CO2 in an incubator, and passaged with 0.25 % trypsin (Sigma, St Louis, MO, USA) in 0.2 mol/l phosphate-buffered saline (PBS; Sigma). The study was approved by the ethics committee of the Cancer Institute, The Affiliated Hospital, Zhengzhou University School of Medicine, Zhengzhou, China.
Total RNA was extracted from colorectal tumor samples and CRC cell lines using TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. Complementary DNA was synthesized from total RNA with the Revert Aid™ First Strand cDNA Synthesis Kit (Thermo Scientific, USA). The primer sequences were as follows: TUG1 forward primer: 5c-TAGCAGTTCCCCAATCCTTG-3′, reverse primer: 5′-CACAAATTCCCATCATTCCC-3′; GAPDH forward primer: 5′-CGCTCTCTGCTCCTCCTGTTC-3′, GAPDH reverse primer: 5′-ATCCGTTGACTCCGACC-TTCAC-3′. The PCR was performed in a total reaction volume of 20 ml and was completed in the ABI PRISM 7000 Fluorescent Quantitative PCR System (Applied Biosystems, Foster City, CA, USA). GAPDH was used as an internal control. The PCR cycling parameters were: one denaturation step of 10 min at 95 °C; 40 cycles, with one cycle consisting of 15 s at 95 °C, 20 s at 55 °C, and 30 s at 70 °C. The median in each triplicate was used to calculate the relative TUG1 expression level using the comparative DCt method (value of 2−DCt(TUG1-GAPDH)). Expression fold changes were calculated using 2−DDCt methods.
Protein isolation and western blotting
For the protein expression analyses, standard western blot assay was carried out. Cultured or transfected cells were washed twice with cold phosphate-buffered saline (PBS) and were lysed with iced RIPA buffer containing 1 % PMSF (KeyGen, Nanjin, China). After total protein detection using a BCA kit (Beyotime, Shanghai, China), protein lysates were separated on 10 % SDS polyacrylamide gel, transferred to PVDF membranes, and blocked in 0.1 % Tween 20 and 5 % skim milk protein in Tris Buffer Saline at room temperature for 2 h. Target proteins were probed with rabbit anti-HDAC1 antibody (1:800; Proteintech Corporation, USA), rabbit anti-HDAC2 antibody and rabbit anti-HDAC3 monoclonal antibody (1:1000; Bioworld Corporation, USA), and mouse anti-E-cadherin, N-cadherin, fibronectin, vimentin (1:1000; Abcam, USA), and rabbit anti-β-Actin antibody (1:2000; acted as an internal control, Zhongshan, Inc., Beijing, China) overnight at 4 °C. The membranes were washed twice and visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. Tagged proteins were detected by enhanced chemiluminescence (KeyGen, Nanjing, China).
Plasmid and lentivirus vector
Ectopic expression of TUG1 in cells was realized through pcDNA-TUG1 transfection. The TUG1 sequence was synthesized and subcloned into the pCDNA3.1 (Invitrogen, Shanghai, China) vector. The empty pcDNA3.1 vector was used as a control. To assess the effect of TUG1 on liver metastasis in vivo, TUG1 was overexpressed by SW480 transfecting with Lenti-GIII-CMV-Human-TUG1 Lentivirus [Applied Biological Materials (ABM) Inc, Canada]. Following this step, the expression levels of TUG1 were detected by quantity real-time PCR.
Plasmid vector (pcDNA-TUG1 and pCDNA3.1) or lentivirus vector (Lenti-GIII-CMV-TUG1 and Lenti-GIII-CMV) were prepared using DNA Midiprep or Midiprep kits (Qiagen, Hilden, Germany), and were transfected into SW480 cells. Two individual siRNAs (TUD1-siRNA and HDAC1-siRNA) and negative control siRNA (silencer negative control siRNA) were purchased from Ambion. siRNA oligonucleotides (10 nmol/L) in Opti-MEM (Invitrogen) were transfected into LOVO cells or SW480 cells, respectively, using Lipofectamine RNAiMAX (Invitrogen) and following the manufacturer's protocol. Forty-eight hours post-transfection, TUD1 and HDAC1 expression levels were measured, and/or cell metastasis ability was assessed. Target sequences for siRNAs were as follows: TUG1-siRNA1 (sense 5′-GGGAUAUAGCCAGAGAACAAUUCU-A-3′, antisense 5′-UAGAAUUGUUCUCUGGCUAUAUCCC-3′) and HDAC1-siRNA, sense 5′-CCAAGTACCACAGCGATGAC-3′, and antisense 5′-TGGACAGTCCTCACCAACG-3′.
Plate clone formation assay
Each well in a 6-well culture plate was seeded with 102 cells and each group contained three wells. The cells were incubated at 37 °C with 5 % CO2 for 2–3 weeks and the medium was replaced every 3 days. The incubation was stopped when colony formation was visible and the cells were then washed twice with PBS and stained with Giemsa solution for 20 min. The number of colonies containing 50 cells was counted under a microscope using the formula: plate clone formation efficiency = (number of colonies/number of cells inoculated) × 100 %.
Wound healing assay
SW480pcDNA-TUG1, LOVOsi-TUG1 and their corresponding control cells were separately seeded into 6-well plates, and were cultured until they were nearly 80 % confluent. Artificial wounds were created by scraping the monolayers with a sterile 10 ml tip, and the cells were washed with PBS several times to remove floating cells. Representative images of cells migrating into the wounds were captured after 0 and 24 h under a microscope (200×).
Cell migration assays
Cell migration assays were performed using 24-well Transwell plates (8.0-μm pore membranes; Costar). About 1 × 104 cells (SW480pcDNA-TUG1 or LOVOsi-TUG1) were loaded into the upper chambers. The lower chambers were filled with a medium (plus 1 % FBS) in the absence (DMSO 0.2 %) or presence of SecinH3 (10, 20, or 40 mM). The Transwell plates were then incubated in a 37 °C, 5 % CO2 incubator for 48 h. After cleaning the cells from the upper side of the polycarbonate membrane and following hematoxylin–eosin staining, the polycarbonate membrane was cut and placed on a microscope slide, cover slipped, and examined under the microscope. The migrated cell numbers and percentages were then counted.
Matrigel invasion assay
The Matrigel invasion assay was done using the BD Biocoat Matrigel Invasion Chamber (pore size: 8 mm, 24-well; BD Biosciences, San Jose, CA, USA) following the manufacturer's protocol. Cells (5 × 104) were plated in the upper chamber in a serum-free medium. The bottom chamber contained a medium with 10 % FBS. After the 48 h incubation, the bottom of the chamber insert was stained with Calcein AM (Invitrogen). The cells that had invaded through the membrane to the lower surface were evaluated in a fluorescence plate reader at excitation/emission.
Experimental in vivo liver metastasis model
Female athymic BALB/c nude mice (aged 6 weeks) were purchased from the Shanghai Laboratory Animal Center Co. Ltd. (Shanghai, China) and maintained in a pathogen-free animal facility at the Laboratory Animal Research Centre of Zhengzhou University. For liver metastatic capacity, the spleen of BALB/c nude mice was injected with 1 × 106 cells per mouse. Briefly, BALB/c nude mice were anesthetized by i.p. injection of Pelltobarbitalum Natricum, and 1 × 106 SW480pcDNA3.1 or SW480pcDNA-TUG1 tumor cells in 25 ml were injected into the exteriorized spleen using an insulin syringe and following abdominal incision. Five minutes after injection, spleen blood vessels were ligated, and the spleen was removed. Finally, the abdominal wound was closed with staples. After 5 weeks, mice were sacrificed and their livers were removed and tumor nodules were numbered.
Statistical analysis was performed using GraphPad Prism 5.01 software. Statistical tests for data analysis included the log-rank test, the Chi square test. Multivariate statistical analysis was performed using a Cox regression model. The quantitative data were presented as the mean ± standard deviations (SD). Differences were considered to be statistically significant at values of P < 0.05.
Upregulation of TUG1 is correlated with CRC progression
To investigate the clinical relevance of abnormal TUG1 expression in CRC, we collected 120 pre-frozen primary CRC tissue samples (Table 1), and determined TUG1 expression levels in all of these samples using quantitative real-time PCR. By data statistics and analysis, we observed that TUG1 expression was significantly elevated in tumor tissues compared with para-tumor tissue (Fig. 1a), and univariate analysis revealed a close association between high TUG1 expression and patient survival (Fig. 1b). These data suggest that TUG1 upregulation is strongly associated with the clinical progression of human CRC.
Table 1 TUG1 (taurine upregulated gene 1) expression and clinicopathologic factors (n = 120)
TUG1 is upregulated in primary human CRC samples. a Statistical analysis was performed to present TUG1 expression in paired para-tumor/tumor samples in each clinical sample test. b Kaplan–Meier curves of CRC patients with low versus high expression of TUG1 (n = 120, P < 0.001, log-rank test); high expression showed that tumor TUG1 expression is 5 times higher than para-tumor tissue; low expression showed that tumor TUG1 expression is 5 times lower than para-tumor tissue. Values represent mean ± SD. * P < 0.05 compared with normal tissues
Possible negative regulating TUG1 expression by histone deacetylase
Next, we attempted to investigate the possible mechanism underlying aberrant TUG1 expression in CRC. The five representative CRC lines of SW480, HCT116, HT29, SW620, and LOVO were used for following analysis. We observed that TUG1 expression was significantly elevated in all CRC lines compared to normal colorectal cells (Fig. 2a). Interestingly, TUG1 expression was found to be 4–6 times higher than in corresponding cell lines (marked as control with no TSA treatment in presence of TSA, an inhibitor for histone deacetylase (Fig. 2b). We then evaluated HDAC1, HDAC2, and HDAC3 expression in these CRC lines in comparison to control and we found that HDAC1 expression was upregulated, while the expression levels of HDAC2 and HDAC3 protein remained unchanged in CRC cells (Fig. 2c). These data suggested that TUG1 expression was possibly regulated by abnormal expression HDAC1 expression. Thus, the CRC lines were used to create sub-lines with HDAC1 stably knocked down by shRNA focusing on analysis of TUG1 expression. From the result, we observed that HDAC1 silencing induced promotion (3.4–4.0 times the level of the si-control) of TUG1 expression (Fig. 2d).
Enhanced expression of TUG1 by histone deacetylase inhibition. a Relative ectopic expression of TUG1 in normal colorectal cell (control) and colorectal cancer cell lines by using Q-RT-PCR. b Relative expression of TUG1 in TSA treated normal tissue (control) and colorectal cancer cell lines. c Western blot analysis of HDAC1, HDAC2, and HDAC3 expression in normal colorectal cell (control) and colorectal cancer cell lines; β-actin served as a loading control. d Knock down of HDAC1 by si-HDAC transfection in normal colorectal cells (control) and colorectal cancer cell lines. e Relative expression of TUG1 in si-HDAC treated normal colorectal cells (control) and colorectal cancer cell lines. The colorectal cancer cell lines containing SW480, HCT116, HT29, SW620, and LOVO. Values represent mean ± SD. * P < 0.05 compared with control or si-control
TUG1 promotes the aggressiveness of CRC cells in vitro
Considering that TUG1 has a strongly correlation with CRC pathological courses, we transfected the SW480 cell line with pcDNA-TUG1 and detected the effect of overexpressed TUG1 on CRC cell metastasis capacity. The results showed that TUG1 overexpression induced an increase in colony formation (Fig. 3a) and caused a significant facilitation of cell migration in a wound-healing assay (Fig. 3b). We also utilized the classical Transwell assay to assess the contribution of TUG1 to cell migration and invasion. As shown in Fig. 3c and d, overexpressed TUG1 increased migration of SW480 cells and enhanced cell invasion ability.
Enhanced metastasis of CRC cells with overexpressed TUG1. a Representative image and number statistics for colony formation in SW480pcDNA and SW480pcDNA-TUG1 cells. b Wound-healing assay for motility of SW480pcDNA and SW480pcDNA-TUG1 cells. Representative pictures of one field at the beginning (t = 0) (left panel) and at the end of the recording (t = 12 h) (right panel) in each condition. c Representative images of transwell migrated cells and d invaded cells in stably transfected SW480pcDNA and SW480pcDNA-TUG1 cells and average number of migrated cells and invaded cells are shown in the right of (c) and (d). Values represent mean ± SD. * P < 0.05 compared with pcDNA
Knock-down TUG1 suppresses CRC cell metastasis in vitro
Another CRC cell line- LOVO was used to establish cell subline with TUG1 stably knocked down by TUG1-shRNA transfection with the purpose of evaluating CRC cell metastasis ability. The results showed that, compared with the si-control, TUG1 silencing reduced the number of colony formations (Fig. 4a). TUG1 silencing also promotes cell migration showed by a wound-healing assay (Fig. 4a). Additionally, by classical Transwell assay, we observed that TUG1-shRNA interference caused significant suppression in ability of cell migration and invasion as shown in Fig. 4c and d.
Silenced TUG1 inhibited metastasis of CRC cells. a Representative image and number statistics for colony formation in LOVOsi-control and LOVOsi-TUG1 cells. b Wound-healing assay for motility of LOVOsi-control and LOVOsi-TUG1 cells. Representative pictures of one field at the beginning (t = 0) (left panel) and at the end (t = 12 h) (right panel) of the recording in each condition are shown. c Representative images of transwell migrated cells, and d invaded cells in stably transfected LOVOsi-control and LOVOsi-TUG1 cells and the average number of migrated cells and invaded cells are shown in the right of (c) and (d). Values represent mean ± SD. * P < 0.05 compared with si-control
Inhibition of mice CRC progression by enhancing tumor-expressing TUG1
Liver metastasis of colon cancer is a common clinical phenomenon. Considering the identification of the key regulator role of TUG1 on CRC cell metastasis in vitro, we next established an in vivo experimental liver metastasis model by injecting human SW480 CRC cells into the spleens of BALB/c nude mice and followed their ability to invade-via the portal vein-the liver to form metastases and then investigated whether TUG1 plays an important role in the liver metastasis of CRC. To define the relationship between TUG1 and CRC liver metastasis in vivo, six, 7-week-old female BALB/c nude mice in each group were injected with SW480pcDNA-TUG1 or SW480pcDNA cells into the spleen before splenectomy. After 5 weeks, the mice were sacrificed, and the metastatic tumor nodules that formed in the liver were numbered. The data showed that metastatic tumor nodules were more frequently found in the livers of the SW480pcDNA-TUG1 group than in the SW480pcDNA group (Fig. 5).
Statistics for mice metastatic nodules in vivo. Nude mice were injected with SW480pcDNA or SW480pcDNA-TUG1 cells and tumor nodules were numbered 7 days post-transplantation. Values represent mean ± SD. * P < 0.05 compared with SW480-control
TUG1 facilitates epithelial-mesenchymal transition (EMT) in CRC cell lines
EMT is an important factor in cell invasion. Thus, we next determined whether EMT markers were altered in our model. The expression of E-cadherin, N-cadherin, vimentin, and Fibronectin protein level was analyzed by Western blot. By data, we found that the expression of N-cadherin, vimentin and Fibronectin was increased while E-cadherin expression was decreased, in SW480-TUG1 cells (Fig. 6a), whereas the opposite results were obtained when TUG1 was knocked down in LOVO cells (Fig. 6b).
Effect of TUG1 on EMT related-gene expression in colorectal cancer cell line of SW480 and LOVO. a Western blot analysis of EMT-related gene expression in pcDNA-TUG1-transfected SW480 cells. b Western blot analysis of EMT-related gene expression in si-TUG1-transfected LOVO cells. Values represent mean ± SD. * P < 0.05 compared with pcDNA or si-control
Recent evidence has shown that lncRNAs play an important role in cancer pathogenesis, and could provide new insights into the biology of this disease [7]. Although lncRNAs may have an impact on various human diseases, including cancer, the basis of their molecular mechanism is still beginning to be elucidated [16]. Likewise, no data are available for the role of them played during the CRC pathological process. One of these lncRNAs is metastasis-associated TUG1. Although TUG1 is recognized as the highly conserved nuclear lncRNA and is a predictive marker for metastasis development in human cancer [17], its role in CRC metastasis remains unclear.
In our current study, we analyzed the behavior of TUG1 in CRC metastasis. Initially, we observed that TUG1 expression levels in CRC tissues were higher than those in corresponding para-carcinoma tissues. As for clinicopathologic variables, TUG1 expression levels were speculated to be linked to colonic carcinogenesis. We thus chose five representative cell lines of CRC and investigated TUG1 levels with a non-tumoral colorectal cell line as control. By in vitro data, we then showed that TUG1 overexpression significantly enhanced tumor-like characteristics by increasing the colony formation, migration, and invasion of CRC cells. Accordingly, the opposite results were obtained when TUG1 was knocked down. These results indicate that TUG1 might play a key role in promoting metastasis of CRC, which was further proven by a mice liver metastasis model in which TUG1 overexpression significantly increased the number of metastatic tumor nodules in the liver. Our study is consistent with previous research revealing that the high expression of TUG1 in primary CRC was strongly associated with lung metastases [17]. Moreover, our data showed that high TUG1 expression in CRC tissues was closely associated with a decreased survival time in CRC patients. These multivariate analyses suggested that TUG1 might be an independent risk factor for CRC metastasis.
Knowledge of how lncRNAs are regulated in complex gene regulatory systems has attracted a lot of attention. Previously, hypermethylation of the promoter or the intergenic differentially methylated region has been found to contribute to reduced expression of lncRNA MEG3 in tumors, indicating that epigenetic regulation is also involved in the expression of these genes [18]. The fact that whether histone deacetylation that functioned as epigenetic regulatory factors manipulate the expression of TGU1 remains unknown. Our findings emphasize that histone deacetylase is a key factor in controlling the expression of the lncRNA TUG1. We observed that both TSA (an inhibitor for histone deacetylase) and HDACs knockdown enhanced THG1 expression. These results, along with those from a recent study [19], highlight the role of epigenetics in regulating lncRNA transcription.
To explore the molecular mechanism through which TUG1 contributes to the invasion and metastasis of CRC cells, we investigated potential target proteins involved in cell motility and matrix invasion, such as EMT-related gene expression. EMT is essential for cancer cell metastasis and it enhances tumor cell invasion in response to environmental triggers, and augments invasive functions and also contributes to cell growth and survival [20, 21]. Important hallmarks of EMT include the loss of E-cadherin expression and increased expression of non-epithelial cadherins, such as N-cadherin and vimentin. The loss of E-cadherin expression is a fundamental event in EMT, and a crucial step in the progression of papillomas to invasive carcinomas [13]. Therefore, we determined the protein levels of these EMT-induced markers following ectopic expression of TUG1. Our results indicated that TUG1 overexpression reduced E-cadherin expression and enhanced the expression levels of N-cadherin, vimentin, and Fibronectin, whereas knockdown of endogenous TUG1 expression significantly abrogated these capacities. These data indicated that TUG1 might influence CRC metastasis by mediating EMT-related gene expression.
In summary, the expression of TUG1 was significantly increased in CRC tumor tissues, suggesting that its downregulation may be a negative prognostic factor for CRC patients, and indicative of poor survival rates and a higher risk for cancer metastasis. We showed that TUG1 possibly regulates the invasive and metastatic ability of CRC cells, partially through regulation of EMT. Our findings promote further the understanding of CRC pathogenesis and development, and facilitate the development of lncRNA-directed diagnostics and therapeutics against cancers. However, the molecular mechanisms by which HDAC1 controls TUG1 expression and TUG1 regulates EMT require further investigation.
An amendment to this paper has been published and can be accessed via the original article.
lncRNAs:
long intergenic non-coding RNAs
TUG1:
taurine up-regulated gene 1
EMT:
epithelial-mesenchymal transition
RNA pol II:
RNA polymerase II
kb:
kilobases
PBS:
phosphate-buffered saline
HRP:
horseradish peroxidase
ABM:
applied biological materials
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JS carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. CD carried out the immunoassays. ZY participated in the sequence alignment. TL participated in the design of the study and XZ performed the statistical analysis. CZ and JW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
This study is supported by 2013 Science and Technology Cooperation Programme of Henan Province (No.132106000071).
Gastrointestinal Surgery, The First Affiliated Hospital of Zhengzhou University, No.1 Jianshe East, Zhengzhou, 450052, China
Junfeng Sun, Chaohui Ding, Zhen Yang, Tao Liu, Xiefu Zhang & Chunlin Zhao
Pediatric Surgery, The First Affiliated Hospital of Zhengzhou University, No.1 Jianshe East, Zhengzhou, 450052, China
Jiaxiang Wang
Junfeng Sun
Chaohui Ding
Zhen Yang
Xiefu Zhang
Chunlin Zhao
Correspondence to Chunlin Zhao or Jiaxiang Wang.
The original version of this article was revised: errors in Figures 3c-d and 4c-d were corrected.
Sun, J., Ding, C., Yang, Z. et al. The long non-coding RNA TUG1 indicates a poor prognosis for colorectal cancer and promotes metastasis by affecting epithelial-mesenchymal transition. J Transl Med 14, 42 (2016). https://doi.org/10.1186/s12967-016-0786-z
DOI: https://doi.org/10.1186/s12967-016-0786-z
Colorectal cancer cell lines
Taurine upregulated gene 1
Cancer microenvironment

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Hans Gildemeister was the defending champion, but did not compete this year.

Pablo Arraya won the title by defeating Juan Aguilera 7–5, 7–5 in the final.

Seeds

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Bottom half

References

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1983 Grand Prix (tennis)
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Phay Mutepa's acceptance speech is a Spoken Word Classic!
Africa Web TV's Phay Mutepa delivers her acceptance speech in spoken words as she became the winner of the Africa Media Award 2017.
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text:
Az aranymetszés vagy aranyarány egy olyan arányosság, ami a természetben és művészetben is gyakran megjelenik, természetes egyensúlyt teremtve a szimmetria és az aszimmetria között.

Aranymetszési arányok találhatók számos ókori épületen, középkori és reneszánsz képzőművészeti alkotásokon. Az ókori püthagoreusok (Püthagorasz és követői), akik szerint a valóság matematikai alapokon nyugszik, az aranymetszésben a létezés egyik alaptörvényét vélték felfedezni, ugyanis ez az arány felismerhető a természetben is (például az emberi testen vagy csigák mészvázán).

Az aranymetszés arányait tartalmazó formák máig nagy esztétikai értékkel bírnak, számos területen (például a tipográfiában vagy a fényképészetben) alkalmazzák őket.

Az aranyarányt numerikusan kifejező irracionális Φ ≈ 1,618 számnak (görög nagy fí) számos érdekes matematikai tulajdonsága van.

Matematikai definíció

Két rész (a és b, a>b) az aranymetszés szerint aránylik egymáshoz, ha az egész (a+b) úgy aránylik a nagyobbik részhez (a), ahogy a nagyobbik rész (a) a kisebbik részhez (b):
.

Vagyis a nagyobbik rész egyenlő az összeg és a kisebbik rész mértani közepével:
.

A fentiekkel egyenértékű az a megfogalmazás, hogy a nagyobbik rész úgy aránylik a kisebbik részhez, mint a kisebbik rész a két rész különbségéhez:
 azaz:
.

Története
Gyakori megjelenése miatt a geometriában már ókori matematikusok is tanulmányozták az aranymetszést. Bizonyíthatóan az ókori Egyiptomban is értették és használták ezt a törvényszerűséget. Az i. e. 2600 körül épült gízai nagy piramis arányaiban is felfedezhető az aranymetszés aránya. A piramis alapélének a fele (átlag 115,18 m) és oldallapjainak a magassága (kb. 186,42 m) az aranymetszés szerint aránylik egymáshoz (0,03%-os eltéréssel, ami hibahatáron belülinek tekinthető).

Az ókori görögök is ismerték ezt az arányt. Püthagorasz, Theodórosz és Eukleidész is foglalkozott vele. Az aranymetszés jelölése, a Φ (görög nagy fí betű) Pheidiász görög szobrász nevéből származik, aki gyakran alkalmazta munkájában. (További jelölések lejjebb.)

Adolf Zeising (1810–1876) Aus experimenteller Ästhetik (A kísérleti esztétikából) című művében ír nagyszámú emberen végzett méréseiről. A jól kifejlett emberi alaknak első osztási pontját a köldökre tette és megállapította, hogy a test törzsének és főbb tagjainak illeszkedési pontjai szintén az aranymetszés szerint aránylanak. Kétségtelen, hogy a korábbi, különösen a görög szoborművek arányai is megfelelnek Zeising elméletének: ha a test magassága 1000, a test alsó része a köldöktől 618, a test felső része a köldöktől 382, a fej hossza pedig 146. Ezek mind az aranymetszési szabály szerint viszonyulnak egymáshoz. Zeising azonkívül megkísérelte az ókor és a középkor legkiválóbb építményein kimutatni, hogy azoknak egészén és egyes részeinek méreteiben az aranymetszés elve uralkodik, ahogy a festészet legismertebb alkotásainak elrendezésében is ugyanez az elv érvényesül.

Az ókorban isteni számnak is nevezték, ugyanis az emberek nem csak matematikai tényként tekintettek rá, hanem az istenség földi jelenlétének és a teremtésnek a kifejezőjeként is értelmezték.

Művészet
Több neves művész, illetve műalkotás épít az aranymetszés szabályaira. Például a magyar Szent Korona, Bartók Béla bizonyos zeneművei, Dante Alighieri Isteni színjátéka, Kassák Lajos A ló meghal… kezdetű költeménye, Leonardo da Vinci és Michelangelo festményei.

Tipográfia
A tipográfia – avagy a betűk művészete – is épít az aranymetszés szabályaira: a címek, alcímek és a szövegtörzs betűméretének viszonyát általában az aranyarányban szokás megállapítani.

Az aranyarány tényezője, a fí

Jelölése
Az aranymetszés szerinti a>b számok arányának jelölése nem egységes.
 a/b jelölésére használatos a  (nagy fí) jelölés (ebben a cikkben is így szerepel).
 Szokás ugyanezt a számot -del vagy -del (a kis fí változatai) jelölni, ám ekkor a nagy  b/a-t jelöli.
 Szokás -del jelölni az  egyenlet másik megoldását, azaz a −b/a-t.
 Ritkábban a  (kis tau) is előfordul az a/b hányados jelölésére.

Kiszámítása
A definícióból kiszámolható, hogy a nagyobb rész (a) hányszorosa a kisebb résznek (b), tehát megkapható az a  szám, amelyre , másképpen:  teljesül.

A definíció szerint:

Innen

Ebbe -t behelyettesítve kapjuk, hogy

-vel szorozva, majd 0-ra rendezve:

Ezt a másodfokú egyenletet megoldhatjuk a megoldóképlettel:

Az egyenlet negatív gyöke (≈ - 0,618) a feladat jellege miatt nem megoldása a problémának, így:

 irracionális szám, tehát nem írható fel két egész szám hányadosaként, ami a  irracionalitásából is látható. Algebrai szám, sőt, algebrai egész, hiszen megoldása a fenti polinomegyenletnek.

Kapcsolata a Fibonacci-sorozattal

A Fibonacci-sorozat első két tagja a 0 és az 1. A következő tagok mindig az őket megelőző két tag összegével egyenlők. (0, 1, 1, 2, 3, 5, 8, 13, 21, 34, 55, 89, 144, 233, 377, 610, …)

A Fibonacci-sorozat egymást követő tagjainak hányadosából képzett sorozat (1/1, 2/1, 3/2, 5/3, …) határértéke éppen az aranymetszés aránya, a .

Törtelőállítások

Végtelen lánctört-előállítás
Mivel

ezért

továbbá

és így tovább.

Ezzel az arányszám ún. (végtelen) lánctört-előállítását kapjuk:

Előállítása lineáris törtfüggvény-sorozat tagjai alakjában
Ha a fenti lánctört-sorozat tagjait egyszerűsítjük, úgy hogy a bennük szereplő törteket közös nevezőre hozzuk, érdekes dologra juthatunk. Ezt azonban egyszerűbben is megtehetjük: Mivel

 a jobb oldalon álló Φ-kbe behelyettesítve a bal oldalon álló Φ jobb oldali alakját:

Most az így kapott kifejezéssel ugyanazt csinálva, mint előbb, azaz beírva a legelső egyenlet jobb oldalát, adódik:

Észrevehető, hogy a számláló és nevező együtthatói az  Fibonacci-sorozat szomszédos elemei. Teljes indukcióval bizonyítható, hogy általában is:

Közelítés
A szám irracionális, közelíthetőség szempontjából pedig meglehetősen rosszul viselkedik. Egyrészt végtelen sok olyan p/q racionális szám van, amire
,
hiszen ez minden irracionális számra teljesül.

Mivel  algebrai, így ez semmilyen  esetén nem igaz már -ra (Roth tétele).

Az is igaz, hogy végtelen sok olyan p/q racionális szám van, amire

teljesül, mert Hurwitz approximációs tétele miatt ez is teljesül minden irracionális számra.

Mivel  olyan szám, aminek lánctörtalakja egy küszöbtől kezdve csupa 1-es, ezért itt  helyére nem lehet nagyobb számot írni. Mivel minden olyan szám esetén, amikor a számra nem igaz, hogy egy küszöbtől kezdve a lánctört alakjában csupa 1-es áll, akkor -cal is igaz az állítás.

Vagyis ilyen értelemben  azok közé a számok közé tartozik, amik a lehető legrosszabbul közelíthetők.

Grafikus megállapítása

Ha az aránypárban  adott, akkor  is egyértelműen meghatározott, ekkor -nek a szerkesztése a következőképpen történik.
Felveszünk egy tetszőleges  szakaszt, amely az aranymetszés arányai szerint a nagyobbik rész, és ehhez szerkesztjük meg az  szakaszt, amely a kisebbik rész lesz.
Az  szakasz  végpontjába merőleges félegyenest állítunk -ra, erre felmérjük az  távolságot. Legyen ennek végpontja az  pont. -ból  sugárral körívet húzunk, amely az  szakaszt -hoz közelebb eső  pontban metszi. Az  távolság lesz az arány kisebbik része, ugyanis a külső pontból húzott érintő- és szelőszakaszok tétele alapján:
.

Az aranymetszés a szabályos tízszög szerkesztése, illetve a szabályos ötszög szerkesztése során nagy segítséget nyújt.

Kapcsolódó szócikkek
 Fibonacci-számok
 Pentagramma
 Vitruvius-tanulmány
 Penrose-féle csempézés
 Arany spirál

Jegyzetek

Források

 Az aranymetszés algebrája
 Spirálok és aranymetszés
 Mi az aranymetszés – példákkal műalkotásokból
 Fibonacci-számok és aranymetszés
 A Φ arányossági tényező: matematika és egyéb
 Phi the Golden Number
 Szoftveres megvalósítás
 Goldenmuseum - kulturális vonatkozások  és
 Ókori történelme  és
 Híres kutatók
 Matematika
 Rövid lexikoncikk

Euklideszi geometria
Elemi matematika
Irracionális számok


My research area covers advanced MRI techniques for improved diagnosis and monitoring of treatment response in paediatric cancer. I specialize in the development of new imaging techniques for measuring the cellular and vascular structure of paediatric brain tumours. I am currently leading the integration of these techniques into clinical practice at Great Ormond Street Children's Hospital. Using these techniques, we can monitor the blood supply to a tumour, which will have a strong influence on the efficacy of drug delivery. Changes in the tumour microstructure as a response to treatment can also be measured.
Hales PW et al. A multi-Gaussian model for apparent diffusion coefficient histogram analysis of Wilms' tumour subtype and response to chemotherapy. NMR in Biomedicine, 2015 Aug, 28(8): 948-957.
Grech-Sollars M, Hales PW et al. Multi-centre reproducibility of diffusion MRI parameters for clinical sequences in the brain. NMR in Biomedicine, 2015 Apr, 28(4): 468–485.
Hales PW et al. A two-stage model for in vivo assessment of brain tumor perfusion and abnormal vascular structure using arterial spin labeling. PLoS ONE 2013 Oct; 8(10): e75717.


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text:
#include "BoxImage.h"

#include <gif_lib.h>
#include <jpeglib.h>
#include <png.h>
#include <stdio.h>

#include <GL/gl.h>

#include <boost/bind.hpp>

#include "Bmp.h"

#include "utf.h"
#include "http/HTTPRequest.h"

namespace org { namespace w3c { namespace dom { namespace bootstrap {

// image/png - 89 50 4E 47 0D 0A 1A 0A
// image/gif - "GIF87a" or "GIF89a"
// image/jpeg - FF D8
// image/x-bmp - "BM"
// image/vnd.microsoft.icon - 00 00 01 00 (.ico), 00 00 02 00 (.cur)

namespace {

unsigned char* readAsPng(FILE* file, unsigned& width, unsigned& height, unsigned& format)
{
    png_byte header[8];
    if (fread(header, 1, 8, file) != 8 || png_sig_cmp(header, 0, 8))
        return 0;

    png_structp png_ptr = png_create_read_struct(PNG_LIBPNG_VER_STRING, NULL, NULL, NULL);
    if (!png_ptr)
        return 0;
    png_infop info_ptr = png_create_info_struct(png_ptr);
    if (!info_ptr) {
        png_destroy_read_struct(&png_ptr, NULL, NULL);
        return 0;
    }
    png_infop end_info = png_create_info_struct(png_ptr);
    if (!end_info) {
        png_destroy_read_struct(&png_ptr, &info_ptr, NULL);
        return 0;
    }
    if (setjmp(png_jmpbuf(png_ptr))) {
        png_destroy_read_struct(&png_ptr, &info_ptr, &end_info);
        return 0;
    }

    png_init_io(png_ptr, file);
    png_set_sig_bytes(png_ptr, 8);

    png_read_info(png_ptr, info_ptr);

    width = png_get_image_width(png_ptr, info_ptr);
    height = png_get_image_height(png_ptr, info_ptr);
    unsigned bit_depth = png_get_bit_depth(png_ptr, info_ptr);
    unsigned color_type = png_get_color_type(png_ptr, info_ptr);

    if (color_type == PNG_COLOR_TYPE_PALETTE)
        png_set_palette_to_rgb(png_ptr);
    if (color_type == PNG_COLOR_TYPE_GRAY && bit_depth < 8)
        png_set_expand_gray_1_2_4_to_8(png_ptr);
    if (color_type == PNG_COLOR_TYPE_GRAY || color_type == PNG_COLOR_TYPE_GRAY_ALPHA)
        png_set_gray_to_rgb(png_ptr);
    if (png_get_valid(png_ptr, info_ptr, PNG_INFO_tRNS)) {
        png_set_tRNS_to_alpha(png_ptr);
        color_type = GL_RGBA;
    }
    if (bit_depth == 16)
        png_set_strip_16(png_ptr);
    switch (color_type) {
    case PNG_COLOR_TYPE_RGB:
    case PNG_COLOR_TYPE_GRAY:
    case PNG_COLOR_TYPE_PALETTE:
        format = GL_RGB;
        break;
    default:
        format = GL_RGBA;
        break;
    }
    png_set_interlace_handling(png_ptr);

    png_read_update_info(png_ptr, info_ptr);

    unsigned rowbytes = png_get_rowbytes(png_ptr, info_ptr);
    png_bytep data = (png_bytep) malloc(rowbytes * height);
    png_bytep* row_pointers = (png_bytep*) malloc(sizeof(png_bytep) * height);
    for (unsigned i = 0; i < height; i++)
        row_pointers[i] = &data[rowbytes * i];
    png_read_image(png_ptr, row_pointers);
    free(row_pointers);

    png_read_end(png_ptr, end_info);
    png_destroy_read_struct(&png_ptr, &info_ptr, &end_info);

    return data;
}

unsigned char* readAsJpeg(FILE* file, unsigned& width, unsigned& height, unsigned& format)
{
    unsigned char sig[2];
    if (fread(sig, 1, sizeof sig, file) != sizeof sig || sig[0] != 0xFF || sig[1] != 0xD8)
        return 0;
    fseek(file, -(sizeof sig), SEEK_CUR);

    JSAMPARRAY img;
    struct jpeg_decompress_struct cinfo;
    struct jpeg_error_mgr jerr;

    cinfo.err = jpeg_std_error(&jerr);  // TODO: set our own error handdler
    jpeg_create_decompress(&cinfo);

    jpeg_stdio_src(&cinfo, file);
    jpeg_read_header(&cinfo, true);
    width = cinfo.image_width;
    height = cinfo.image_height;
    jpeg_start_decompress(&cinfo);

    unsigned char* data = (unsigned char*) malloc(height * width * cinfo.out_color_components);
    img = (JSAMPARRAY) malloc(sizeof(JSAMPROW) * height);
    for (unsigned i = 0; i < height; ++i)
        img[i] = (JSAMPROW) &data[cinfo.out_color_components * width * i];

    while(cinfo.output_scanline < cinfo.output_height)
        jpeg_read_scanlines(&cinfo,
                            img + cinfo.output_scanline,
                            cinfo.output_height - cinfo.output_scanline);
    jpeg_finish_decompress(&cinfo);
    jpeg_destroy_decompress(&cinfo);

    free(img);

    if (cinfo.out_color_components == 1)
        format = GL_LUMINANCE;
    else
        format = GL_RGB;
    return data;
}

uint16_t readUint16(const char* p)
{
    return static_cast<uint8_t>(p[0]) + (static_cast<uint8_t>(p[1]) << 8);
}

unsigned char* expandColors(unsigned char* dst, unsigned char* src, unsigned count, int transparentIndex, GifColorType* colors)
{
    for (unsigned i = 0; i < count; ++i, ++src) {
        if (*src == transparentIndex) {
            *dst++ = 0;
            *dst++ = 0;
            *dst++ = 0;
            *dst++ = 0;
        } else {
            GifColorType* color = &colors[*src];
            *dst++ = color->Red;
            *dst++ = color->Green;
            *dst++ = color->Blue;
            *dst++ = 255;
        }
    }
    return src;
}

unsigned char* readAsGif(FILE* file, unsigned& width, unsigned& height, unsigned& format, unsigned &frameCount, std::vector<uint16_t>& delays, unsigned& loop)
{
    unsigned char sig[6];
    if (fread(sig, 1, sizeof sig, file) != sizeof sig)
        return 0;
    if (memcmp(sig, GIF87_STAMP, 6) && memcmp(sig, GIF89_STAMP, 6))
        return 0;
    fseek(file, -(sizeof sig), SEEK_CUR);
    long pos = ftell(file);

    int fd = fileno(file);
    lseek(fd, pos, SEEK_SET);
    GifFileType* gif = DGifOpenFileHandle(fd);
    if (!gif)
        return 0;
    if (DGifSlurp(gif) != GIF_OK || gif->ImageCount < 1) {
        DGifCloseFile(gif);
        return 0;
    }

    width = gif->SWidth;
    height = gif->SHeight;
    frameCount = gif->ImageCount;
    loop = 1;
    unsigned char* data = (unsigned char*) malloc(width * height * 4 * frameCount);
    if (!data) {
        DGifCloseFile(gif);
        return 0;
    }

    if (1 < frameCount)
        delays.resize(frameCount, 10);

    format = GL_RGBA;
    for (unsigned frame = 0; frame < frameCount; ++frame) {
        SavedImage* image = &gif->SavedImages[frame];
        int transparentIndex = -1;
        for (int i = 0; i < image->ExtensionBlockCount; ++i) {
            ExtensionBlock* ext = image->ExtensionBlocks + i;
            switch (ext->Function) {
            case GRAPHICS_EXT_FUNC_CODE:
                if (ext->ByteCount == 4) {
                    if ((ext->Bytes[0] & 1))    // transparent?
                        transparentIndex = static_cast<unsigned char>(ext->Bytes[3]);
                    delays[frame] = readUint16(ext->Bytes + 1);
                }
                break;
            case APPLICATION_EXT_FUNC_CODE:
                if (ext->ByteCount == 11 && (!memcmp(ext->Bytes, "NETSCAPE2.0", 11) || !memcmp(ext->Bytes, "ANIMEXTS1.0", 11))) {
                    ++ext;  // Move to the sub-block
                    if (ext->ByteCount == 3) {
                        loop = readUint16(ext->Bytes + 1);
                    }
                }
                break;
            default:
                break;
            }
        }
        unsigned char* p0 = data + frame * width * height * 4;
        ColorMapObject* colorMap = image->ImageDesc.ColorMap ? image->ImageDesc.ColorMap : gif->SColorMap;
        if (!image->ImageDesc.Interlace)
            expandColors(p0, image->RasterBits, width * height, transparentIndex, colorMap->Colors);
        else {
            unsigned char* index = image->RasterBits;
            for (unsigned row = 0; row < height; row += 8)
                index = expandColors(p0 + width * row * 4, index, width, transparentIndex, colorMap->Colors);
            for (unsigned row = 4; row < height; row += 8)
                index = expandColors(p0 + width * row * 4, index, width, transparentIndex, colorMap->Colors);
            for (unsigned row = 2; row < height; row += 4)
                index = expandColors(p0 + width * row * 4, index, width, transparentIndex, colorMap->Colors);
            for (unsigned row = 1; row < height; row += 2)
                index = expandColors(p0 + width * row * 4, index, width, transparentIndex, colorMap->Colors);
        }
    }
    DGifCloseFile(gif);
    return data;
}

unsigned char* readAsBmp(FILE* file, unsigned& width, unsigned& height, unsigned& format)
{
    BitmapFileHeader fileHeader;
    if (!fileHeader.read(file))
        return 0;
    BitmapInfoheader header;
    if (!header.read(file))
        return 0;
    RGBQuad colorTable[header.usedColors ? header.usedColors : 1];
    if (0 < header.usedColors && !header.readColorTable(file, colorTable))
        return 0;
    width = header.getWidth();
    height = header.getHeight();
    format = GL_RGBA;
    unsigned char* data = static_cast<unsigned char*>(malloc(width * height * 4));
    if (!data)
        return 0;
    if (std::fseek(file, fileHeader.offset, SEEK_SET) == -1)
        return 0;
    bool result = header.readPixels(file, colorTable, data);
    if (!result) {
        free(data);
        return 0;
    }
    return data;
}

// file must points to BitmapInfoheader
unsigned char* readAsIco(FILE* file, unsigned& width, unsigned& height, unsigned& format)
{
    BitmapInfoheader header;
    if (!header.read(file))
        return 0;
    RGBQuad colorTable[header.usedColors ? header.usedColors : 1];
    if (0 < header.usedColors && !header.readColorTable(file, colorTable))
        return 0;
    width = header.getWidth();
    height = header.getHeight();
    if (width * 2 == height) {   // has AND plane?
        header.height /= 2;
        height /= 2;
    }
    format = GL_RGBA;
    unsigned char* data = static_cast<unsigned char*>(malloc(width * height * 4));
    if (!data)
        return 0;
    // TODO: Process AND plane.
    // TODO: Support JPEG and PNG.
    bool result = header.readPixels(file, colorTable, data);
    if (!result) {
        free(data);
        return 0;
    }
    return data;
}

}  // namespace

BoxImage::BoxImage(unsigned repeat) :
    state(Unavailable),
    flags(0),
    pixels(0),
    naturalWidth(0),
    naturalHeight(0),
    repeat(repeat),
    format(GL_RGBA),
    frameCount(1),
    delays(1),
    total(0.0f)
{
}

void BoxImage::open(FILE* file)
{
    assert(file);
    long pos = ftell(file);
    pixels = readAsIco(file, naturalWidth, naturalHeight, format);
    if (!pixels) {
        fseek(file, pos, SEEK_SET);
        pixels = readAsPng(file, naturalWidth, naturalHeight, format);
    }
    if (!pixels) {
        fseek(file, pos, SEEK_SET);
        pixels = readAsJpeg(file, naturalWidth, naturalHeight, format);
    }
    if (!pixels) {
        fseek(file, pos, SEEK_SET);
        pixels = readAsGif(file, naturalWidth, naturalHeight, format, frameCount, delays, loop);
    }
    if (!pixels) {
        fseek(file, pos, SEEK_SET);
        pixels = readAsBmp(file, naturalWidth, naturalHeight, format);
    }
    if (!pixels) {
        state = Broken;
        return;
    }
    state = CompletelyAvailable;
    total = 0.0f;
    for (size_t i = 0; i < delays.size(); ++i)
        total += delays[i];
}

unsigned BoxImage::getCurrentFrame(unsigned t, unsigned& delay, unsigned start)
{
    if (frameCount <= 1 || total == 0.0f)
        return 0;
    if (loop) {
        t -= start;
        if (total * loop <= t)
            return 0;
    }
    t %= total;
    unsigned d = 0.0f;
    for (unsigned i = 0; i < delays.size(); ++i) {
        d += delays[i];
        if (t < d) {
            delay = d - t;
            return i;
        }
    }
    return 0;
}

//
// HttpRequest
//

BoxImage* HttpRequest::getBoxImage(unsigned repeat)
{
    if (!boxImage)
        boxImage = new(std::nothrow) BoxImage(repeat);
    if (!boxImage)
        return 0;

    switch (getReadyState()) {
    case UNSENT:
        boxImage->setState(BoxImage::Unavailable);
        break;
    case OPENED:
    case HEADERS_RECEIVED:
    case LOADING:
    case COMPLETE:
        boxImage->setState(BoxImage::Sent);
        break;
    case DONE:
        if (boxImage->getState() < BoxImage::PartiallyAvailable) {
            if (getError()) {
                boxImage->setState(BoxImage::Unavailable);
                break;
            }
            if (FILE* file = openFile()) {
                boxImage->open(file);
                fclose(file);
            }
        }
        break;
    default:
        boxImage->setState(BoxImage::Unavailable);
        break;
    }
    return boxImage;
}

}}}}  // org::w3c::dom::bootstrap

redpajama_set_name: RedPajamaGithub


Hey everyone, the two wire connector beside the rad cap, what is it for, low coolant warning? Can someone tell me how it is tested to see if it's working? I'd love to rig up a warning light on my gauge panel. Ignore the green circled thing.
Why Yes it IS a level warning.
But on a stock car it only throws an error code 54.
Wire it it up as you please if nit going to the stock ST185 RC/CS or ST205 ecu.
You can test the senor with a VOM meter.
Awesome, I'm going to toss a 12v bulb to it, fill it up and see if it turns off once full. Great peace of mind/cheap insurance.
And thanks Gman for the quick response.
You can test it by flipping the intercooler upside down to simulate "full". I believe open loop is low coolant and closed is full.


Since the Supreme Court of the United States handed down its opinion on June 4 in Masterpiece Cakeshop, Ltd. v. Colo. Civil Rights Comm'n, 138 S. Ct. 1719, 1723 (2018), many an LGBT rights attorney have been asked what does it all mean. Certainly, media outlets have tried their hands at distilling it down into 30-second sound bites, some better than others. At least to this LGBT rights attorney, several members and allies of the LGBT community have expressed concern that, despite what the technical legal import may be, some anti-LGBT individuals in their communities will be emboldened and act accordingly. Interestingly, all sides of the political spectrum appear to have become emboldened. We have seen recent news stories of employees and owners of establishments open to the public deny service to patrons with whom they disagree politically. Woe to the patron wearing a MAGA hat or the gay couple in a dive bar.
Many of us have heard that the Masterpiece Cakeshop decision was narrow and limited to its facts. That is essentially correct. Former Justice Anthony Kennedy, writing for the majority, ruled in favor of the Colorado baker, who refused to create a cake for the wedding of a gay couple. Justice Kennedy reasoned that, because one or two of the commissioners at the Colorado Human Rights Commission (where the lawsuit began) made biased comments against the baker's assertion of a sincerely held religious belief against gay marriage, the government acted with hostility to the religious beliefs of the baker and in a manner that passed judgment upon the legitimacy of the baker's religious belief. This is contrary to the requirements of the free exercise clause of the First Amendment. Additionally, Justice Kennedy reasoned that the Colorado Commission decided other similar cases under different standards, resulting in dissimilar rulings. For these procedural reasons, the Court found in favor of the baker (concurring opinions by other justices agreed with the ultimate ruling, but not necessarily the detailed reasoning).
Many of us may wonder how this is even an issue. Several of you readers may have grown up during the civil rights era of the 1950s and 1960s. And several of you readers are at least well aware of that history. The Supreme Court decided the religious expression versus public accommodation issue many decades ago, ruling that non-religious entities open to the general public cannot deny services to individuals based on the color of their skin, despite any sincerely held religious beliefs. Freedom from discrimination trumps a religious right to discriminate. The same logic would seem to apply to the category of LGBT individuals. But, as many in the legal profession know, the Court tends to avoid ruling directly on such hot button issues if it can (although many recent and not-so-recent exceptions come to mind).
However, the Court wholly deflected the issue of whether creating a wedding cake constitutes artistic expression protected as speech under the First Amendment. This author, for one, finds this issue to be a much greater legal minefield for the Court, which deftly sidestepped the issue entirely. But this battle will likely continue for advocates who see a more favorable Court in the future.
So what do we as lawyers take from this decision? First, on both sides of the bar, lawyers should look at the procedural record for any religious animus to bolster their position in these types of public accommodation suits. Second, the ultimate issue of freedom from discrimination versus a religious right to discriminate against the non-heterosexual community is still technically up in the air. Third, the issue of protected speech versus commercial activity remains at the status quo, that is, vague and fact specific. Of note, in Washington's own 'public accommodation versus religious freedom case,' State v. Arlene's Flowers, Inc., 187 Wn.2d 804, 820, 389 P.3d 543, 551 (2017), which has been remanded by SCOTUS in light of its Masterpiece Cakeshop ruling, an attorney for the gay plaintiffs has informed certain WSBA members that they feel confident the WA Supreme Court ruling in favor of the gay couple seeking flowers for their wedding will still hold despite the Masterpiece Cakeshop ruling.
And what do we as human beings take from this decision? It is this author's opinion that this decision presents a larger lesson pertinent to this current political climate. Whether or not one agrees that certain Colorado Commissioner comments demonstrated bias and hostility, we should now better understand that there is no place in reasoned and open debate for vitriol and hostility. If we wish to change hearts and minds, to any position, personal attacks and lack of empathy never work. Anger is easy and ultimately unsuccessful. Compassion and open-minded discussion are hard but often fruitful.


text:
Review: 'Family' too familiar
The Film Arcade, 2019.
This forgettable, throw-away indy comedy has some funny moments, but they're sparse.
Grade: C-
Taylor Schilling (Piper from "Orange is the New Black") stars as a walking cliché in "Family," a new film that will mostly be remembered for featuring Juggalos. What's a Juggalo, you might ask? It's a fan of the psychopathic rap group known as The Insane Clown Posse (or "ICP"), a crazy hardcore hip-hop group that started in Detroit in the late 80s and caught fire in the early 90s. Think "Parrot-head" but with everyone's face painted like a clown, where evil and chaos is pedaled instead of margaritas and sandals.
Kate (Schilling) is a socially-awkward business woman who is all work and no play. She cares for nobody and doesn't seem to have an empathetic bone in her body. When her brother (Eric Edelstein) calls her up in a panic, she reluctantly agrees to watch her young niece, Maddie (Bryn Vale) as a favor. Kate has no such skills, and Maddie has her own troubles with bullies and whatnot, and...well...you can sort of see where this is headed.
Kate learns from Maddie and Maddie learns from Kate. Yawn. This universe is populated with a sea of semi-interesting side characters, with Kate McKinnon, Brian Tyree Henry and Matt Walsh showing up, but it's the young Fabrizio Zacharee Guido who brings the real laughs every time he appears (he's the lone stand-out). None of these characters are around long enough to shift the movie towards "worth seeing." Instead, we're stuck with the miserable Kate, who eventually learns to open up her heart when she chases Maddie through a Juggalo gathering, with the real ICP on stage.
Nothing really comes together, nothing really works in the dysfunctional "Family." You might chuckle, or smile a few times, or if you self-identify as a Juggalo you may just be happy that your "people" are getting film recognition. But for everyone else? Real friends don't let friends see "Family."
Genre: Comedy.
Run Time: 1 hour 25 minutes.
Rated R.
Starring: Taylor Schilling, Kate McKinnon, Bryn Vale, Brian Tyree Henry, Matt Walsh, Jessie Ennis, Eric Edelstein.
Written and Directed by Laura Steinel (feature-film directorial debut).
"Family" opens in limited release on Friday, April 26th, 2019.


Go Windsurfing is 1 year old!
To celebrate this year we will be offering a free windsurfing holiday in Fiji as our major prize in a season long competition. All you have to do to enter is enroll on any Go Windsurfing course in between October and March 2004/2005 and your name will be entered into a raffle at the end of the season. The prize includes everything in the land package (see Fiji below) , all you will have to do is get yourself to Ellington Wharf.We will be offering some consolation prizes as well in the form of windsurfing clinics in WA, NSW or New Zealand.More of this in my October newsletter.
Fiji was a resounding success, with six weeks of fantastic conditions on the island of Nananu-I-Ra(see photo below).What a place!!! We were the guests of John and Phyl Morrison who run a small resort right on the beach of this island paradise. To go sailing it was a simple case of walking 20 feet to the waters edge (from your hammock) and blasting off. The normal wind in Fiji at this time of year is 15-30 knots south east Trade winds, although this year we had a couple of weeks with unseasonable rain and light wind, but this was very unusual.
Next year our Fiji clinics will be up and running from May through to November. Virgin Blue have just started to fly to Fiji from Sydney and Brisbane for as little as $600 return ,so the cost of getting there next year will be even cheaper.
Our package prices for next season are as follows.
Taxi from Nadi Airport to Ellington wharf approx $85 FJ, we will send a taxi down to meet you at Nadi airport.
Beer and any tipple you fancy whilst on your windsurf holiday.
All costs are in Australian dollars unless shown otherwise.
Safety Bay will be up and running again from the 2nd October, with a Have a Go Day on the 4th October.
As well as offering free lessons to novices ,there will also be demo boards from as many of the major brands as i can muster. I will you updated in the next few weeks as to what.Go Windsurfing will also offering a free sausage sizzle and rumor has it, there will be a swap meet as well.
The school has grown from last year, with a heap of new boards from Mistral,F2 and and JP. Boards will include the F2 Powerglides and Styles, and Mistral Syncros. Rigs for this year will be from North Sails and Neil Pryde, with sizes from 4m up to 7m .
For more details on course check out our newly updated website.
New for the coming year, Go Windsurfing will also be offering Kitesurfing lessons. We have two new coaches from the UK who as well as being great windsurfing instructors are also kite instructors. For those who do not want to see more kites in the bay, do not worry, as we have negotiated with CALM to operate out of Shoalwater Bay, so the two activities will stay separate.
New for 2004/2005 season is Go Windsurfng New Zealand . Our head honcho in Auckland is Peter Head. Peter is originally from the Uk and has been working in the watersport and ski industry for the last ten years. He will be running windsurfing clinics to all of New Zealand's windsurfing hotspots, including Taranaki, whangaparaoa, Raglan, Bay Of Islands/ far North, Mt. Maunganui, Christchurch/ Southern Lakes and Queens Town/ Wanaka, etc ,etc, as well as running climbing, snow boarding trips and mountain biking . Having just spent a few days snow boarding, only 24 hours after windsurfing on a tropical beach in Fiji, I have to admit it is a bit a mind bender!!! For more details on what Pete is up to check out our New Zealand page on our website.
I will be looking at doing one, possibly two east coast windsurfing clinics this summer, probably in NSW. I am in the process of finding the right location, but will let you know asap, but in the mean time if any of you east coasters have any good spots and would like me to come over please let me know.


text:
BromsaMS
Finns möjligheten att bromsa MS
example group
example item 1
Vitamin D And Multiple Sclerosis
Here's more good news about vitamin D: researchers at the Harvard School of Public Health have found that the risk of MS is lowest among people whose vitamin D levels are high. While other research has suggested this link, the Harvard study is the first to indicate that increasing vitamin D levels could help prevent MS, a chronic degenerative neurological disease that affects some 350,000 people in the United States and two million worldwide. Working with researchers from the U.S. Army and Navy, the Harvard team analyzed stored serum samples from more than seven million individuals for levels of 25-hydroxyvitamin D. They found that the risk of MS among whites whose vitamin D levels were highest was 62 percent lower than among those whose levels were lowest. No significant associations were found among blacks and Hispanics, perhaps because there were fewer of their serum samples available or because these groups tend to have low vitamin D levels. More study is needed to establish whether increasing vitamin D levels with supplements will reduce the risk of MS. The study was published in the December 20, 2006 issue of the Journal of the American Medical Association
Rhye's of Wiki
Wiki for Rhye's mods and general awesomeness
CS 101C
Predator-Prey Model
Balchipedia
The Encyclopedia of Balch History


UK: DB Schenker Rail (UK) Ltd announced a review of options for Piggyback rail freight services in Britain in June 8, following independent enquiries from a number of freight customers seeing to cut distribution costs.
The freight operator said ongoing work to increase clearances is opening up more rail routes which could be suitable for Piggyback operations, where lorry trailers are loaded onto suitable wagons to provide quick modal integration. Services could be offered later this year if the review finds a viable commercial case.
Interested parties are invited to contract DB Schenker for further discussions of the concept.


Motion-Activated Light That's Ready Where and When You Need It.
The NEBO EYE Smart Sensor Light detects motion that automatically activates the 260 lumen C•O•B area light. The daylight sensor allows the light to remain off when enough light is already present. The 3 light modes (high, low & security flash), help you illuminate any space with the perfect amount of light. The EYE magnetically rests inside of the base, which allows for multi-directional rotation or easy removal for hand-held use.
Each package contains two EYE Smart Sensor Lights.


text:
if(NOT DEFINED CMAKE_CROSSCOMPILING)
  set(CMAKE_SYSTEM_NAME Linux)
  set(CMAKE_C_COMPILER   /Users/robert/Work/my_own-gcc)
endif()

#set(CMAKE_FIND_ROOT_PATH ${CMAKE_SOURCE_DIR})

#here is the fun part compiler verification only looks at CMAKE_CXX_FLAGS
#and than cmake will clear both CMAKE_CXX_FLAGS and CMAKE_CXX_FLAGS_INIT,
#but after that is done if it detects a CMAKE_USER_MAKE_RULES_OVERRIDE file
#it will re-parse that file and set CMAKE_CXX_FLAGS to be CMAKE_CXX_FLAGS_INIT
#it is done all processing. So to get this file to be a toolchain and a
#override we set both
set(CMAKE_CXX_FLAGS_INIT "-g")
set(CMAKE_CXX_FLAGS "${CMAKE_CXX_FLAGS_INIT}")


text:
Kim Petras (Colônia, 27 de agosto de 1992) é uma cantora e compositora alemã, atualmente residindo em Los Angeles, Califórnia. Desde 2017, Petras lança suas músicas e projetos em seu próprio selo, a BunHead Records, e em 2021, ela assinou com a gravadora Republic Records.

Petras começou a gravar músicas quando adolescente, lançando seu primeiro extended play (EP), One Piece of Tape, em 2011. Petras lançou seu single de estreia de forma independente em 2017, "I Don't Want It at All", produzido por Dr. Luke e Cirkut. A canção ficou no topo das paradas internacionais do Spotify, resultando na parceria de Petras com a empresa. Desde então, lançou os singles "Hills", "Can't Do Better", "Hillside Boys", "Slow It Down", "Heart to Break", "Faded", "All the Time", "Homework", "If U Think About Me", "1, 2, 3 Dayz Up" (com a produtora musical Sophie) e "Feeling of Falling" (com o trio eletrônico Cheat Codes); cada um dos quais acumula mais de um milhão de transmissões. Todas essa canções seriam incluídas em seu primeiro projeto, intitulado Era 1.

Em outubro de 2018, Petras lançou o extended play Turn Off the Light, Vol. 1, um projeto com temática de halloween, feriado favorito da artista. Seu álbum de estréia, Clarity (2019), recebeu críticas positivas da mídia especializada e incluía letras sobre amor, festas, relacionamentos, sexualidade, entre outros, embalados por melodias de dance-pop, electropop, hip hop e R&B. Em outubro, Petras lançou seu segundo álbum de estúdio, Turn Off the Light, continuação do extended play homônimo, consistindo em canções novas e canções do primeiro volume, todos embalados por melodias dançantes e sombrias.

Em 2021, Petras assinou contrato com a Republic Records e começou a trabalhar em novas musicas para o seu primeiro álbum de estúdio, dentre elas os singles "Future Starts Now" e Coconuts. Ela também anunciou o lançamento de um novo EP, intitulado Slut Pop para fevereiro de 2022.

Sua colaboração com Sam Smith intitulada "Unholy" rendeu a Petras uma série de recordes históricos, fazendo dela a primeira mulher abertamente transgênero a ganhar um Grammy Award, assim como a primeira que atingiu o topo das paradas ao redor do mundo em diferentes charts musicais.

Biografia
Kim Petras nasceu em 27 de agosto de 1992 em Colônia, na Renânia do Norte-Vestfália, Alemanha. Kim nasceu sob o gênero masculino, mas quando tinha dois anos de idade, começou a insistir que era uma menina. Seus pais, Lutz e Konni, achavam que isso era uma "fase" que se encerraria, porém durante a sua adolescência, ela confirmou que era transgênero. No começo, a ideia de que Kim era uma criança transgênero não foi bem aceita, mas os seus pais começaram a buscar tratamento em diversos lugares da Alemanha, até que conheceram o psiquiatra Bernd Meyenburg, do hospital universitário da Universidade de Frankfurt, que foi chefe de uma clínica para crianças e estava estudado a transexualidade desde a década de 1970.

Em 2006, Petras, então com treze anos, fez sua primeira aparição significativa na mídia em um programa na televisão local Stern. Nele, ela descreveu sua transição de gênero e o tratamento médico que recebeu no Centro de Endocrinologia da Universidade de Hamburgo, ministrado pelo médico Achim Wusthof. No entanto, foi somente no ano seguinte, com quatorze anos, que Petras acabou ganhando notoriedade mundial pela sua identidade de gênero, logo após participar de diversos programas de televisão de seu país, por lutar para que quando completasse dezesseis anos para fazer a cirurgia de readequação sexual, porém de acordo com a lei alemã, a pessoa deveria ter no mínimo dezoito anos para ser autorizada a submeter-se a esse tipo de cirurgia. Em 2007, a publicidade gerada resultou em aparições na televisão em matérias sobre sua transição de gênero aos doze anos, e descrevendo-a como "a mais jovem transgênero do mundo". Esta afirmação, no entanto, era imprecisa, já que diversas disforias de gênero podem ser diagnosticados na infância e a transição em menores de doze anos, embora seja incomum,não é inédita (um outro exemplo conhecido é o da blogueira Jazz Jennings). No entanto, Petras pode ser considerada uma das pessoas mais jovens do mundo a receber o tratamento. Em setembro de 2007, Kim foi contratada como garota propaganda para uma rede nacional de salões de beleza alemã. Em novembro de 2008, Kim Petras anunciou em seu blog que tinha feito finalmente a cirurgia de readequação sexual. A história chegou aos meios de comunicação em 4 de fevereiro de 2009 e apareceu nos jornais de todo o mundo, inclusive na primeira página de alguns. O que atraiu a atenção da mídia mundial é que até então ela poderia ter sido a pessoa mais jovem do mundo a submeter-se à cirurgia de redesignação sexual. No ano seguinte, Petra engrenou sua carreira como modelo, foi fotografada por Philipp Paulus, para a grife de Lennart Wronkowitz. Em meados de 2011, foi contrata pelo YouTube, transformando sua vida em um reality show, viagens realizadas, cobrindo eventos, entrevistando artistas como Justin Bieber e a banda Tokio Hotel. Ao passar dos anos, Kim também gravou vídeos com covers de artistas como Britney Spears, Chris Brown, Demi Lovato e Lady Gaga  chamando atenção nas redes sociais e conseguindo também muitos fãs e seguidores.

Carreira

200814: Início de carreira

Entre 2008 e 2009, Petras lançou uma série de singles pela Joyce Records: "Fade Away" (2008), "Last Forever" (2009), "Die for You" (2009) e "Boomerang" (2009). Em 2009, Petras foi destaque em "Taste" de Sobertruth com Kim Petras e em 2011 ela foi destaque em "Magnetic". Mais tarde, em 2011, Petras lançou seu primeiro extended play, One Piece Of Tape, sob o selo independente Bionic Ballroom.

Em 2013, Petras participou de dois singles chamados "Flight to Paris" e "Hearbeat" do DJ alemão Klaas. Ao longo dos próximos anos, Petras trabalhou com produtores, incluindo The Stereotypes e Aaron Joseph em uma carreira musical, lançando demos em sua página SoundCloud, incluindo uma intitulada "STFU". Por suas contribuições para a mídia social, Petras foi classificada em 19º lugar no Quadro de Artistas da Billboard, listando artistas em desenvolvimento, em julho de 2013. Em 2015, Petras apareceu em "You", de Isaac Phase, em seu álbum, Phase 00001.

Em 2013, realizou alguns duetos com Klaas e anunciou em suas redes sociais uma "pausa na carreira" para cuidar de assuntos pessoais. Em 2014, com a pausa em sua carreira para cuidar de assuntos pessoais, esteve focada em buscar inspirações para um novo projeto. No segundo semestre de 2015, anunciou que esteve ausente de suas redes sociais, pois esteve de mudança para Los Angeles, Califórnia, juntamente com a sua irmã mais velha para viver novas experiências. Em 2016, esteve realizando várias composições, em busca de gravadoras e produtores para a apresentação de suas canções. Neste ano, fundou seu próprio selo, a BunHead Records.

20172019: Avanços musicais, Era 1 e Turn Off the Light Vol. 1

Após um hiato de três anos, Kim voltou ao universo musical em agosto de 2017 com o single, "I Don't Want It at All", uma faixa produzida por Dr. Luke, conhecido pelas suas produções para Kesha, Katy Perry, entre outros artistas de grande porte. O single foi disponibilizado nas plataformas digitais e ficou em primeiro lugar na playlist "United States Viral 50" do Spotify. No mesmo mês, Petras foi configurada pela plataforma como um dos quatro artistas globais com o selo RISE (crescer), que era "programa projetado para identificar quais seriam os próximos grandes nomes da música mundial". No final de 2017, Kim foi considerada pela revista Paper, uma das "artistas que provavelmente iriam dominar as paradas de músicas nos próximos anos". Ainda em 2017, ela ainda apareceria na mixtape Pop 2, da cantora inglesa Charli XCX, na faixa "Unlock It", ao lado do rapper Jay Park.

Em janeiro de 2018, o vídeo dirigido por Nicholas Harwood para o single "Faded" de Petras, que apresenta Lil Aaron, estreou no Noisey. Nesse mesmo mês, ela apareceu na edição de janeiro da Galore. Em fevereiro de 2018, Petras lançou o single "Heart to Break", sendo alusivo ao Dia dos Namorados. A música estreou no rádio na BBC Radio 1 em 19 de fevereiro. Bryan Kress, da revista Billboard, observou que, enquanto o single "ainda mantém o som pop otimista de Petras...[ele] se aventura em um novo território como compositora". No dia 21 do mesmo mês, Petras lançou mais um single para o projeto, Spotify Singles, sendo disponibilizado exclusivamente online, com uma versão ao vivo de "Hills" e um cover de "Human", canção da banda The Killers.

Em 1 de outubro de 2018, Petras lançou Turn Off the Light, Vol. 1, o seu primeiro EP com temática de halloween. Com a participação de Elvira, a Rainha das Trevas  em uma das faixas, o EP foi visto pelos críticos como um desvio do som típico de Petras, apresentando um som dançante, e ao mesmo tempo, sombrio. Posteriormente, Petras anunciou planos de lançar um single por mês, como preparação para seu álbum de estréia. Ela colaborou com o grupo eletrônico Cheat Codes na canção "Feeling of Falling" em novembro de 2018. Petras ainda compôs a música "Young & Wild, que foi selecionada para o EP Yes or Yes, do  grupo de k-pop  Twice.

Em fevereiro de 2019, Petras lançou três singles intitulados "1,2,3 Dayz Up", com a produtora Sophie, "If U Think About Me" e "Homework", com o rapper Lil Aaron. Petras não expressou publicamente planos de lançar um álbum com nenhum de seus singles digitais de 2016 até o início de 2019, mas sempre se referia a estes,como os projetos da chamada Primeira Era de sua carreira.

 20192020: Clarity e Turn Off the Light

Petras lançou seu álbum de estréia, Clarity, em 28 de junho de 2019. Durante os dois meses anteriores ao lançamento, Petras lançou uma música do álbum por semana como preparação para o lançamento do álbum, como "Got My Number", "Broken", "Clarity", "Personal Hell", "Do Me", "Another One" e "Blow It All". Em junho, Petras embarcou na Broken Tour, passando pela América do Norte e Europa. Petras ainda estampou a capa das revistas Galore e Notion naquele mesmo mês. O álbum ainda teve como  singles promocionais,  "All I Do Is Cry" e "Sweet Spot", e foi acompanhado pelo single principal, "Icy", que foi lançado junto do álbum. Petras fez uma série de aparições em podcasts e séries da internet para promover o disco, incluindo uma entrevista no Zach Sang Show.

Em agosto do mesmo ano, Petras anunciou que lançaria uma prensagem de vinil de edição limitada de Turn Off the Light, Vol. 1 por meio da Urban Outfitters. Petras anunciou que Turn Off the Light seria lançado exatamente um ano após o lançamento da primeira parte. Em 1 de outubro de 2019, o segundo álbum de estúdio de Petras, Turn Off the Light, foi lançado. O álbum traz todas as músicas do Vol. 1 e também nove novas faixas. Em 11 de fevereiro de 2020, ela lançou o single "Reminds Me". No mesmo mês, ela anunciou que seria o ato de abertura na parte europeia da turnê The Romance Tour de Camila Cabello, que foi adiada devido à pandemia de COVID-19.

Em 7 de maio, Petras lançou o single "Malibu" como o single principal de seu primeiro álbum de estúdio. Kim Petras analisa a criação de 3 anos de seu novo single 'Malibu' e provoca seu colorido álbum de estréia: 'Eu não me sinto mais com o coração partido'. Ela descreveu a música como "pop escapista" e comentou que "Este é realmente um novo começo, o início de um novo capítulo e um novo ciclo de álbum", diz ela. "Eu não me sinto mais com o coração partido. Não me sinto presa a isso. Eu só quero fazer música que me faça querer dançar e me faça esquecer todas as coisas estúpidas agora." A música foi promovido por uma apresentação no The Tonight Show Starring Jimmy Fallon. Petras foi posteriormente apresentada na música "Broken Glass" de Kygo, de seu álbum Golden Hour (2020), lançada em 29 de maio. A música se tornou o avanço comercial de Petras, fazendo sucesso em vários países europeus e também no Canadá.

Em 23 de outubro de 2020, Petras anunciou a terceira parte do Turn Off the Lights para outubro de 2021, junto com lançamento da música "Party Till I Die", que foi incluída na nova versão do álbum homônimo. A canção "Reminds Me" foi usada no sample de "Reminds Me of You", de The Kid Laroi com a participação de Juice Wrld, sendo um lançamento póstumo do último.

 Arte
Kim Petras é uma artista pop que também faz música eletrônica dance (EDM), dance-pop e eletropop. Ela credita a cena pop do final dos anos 1990 / início dos 2000 e a Italo disco dos anos 1980 como a base primária para seu som. As inspirações de Petras incluem Lady Gaga, Beyoncé, Britney Spears, Christina Aguilera, Kylie Minogue, Madonna e Spice Girls, bem como Boy George, Debbie Harry, Queen, Freddie Mercury, Judy Garland, Baby E, Lil Aaron e Liz Y2K. Em uma entrevista, Petras afirmou que "Para mim, a música pop é uma fuga dos meus problemas. Posso colocar meus fones de ouvido e ouvi-la por três minutos e meio para esquecer tudo que está me incomodando. Sempre foi assim durante Eu sinto que o pop definitivamente salvou minha vida de muitas maneiras."

Sobre sua composição, Petras disse a Noisey que "há algo em fazer uma música que todos possam cantar e lembrar, e quando você a ouve pela primeira vez, já conhece a letra do segundo refrão, como se você sempre conhecesse a música. Estou obcecada com essa ideia." Ela comentou sobre os temas, afirmando" Eu escrevo sobre meninos, desgosto, sexo, diversão e as coisas pelas quais eu passo."

 Imagem pública
Após sua estreia, Petras foi apelidada de "a nova princesa do pop" por Nasty Galaxy. Este título foi posteriormente repetido por outras publicações, incluindo a Billboard, ABC News, V, e Idolator.

Após o lançamento de seu álbum de estreia Clarity em 2019, muitos críticos elogiaram o álbum, mas criticaram seu envolvimento com o Dr. Luke, que havia sido acusado de agredir sexualmente e verbalmente a cantora e compositora americana Kesha em 2015. Para evitar polêmica, Petras se absteve de mencionar Luke em entrevistas e não deu ao Dr. Luke um crédito formal de produção em seu álbum.

 Filmografia

 Filme

 Série

 Discografia

Álbuns de estúdio
 Clarity (2019)
 Turn Off the Light (2019)Extended play (EP)
 One Piece Of Tape (2011)
 Spotify Singles (2018)
 Turn Off the Light, Vol. 1 (2018)
 Slut Pop'' (2022)

Ligações externas

Kim Petras no Spotify
Kim Petras no Youtube

Cantores da Alemanha
Compositores da Alemanha
Mulheres transgênero
Cantores de música pop
Músicos LGBT da Alemanha
Mulheres na música electrónica
Cantores de língua inglesa da Alemanha
Músicos transgênero
Vencedores do Grammy Awards


text:
Az All Things Must Pass George Harrison harmadik szólóalbuma, amely 1970. november 27-én jelent meg. Ez volt Harrison első albuma, amit a Beatles feloszlása után készített el. Az album még egy szempontból is első: ez volt a rocktörténelem első tripla nagylemez-albuma egyazon előadótól. Két nagyobb részből állt: egy "hagyományos" dupla albumból és az Apple Jam című örömzenélés felvételéből. A tripla albumot 1970-es megjelenése idején igazi mesterműnek tartották, manapság pedig az egyik legjobb olyan szólóalbumnak tartják, amit egy volt Beatles-tag készített.

A dalok mennyisége sok kritikust meglepett, mert a Beatles idején John Lennon és Paul McCartney sokkal több dallal szerepelt az albumokon, bár 1968-tól Harrison dalainak színvonala egyenértékű volt a Lennon-McCartney szerzőpároséval (While My Guitar Gently Weeps, Something és Here Comes the Sun). Mivel a Beatles albumain Harrison csak egy vagy két dallal szerepelt (állítólag Lennon és McCartney nem engedte, hogy több dala kerüljön az albumokra), a zenekar feloszlása után rengeteg kiadatlan vagy fel sem vett dal gyűlt össze, így nem volt nehéz a tripla album rögzítése.

Az album felvételei 1970. május 26-ától augusztusig az Abbey Road Studiosban, augusztustól szeptemberig pedig a Trident Studiosban zajlottak. Az album társproducere Phil Spector volt; Spector sajátos felvételi technikájának (Wall of Sound) eredménye az albumon hallható erős visszhang. A felvételen részt vevő zenészek a rock legjobbjai közül kerültek ki: Eric Clapton (több, mint harminc évig nem tüntették fel a nevét, különböző szerződések miatt), Ringo Starr, Billy Preston, a Badfinger tagjai, Peter Frampton és a tizenkilenc éves Phil Collins, aki 1970. augusztus 4-én csatlakozott a Genesishez. Harrison barátja, Bob Dylan írta az I'd Have You Anytime szövegét, Harrison pedig Dylan If Not for You című dalát vette fel (Dylan 1970-es, New Morning című albumán szerepelt).

Szerepel az 1001 lemez, amit hallanod kell, mielőtt meghalsz című könyvben.

Az album dalai
Minden dalt George Harrison írt, kivéve, ahol jelölve van.

Az 1970-es kiadás

Dupla stúdióalbum

Apple Jam

A 2001-es kiadás
CD 1

 "I'd Have You Anytime" (George Harrison – Bob Dylan) – 3:00
 "My Sweet Lord" – 4:43
 "Wah-Wah" – 5:39
 "Isn't it a Pity" (Version 1) – 7:13
 "What is Life" – 4:27
 "If Not for You" (Bob Dylan) – 3:33
 "Behind that Locked Door" – 3:10
 "Let it Down" – 5:01
 "Run of the Mill" – 2:52
 "I Live for You" – 3:37
 Egy korábban kiadatlan dal az 1970-es felvételekről.
 "Beware of Darkness" – 3:22
 A dal akusztikus demója 1970. május 27-éről.
 "Let it Down" – 3:55
 A dal akusztikus demója a fenti napról. A szintetizátoros rájátszás 2000-ben készült.
 "What is Life" – 4:27
 A dal zenéjének korai változata 1970. augusztus 9-éről, trombitával és oboával.
 "My Sweet Lord (2000)" – 4:57
 Az eredeti 1970-es felvétel 2000-ben készült rájátszásokkal. A dalban Sam Brown vokálozik.

CD 2

 "Beware of Darkness" – 3:52
 "Apple Scruffs" – 3:09
 "Ballad of Sir Frankie Crisp (Let It Roll)" – 3:52
 "Awaiting on You All" – 2:50
 "All Things Must Pass" – 3:47
 "I Dig Love" – 5:00
 "Art of Dying" – 3:43
 "Isn't it a Pity" (Version 2) – 4:51
 "Hear Me Lord" – 6:00
 "It's Johnny's Birthday" (George Harrison – Mal Evans – Eddie Klein) – 0:49
 "Plug Me In" (Jim Gordon – Carl Radle – Bobby Whitlock – Eric Clapton – Dave Mason – George Harrison) – 3:19
 "I Remember Jeep" (Ginger Baker – Klaus Voormann – Billy Preston – Eric Clapton – George Harrison) – 8:09
 "Thanks for the Pepperoni" (Jim Gordon – Carl Radle – Bobby Whitlock – Eric Clapton – Dave Mason – George Harrison) – 5:32
 "Out of the Blue" (Jim Gordon – Carl Radle – Bobby Whitlock – Eric Clapton – Gary Wright – George Harrison – Jim Price – Bobby Keys – Al Aronowitz) – 11:16

Közreműködők
Gitár
 George Harrison
 Eric Clapton
 Dave Mason
 Peter Frampton
 Badfinger

Pedálos steel-gitár
 Pete Drake

Basszusgitár
 Klaus Voormann
 Carl Radle

Dob és ütőhangszerek
 Ringo Starr
 Jim Gordon
 Alan White
 Ginger Baker
 Phil Collins
 Badfinger
 Ray Cooper
 Mal Evans

Billentyűs hangszerek
 Billy Preston
 Bobby Whitlock
 Gary Wright
 Gary Brooker
 Leon Russel

Fúvósok
 Bobby Keys – tenorszaxofon
 Jim Price – trombita

Vokál
 The George O'Hara-Smith Singers (Eric Clapton és Bobby Whitlock)

Lásd még

 George Harrison-diszkográfia

További információk
 All Things Must Pass Shockwave website
 Az All Things Must Pass hivatalos honlapja
 Beatles Recorded Sound Index

George Harrison-albumok
1970 nagylemezei
Minden idők 500 legjobb albuma (Rolling Stone magazin)
A Billboard 200 lista első helyezettjei
A UK Albums Chart listavezetői
Az ausztrál albumlista első helyezettjei


text:
Jeff Chu
Jeff Chu is a Princeton, New Jersey-based freelance reporter and writer as well as a seminarian at Princeton Theological Seminary. Most recently, I was a contributing editor at Modern Farmer (R.I.P., print edition). I was also on staff at Fast Company for almost eight years. During that time, I edited, wrote, and reported on lots of different things. The ones I liked? International affairs (I led our award-winning coverage of China), fashion and design, the intersection of business and social justice. As far as I know, my stories have only gotten two people fired (I was not one of the two).
My reporting and writing have also appeared in a bunch of other places—The New York Times Magazine, Travel+Leisure, the Wall Street Journal, the Washington Post. When big publishers were still starting magazines and pouring millions of dollars into them, I was an editor on the launch team of the now-defunct Conde Nast Portfolio. And before that, I spent seven years at Time magazine, where I really received my journalistic education. I was a London-based web producer and staff writer—my first cover story was on Britney Spears and her Swedish songwriter, Max Martin—and then a New York-based writer and editor.
I'm from California, and my loyalty to the Bay Area is such that I've sometimes only half-jokingly said that I would be in favor of Northern California seceding so that Los Angeles isn't a part of us anymore. Sometimes, people ask, "But where are you really from?" I was born in California! And yes, racists and xenophobes, I know what you're asking, and the answer is Hong Kong! My family is from Hong Kong!
I feel most at home reporting on religion (though the book was my first—and will probably be my only—major project on faith and sexuality, because you should see the emails people send you when you write about this stuff!). My grandfather was a Baptist preacher, my uncle still is one, and my family is full of Sunday-school teachers, church pianists and organists, deacons, and the like. My high school was an allegedly nondenominational Christian one, though its roots were in the Christian Reformed tradition and we had some zany Bible classes. During my childhood, my family attended Baptist and PCA churches. Then, in college, I did the nondenominational-evangelical thing. In grad school and afterward, I went Anglican (I was living in England, so it seemed like the locally appropriate thing to do) and then stopped doing church altogether. Today, I call the Reformed Church in America (RCA) home. I am a candidate for ordination in the RCA's Rockland-Westchester Classis, and I divide my church time between Old First Reformed Church in Brooklyn and the Reformed Church of Bronxville in Bronxville, New York. Some days, I believe in God; other days, I want to believe in God.
I went to high school in Miami, at Westminster Christian, where I sat behind Alex Rodriguez in Mr. Warner's world history class. A graduate of Princeton and the London School of Economics, I'm a writer today largely because of two people: Carmen Gonzalez, my second- and third-grade teacher at Black Pine Circle Day School in Berkeley, California, who first taught me about storytelling, and Charlotte Grimes, whose one-semester journalism seminar at Princeton—the only journalism course I ever took—taught me that I was not a terrible reporter and might even someday be a good one. I was a 2004 Phillips Foundation journalism fellow (my project examined complaint in American history), a 2011-2012 French-American Foundation Young Leader, and a 2015 International Reporting Project fellow.
You can find a few things I've written here: https://medium.com/@jeffchu
Website: http://byjeffchu.com/
Twitter: @jeffchu
Facebook: https://www.facebook.com/byjeffchu
LinkedIn: https://www.linkedin.com/in/byjeffchu


text:
Martin Peyerl (11 August 1983 – 1 November 1999) was a German student who, on 1 November 1999 (the day of All Saints), fired from his bedroom window, killing four people and wounding seven others before committing suicide.

Biography
Born to Rudolf and Theresa Peyerl on 11 August 1983, Martin Peyerl and his sister Daniela lived with their parents on a busy street in Bad Reichenhall, Germany.

After the shooting, no clear motive has been presented for Peyerl's actions. Peyerl's status as an outsider at school, and that he had an alcoholic father at home who was frequently unemployed, have been regarded as possible influences. Peyerl was an avid gun enthusiast and frequently purchased gun magazines. He told classmates that he sometimes went to the forest looking for birds "to shoot" and sometimes practiced shooting with his father in the garage. Rudolf Peyerl, a twelve-year veteran of the German Army, was himself enthusiastic about firearms, owning as many as nineteen. A few months prior to 1 November, Martin was temporarily expelled from school because of Nazi photographs pasted in his notebook.

Neighbors said that Peyerl was a normal albeit introverted boy, but a psychiatrist who was involved in this case spoke of a loser type. Descriptions of Martin by former classmates were strikingly similar to descriptions given by classmates of Eric Harris and Dylan Klebold (who perpetrated the Columbine High School massacre). He is said to have been a shy loner who preferred playing video games over talking to people. One classmate, Stefanie Hocheder, said "Martin was always nice", but that he was largely ignored and rarely had anything to say. Another said Martin was "a bit of a right-wing".

Given the similarities between the shooters at Columbine and Martin Peyerl and the short amount of time between their respective rampages, it is possible Peyerl was influenced by the massacre at Columbine High School on 20 April 1999. Martin commented one day leading up to 1 November 1999, that it was "completely crazy what these guys have done" and that he believed Harris' and Klebold's actions to be "something we should do".

The shooting

On 1 November 1999, Peyerl's parents left the house to visit the grave of one of Peyerl's grandparents in Piding, something they typically did on All Saints Day. Martin did not go with them. Instead, in their absence, he broke into his father's gun cabinet- which contained more than ten firearms- where he stole a Ruger Mini-14 and positioned himself in his bedroom window, and began shooting at anything that moved. Peyerl killed neighbors Ruth and Horst Zillenbiller, then a 54-year-old patient at the hospital across the street when he stepped outside to smoke. Seven others were ultimately wounded, among them actor Günter Lamprecht.

His sister Daniela, a nanny at the hospital across the street, came home at around noon. According to police, a struggle of some kind occurred between the two, ending when Martin shot his sister five times and killed her. He then fatally shot the family cat, sat down in a bathtub, and committed suicide with a single blast from a shotgun. Police stormed the house at 6:00 p.m., finding the bodies of Daniela and Martin Peyerl along with the cat. An enormous swastika was painted above Peyerl's bed; in his room were a number of additional painted swastikas and other Nazi symbols. A number of videos and CDs with violent content were also discovered. A portrait of Adolf Hitler hung above the bed of Peyerl's sister.

Aftermath
Numerous politicians in Germany called for changes in German firearm legislation following Peyerl's indiscriminate shooting on 1 November 1999. Rudolf and Theresa Peyerl were interviewed as witnesses by police shortly after, and the German Kriminalpolizei started an investigation. Wolfgang Giese, head of the investigation, denied the possibility that drugs, alcohol, or extreme right-wing ideology were behind Peyerl's actions, saying those things played "no role". Instead, Giese asserted, the problem was "in the personality of the offender".

Investigators concluded that Martin Peyerl, like Eric Harris and Dylan Klebold, did not commit murder-suicide as a spontaneous act, but likely planned his actions well in advance. Peyerl left behind no journal or videos as the Columbine killers did, however, leaving his actions for the most part a mystery. Despite this, investigators remained certain that Peyerl's own death was as much a part of whatever plan he devised as the indiscriminate shooting of others.

External links
 Der Martin war immer nett, Der Spiegel (45/1999)
 Fünfter Toter nach Amoklauf, Spiegel Online (2 September 1999)

1983 births
1999 deaths
1999 mass shootings in Europe
1999 suicides
Criminal snipers
Mass murder in 1999
German mass murderers
Deaths by firearm in Germany
Suicides by firearm in Germany
Murder–suicides in Germany
Mass shootings in Germany
German children
Columbine High School massacre copycat crimes