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Published in Oncotarget V8N69, Dec 26, 2017
CDC5L drives FAH expression to promote metabolic reprogramming in melanoma
Oncotarget. 2017; 8:114328-114343. https://doi.org/10.18632/oncotarget.23107
Zhichao Gu, Huafeng Zhang, Yong Li, Susu Shen, Xiaonan Yin, Wei Zhang, Ruimin Cheng, Yong Zhang, Xiaoyan Zhang, Hui Chen, Bo Huang and Yuchun Cao _
Zhichao Gu1, Huafeng Zhang2, Yong Li3, Susu Shen1, Xiaonan Yin4, Wei Zhang1, Ruimin Cheng1, Yong Zhang1, Xiaoyan Zhang1, Hui Chen1, Bo Huang4 and Yuchun Cao1
1Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
2Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
3Department of Oncology, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, China
4National Key Laboratory of Medical Molecular Biology and Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China
Yuchun Cao, email: 963808187@qq.com
Keywords: fumarylacetoacetate hydrolase (FAH); anaplerotic reactions; cell division cycle 5-like protein (CDC5L); tumor metabolic reprogramming; melanoma
Received: August 09, 2017 Accepted: November 15, 2017 Published: December 07, 2017
Metabolic reprogramming allows tumor cells to thrive in the typically hypoxic tumor microenvironment. Using immunodetection and clinical data analyses, we demonstrate here that fumarylacetoacetate hydrolase (FAH) is highly expressed in melanoma and correlates with poor survival. FAH knockdown inhibits proliferation and migration, while promoting apoptosis in melanoma cells, result in prolonged survival in tumor-bearing mice. Molecular analyses using real time RT-PCR, western blot, and 13C tracing showed that these changes are driven by strong stimulation of anaplerotic reactions through the TCA cycle and the pentose-phosphate pathway, resulting in increased fatty acid and nucleotide synthesis. Using bioinformatic, ChIP-PCR, and gene silencing analyses, we determined that cell division cycle 5-like protein (CDC5L) is an important transcription factor regulating FAH expression in melanoma cells. These findings reveal that FAH induces metabolic reprogramming in melanoma and so emerges as both a potentially useful independent prognostic indicator and an attractive therapeutic target.
CDC5L drives FAH expression to promote metabolic reprogramming in melanoma | Gu | Oncotarget
Melanoma, a malignant tumor that arises from melanocytes, is the most dangerous skin tumor, displaying high recurrence and a low survival rate that contribute to more than 50,000 deaths per year worldwide. Most researchers pinpoint excessive ultraviolet light exposure as the primary cause of melanoma, especially in people with low skin pigmentation. Current treatments include surgical removal, chemotherapy, radiotherapy, and immunotherapy, which are, especially at advanced tumor stages, characterized by poor efficacy. Therefore, there is an urgent need to define new therapeutic targets and prognostic indicators for this disease.
Increasing evidence suggests that metabolic reprogramming is a critical hallmark of tumor cells [1–3]. This phenomenon is considered to be largely regulated by oncogenic activation, which might be linked to specific transcription factor activity, signal transduction pathways, and/or epigenetic mechanisms [4]. Unlike normal cells, which have a comparatively low rate of glycolysis and derive their energy mainly from oxidation of pyruvate in the mitochondria, most cancer cells produce energy predominantly by enhanced glycolysis in the cytoplasm, even if oxygen is plentiful (i.e., the Warburg effect) [5–8]. Initially, the Warburg effect was thought to be the result of mitochondrial damage or shut-down mediated by cancer genes or other mechanisms [9]. However, mounting evidence demonstrated that mitochondrial functions, albeit frequently dysregulated in cancer cells, play essential biosynthetic roles in various tumors, especially in melanoma, and contribute to metabolic reprogramming by enhancing anaplerotic reactions [10–13]. Anaplerotic reactions are chemical reactions that form biosynthetic intermediates within metabolic pathways such as the tricarboxylic acid (TCA) cycle. In the TCA cycle, concentrations of intermediates typically remain constant despite multiple biosynthetic reactions that utilize these molecules as substrates. Anaplerosis refers to the process of replenishing TCA cycle intermediates that have been extracted for biosynthesis through cataplerotic reactions [1, 13]. Four main reactions, catalyzed by pyruvate carboxylase (PC), aspartate transaminase, glutamate-dehydrogenase, methylmalonyl-CoA mutase, and adenylosuccinate lyase, have been described during anaplerosis [1]. The most important one among these is considered to be the irreversible carboxylation of pyruvate into oxaloacetate by PC [14–16]. Anaplerotic flux must balance cataplerotic flux to maintain the homeostasis of multiple metabolic pathways, such as the TCA cycle, in normal tissues and in tumors such as melanoma [13, 17].
Fumarylacetoacetate hydrolase (fumarylacetoacetase; FAH) is an enzyme encoded by the FAH gene, which is located on chromosome 15q25.1 and contains 14 exons. FAH is the last enzyme in the sub-pathway of L-phenylalanine and tyrosine degradation, catalyzing the hydrolysis of 4-fumarylacetoacetate into fumarate and acetoacetate [18, 19]. Mutations in the FAH gene cause type I tyrosinemia [20], a hereditary metabolic condition characterized by increased tyrosine levels in the blood and urine of patients [21]. Once produced, fumarate can be converted to malate by fumarase (fumarate hydratase; FH) in the cytoplasm. Then, through transporters such as SLC25A1, SLC25A10, and SLC25A11 [22], or via citrate-malate antiport, malate may be directly transported into the mitochondria to participate in the TCA cycle. Alternatively, malate may be converted to pyruvate by malic enzyme (ME). Pyruvate then enters the mitochondria and is converted to oxaloacetate (OAA) via PC or to acetyl-CoA by the pyruvate dehydrogenase complex (PDC), to fuel the TCA cycle.
Whether FAH participates in anaplerotic reactions in melanoma cells remains unclear. In the present study, we evaluated FAH expression in clinical samples, conducted survival analyses in both patients' data and a mouse melanoma model, and performed bioinformatics, real time RT-PCR, western blot and 13C tracing analyses to assess the role of FAH in human melanoma cells. Our results indicate that FAH transcription, driven by cell division cycle 5-like protein (CDC5L), is essential in the metabolic reprogramming of melanoma by promoting important anaplerotic reactions that sustain tumor growth and potentiate the disease's severity.
FAH is highly expressed in melanoma and correlates with poor clinical outcomes
Analysis of a human melanoma tissue microarray by immunohistochemistry (IHC) with a FAH antibody revealed significantly higher, mainly cytosolic, FAH expression in melanoma than in benign nevus and normal skin (Figure 1A, Supplementary Figure 1, and Supplementary Table 1). Consistently, the same results were found in two different melanoma registries (Haqq and Talantov) listed in the Oncomine database (Figure 1B). To assess whether FAH levels correlate with clinical outcomes in melanoma patients, we obtained detailed FAH mRNA levels and survival information from 278 melanoma patients from TCGA Research Network (http://cancergenome.nih.gov/). The association between FAH levels and overall survival (OS) and disease-free survival (DFS) was estimated by Kaplan-Meier survival analysis. Data showed that patients with high FAH levels exhibited significantly shortened OS and DFS than those in which FAH expression was low (Figure 1C), suggesting the potential of FAH as an independent prognostic indicator for melanoma.
Figure 1: FAH expression is inversely associated with survival in both melanoma patients and melanoma-bearing mice. (A) FAH expression by IHC in representative samples of a human melanoma tissue microarray. Data are expressed as mean IOD values; error bars represent SEM; ***p < 0.001 (Student's t-test). (B) FAH mRNA expression in melanoma, nevus, and skin tissues from two different datasets (Haqq and Talantov) analyzed using the Oncomine database. Error bars indicate SEM; *p < 0.05; ***p < 0.001 (Student's t-test). (C) FAH mRNA and survival information from 278 skin melanoma patients (TCGA database). D. Survival analysis of BALB/c-nude mice inoculated with melanoma cells stably expressing shRNA directed against FAH. Data are mean ± SEM; n = 8; *p < 0.05; ***p < 0.001 (Student's t-test).
To model and test this association, we transduced human A375 and A875 melanoma cells with lentivirus expressing shRNA against FAH (shFAH) or scrambled (control) shRNA and then performed puromycin selection to establish stable clones. Successful construction of the lentiviral vectors was confirmed by direct nucleic acid sequencing (data not shown). FAH expression was confirmed by GFP detection using fluorescence microscopy, real-time RT-PCR, and western blot (Supplementary Figure 2A–2C). A tumor-bearing mouse model was then established by injecting shFAH-A375 or shFAH-A875 cells into the flanks of nude mice, and survival times were recorded. Consistent with the observations in patients, the survival time of FAH-silenced animals was longer than that of the control groups (Figure 1D). These results showed that high FAH expression is common in melanoma and is associated with shortened survival times in both patients and tumor-bearing mice.
Figure 2: FAH knockdown arrests cell cycle, reduces proliferation and migration, and increases apoptosis in melanoma cells. (A) Proliferation assay in melanoma cells transfected with FAH siRNA or scrambled siRNA and cultured in complete medium. Cell counts were obtained at different time points. (B) Transwell migration assay in melanoma cells transfected with FAH siRNA or scrambled siRNA. (C–D) Flow cytometric evaluation of cell cycle staging and apoptosis in melanoma cells transfected with FAH siRNA or scrambled siRNA. Data from 3 independent experiments are expressed as mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001 (Student's t-test).
FAH expression promotes proliferation, migration, and survival in melanoma cells
To investigate the impact of FAH fluctuations on melanoma cell growth and survival, we first knocked down FAH expression using two different siRNAs and analyzed proliferation, migration, apoptosis, and cell cycle staging. The silencing efficiency of both siRNAs was verified using real-time RT-PCR and western blot (Supplementary Figure 2D). Compared with the respective control groups, both proliferation and migration were significantly decreased in FAH-silenced A375, A875, and SK-MEL-1 cells, and these effects correlated with FAH silencing efficiency (Figure 2A–2B). Interestingly, FAH knockdown or overexpression did not influence A375 cell proliferation in serum-free medium, suggesting that impaired migration does not result from decreased proliferation (Supplementary Figure 2E). We also detected the expression of several important proteins involved in epithelial-mesenchymal transition (EMT) in melanoma cells. For these, no noticeable expression changes were seen after FAH knockdown (Supplementary Figure 2F). We next evaluated the consequences of FAH silencing on cell cycle staging and apoptosis using flow cytometry. Results showed that in the melanoma cells the two siFAHs tested effectively reduced the number of cells in G2+S, while increasing the number of cells in G1 (Figure 2C). Meanwhile, Annexin V-FITC staining experiments showed that FAH knockdown was associated with increased apoptosis in the melanoma cell lines (Figure 2D).
Next, we increased the expression of FAH in melanoma cells using a plasmid vector (FAH-pcDNA3.1). Direct DNA sequencing (data not shown) and agarose gel electrophoresis (Supplementary Figure 2G) were used to validate the construct. Transfection efficiency was verified by real-time RT-PCR and western blot (Supplementary Figure 2G). FAH-pcDNA3.1 transfection did not affect proliferation in melanoma cells (Supplementary Figure 3A) but increased, as expected, their rate of migration compared with control cells (Supplementary Figure 3B). Cell cycle analysis showed a decreased G1 population, and an increase in the number of cells in G2+S phase (Supplementary Figure 3C), while no noticeable differences in the rate of apoptosis were observed between the FAH-overexpressing and control cells (Supplementary Figure 3D). We speculated that this phenomenon might be associated with sufficient endogenous FAH expression and limited substrates in melanoma cells. Thus, combined results from FAH silencing and overexpression experiments suggest that FAH promotes proliferation and migration, while inhibiting apoptosis in melanoma cells.
Analysis of FAH-regulated gene expression in melanoma cells
To shed light on the biological processes regulated by FAH, we performed mRNA expression profiling in control and FAH-knockdown melanoma cells. A375 cells were transfected in triplicate with siFAH or a scrambled siRNA for 72 h. Microarray analysis using Affymetrix GeneChip Human Gene 1.0 ST arrays identified 2535 DEGs after FAH silencing (Supplementary Table 2). Gene Ontology (GO) analysis was next employed to interrogate the biological functions of these DEGs. Significantly enriched GO terms were clustered based on their functional annotations. The data showed that for these 2535 DEGs, there were 512 functionally annotated clusters that were significantly enriched. The main clusters were involved in small molecule metabolic process, G1/S transition of cell cycle, cell proliferation, cellular response to hypoxia, cell migration, biosynthetic process, TCA cycle, and cell death (Figure 3A). Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, we further analyzed the pathways in which the DEGs were significantly enriched by applying the PathNet algorithm [23] (Figure 3B–3C). In line with GO analysis results, DEG involvement was detected in 164 pathways, affecting cell cycle, apoptosis, the TCA cycle, tyrosine metabolism, and HIF-1 signaling, among others. Overall, these results suggest that FAH may affect multiple important biological functions and metabolic processes, including the TCA cycle, in melanoma cells.
Figure 3: GO and pathway interaction analyses. (A–B) GO and pathway analyses of differentially expressed genes (DEGs) in A375 cells after siRNA-mediated FAH silencing. GO analysis was applied to analyze the DEGs' main functions. Pathway analysis was predominantly based on the KEGG database. The vertical axis represents categories and the horizontal axis represents -log 10 p values of significant GO terms or pathways. (C) Pathway network (PathNet) was constructed by connecting significant pathways including DEGs identified upon FAH knockdown. Circles represent pathways and lines indicate pathway interactions.
FAH promotes anaplerotic reactions and fatty acids synthesis in melanoma cells
FAH synthesizes fumarate and acetoacetate from L-phenylalanine and tyrosine. In the melanoma cells studied here, both immunostaining and western blot showed that FAH localized mainly in the cytosol, rather than the mitochondria (Figure 4A–4B). We speculated that fumarate formed by FAH activity is converted to malate by fumarase (FH), which is then transported into the mitochondria, where it fuels anaplerotic reactions via the TCA cycle. To assess this hypothesis, we first analyzed the expression of fumarase in melanoma cells. Consistent with previous reports [24], western blots showed both cytosolic and mitochondrial fumarase expression (Figure 4C). On the other hand, and as expected, fumarate levels decreased after FAH knockdown (Figure 4D) and increased after ectopic FAH expression (Supplementary Figure 4A). Then, we accessed the cBioPortal for Cancer Genomics [25, 26] to evaluate and correlate mRNA levels of FAH and the oxoglutarate/malate carrier SLC25A11 in cutaneous melanoma data from TCGA. Results showed that indeed, the mRNA levels of FAH were positively correlated with those of SLC25A11 (Figure 4E).
Figure 4: FAH expression modulates TCA cycle enzyme and transport activity. (A) A375 cells were stained with FAH or COX-IV antibodies and photographed under a two-photon confocal microscope. Scale bars, 5 μm. (B) Western blot analysis of FAH expression in cytosolic and mitochondrial fractions isolated from A375, A875, and SK-MEL-1 melanoma cells. β-actin and COX-IV served as cytosolic and mitochondrial markers, respectively. (C) Western blot analysis of β-actin, COX-IV, and fumarase in cytosolic and mitochondrial fractions of A375, A875, and SK-MEL-1 melanoma cells. (D) Total cellular fumarate levels in siFAH-transfected melanoma cells. (E) FAH and SLC25A11 mRNA data from clinical samples of melanoma (TCGA database). (F) Expression profiling of FAH, FH, SLC25A1, SLC25A10, SLC25A11, ME1, ME2, PC, PDHX, PDPR, PDHB, and PDHA1 mRNAs in FAH-silenced A375 cells. (G) Western blot analysis of FAH, FH, ME1, PC, and PDHB in siFAH-transfected A375 cells. (H) Real-time RT-PCR analysis of FAH, FH, ME1, PC, PDHB, SLC25A1, SLC25A10, and SLC25A11 mRNAs in siFAH-transfected A375 cells. Data are presented as mean ± SEM; n = 3. *p < 0.05; **p < 0.01; ***p < 0.001 (Student's t-test).
Next, we performed mRNA expression profiling in FAH-silenced A375 cells, and found that the expression of multiple enzymes, including FH, malic enzyme (ME1 and ME2), pyruvate dehydrogenase complex (PDHB, PDHX, PDPR, and PDHA1), pyruvate carboxylase (PC), and the mitochondrial carrier proteins SLC25A1, SLC25A10, and SLC25A11, was downregulated after FAH knockdown (Figure 4F). To validate these findings, we performed real-time RT-PCR and western blot in FAH-silenced or FAH-overexpressing A375 cells. Consistently, results showed that the expression of FH, ME1, PC, PDHB, SLC25A1, SLC25A10, and SLC25A11 was largely decreased after FAH silencing (Figure 4G–4H) and slightly increased after FAH overexpression (Supplementary Figure 4B–4C). These results suggested that FAH activity effectively provides intermediates for the TCA cycle.
We next accessed cutaneous melanoma data from TCGA and asked whether FAH can accelerate metabolic flux through the TCA cycle by analyzing, at the mRNA level, the correlation between FAH and the enzymes involved in the TCA cycle. Interestingly, rate-limiting enzymes of the TCA cycle, including citrate synthase (CS), isocitrate dehydrogenase (IDH3A, IDH3B, and IDH3G) and the a-ketoglutarate dehydrogenase complex (OGDH, DLST, and DLD), were all found to be positively associated with FAH (Figure 5A). Moreover, other TCA cycle enzymes, such as malate dehydrogenase (MDH), aconitase (ACO2), succinyl-CoA synthetase (SUCLG1 and SUCLG2), succinate dehydrogenase (SDHA, SDHB, SDHC, and SDHD) and FH, also displayed a positive correlation with FAH (Figure 5A and Supplementary Figure 5A). These results were followed by analyses of mRNA expression profiling in FAH-silenced A375 cells, which showed that the expression of the TCA cycle enzymes mentioned above was indeed downregulated after FAH knockdown (Figure 5B). We next performed real-time RT-PCR and western blot in FAH-silenced or overexpressing A375 cells. As expected, a clear decrease in the expression of rate-limiting enzymes of the TCA cycle was observed after FAH knockdown (Figure 5C–5D) while a slight increase occurred instead in FAH-overexpressing cells (Supplementary Figure 5B-–5C).
Figure 5: FAH knockdown reduces flux through the TCA cycle and inhibits fatty acids synthesis. (A) FAH, CS, IDH3A, IDH3B, IDH3G, OGDH, DLST, DLD, and FH mRNA data from clinical samples of melanoma (TCGA database). (B) Expression profiling of ACLY, ACACA, ACACB and TCA cycle enzymes' mRNAs in FAH-silenced A375 cells. (C) Quantification of CS, OGDH, DLST, and IDH3B mRNAs by real-time RT-PCR in siFAH-transfected A375 cells. (D) Western blot analyses of FAH, CS, OGDH, IDH3A, ACLY, and ACACA expression in siFAH-transfected A375 cells. (E) Analysis of the relative abundance of 13C-labeled citrate, malate, and fumarate in A375 cells transfected with siFAHs. Cells were cultured with 13C-D-glucose for 24 hours and analyzed by LC-MS. (F) Quantification of ACLY, ACACA and ACACB mRNAs by real-time RT-PCR in siFAH-transfected A375 cells. Results are expressed as mean ± SEM of samples assessed in triplicates. Representative data from three separate experiments are shown. *p < 0.05; **p < 0.01; ***p < 0.001 (Student's t-test).
To further validate the contribution of FAH to metabolic flow through the TCA cycle, we performed 13C tracing by Liquid Chromatography Q-Exactive Mass Spectrometry (LC-QE-MS) in A375 cells using 13C-D-glucose. Through this approach, TCA cycle intermediates would obtain after each cycle two 13C atoms from acetyl coenzyme A (acetyl-CoA), derived from glycolysis of 13C-D-glucose. Hence the relative abundance of intermediates containing two (m+2), four (m+4), or six (m+6) 13C atoms represents the metabolic flow rate of the TCA cycle. Results showed that the relative abundance of citrate, malate, or fumarate containing two (m+2) or four (m+4) 13C atoms was decreased after FAH knockdown (Figure 5E) and slightly increased after FAH overexpression (Supplementary Figure 5D), indicating deceleration and acceleration, respectively, of the TCA cycle. This suggests that FAH effectively increases TCA cycle activity. Together, these results indicate that FAH stimulates energy metabolism and biosynthesis, affecting multiple biological behaviors in melanoma.
Citrate is an essential intermediate in the TCA cycle that is transported from the mitochondria to the cytoplasm by SLC25A1 [22] to be broken down into acetyl-CoA by the enzyme ATP-citrate lyase (ACLY). Acetyl-CoA, in turn, is converted into malonyl-CoA by acetyl-CoA carboxylases (ACACA and ACACB), which is the rate-limiting step in fatty acid biosynthesis. Since the above studies have demonstrated that FAH effectively promotes anaplerotic reactions, we speculated that FAH activity may also lead to increased citrate levels in mitochondria and subsequently influence fatty acids synthesis. This hypothesis was tested by analyzing mRNA expression profiling data in FAH-silenced A375 cells. Results showed that the expression of ACLY, ACACA, and ACACB were decreased after FAH knockdown (Figure 5B). These findings were corroborated by real-time RT-PCR and western blot, (Supplementary Figure 5D and 5F) which showed increased ACLY, ACACA, and ACACB levels after FAH overexpression (Supplementary Figure 5C and 5E). These data indicated that FAH promotes fatty acids synthesis from citrate in melanoma cells.
Enhanced FAH expression stimulates glycolysis, the pentose phosphate pathway, and nucleotide synthesis in melanoma cells
We next asked if in addition to the TCA cycle, FAH activity may affect other glucose-utilizing metabolic pathways, such as glycolysis and the pentose phosphate pathway (PPP), an important branch of glycolysis that provides the nucleotide precursor ribose 5-phosphate and is a major source of cytosolic NADPH. To address this question, we further analyzed mRNA expression profiling data in FAH-silenced A375 cells. Results showed that levels of the glucose transporters (SLC2A family) and related enzymes involved in glycolysis, the PPP pathway, and nucleotide synthesis, such as the rate-limiting enzymes hexokinase (HK), phosphofructokinase (PFKL, PFKM, and PFKP), pyruvate kinase (PKM), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PD) and phosphoribosyl pyrophosphate synthetase (PRPS1 and PRPS2), were mostly decreased after FAH knockdown (Figure 6A). In parallel, real-time RT-PCR and western blot analyses confirmed a decrease in the expression of SLC2A1 and the above mentioned enzymes after FAH knockdown (Figure 6B–6C), whereas an increase, in contrast, was observed in A375 cells after FAH overexpression (Supplementary Figure 6A–6C).
Figure 6: FAH knockdown inhibits glycolysis, pentose phosphate pathway activity, and nucleotide synthesis in melanoma cells. (A) Expression profiling of glycolytic, PPP, and nucleotide biosynthetic enzymes' mRNAs in FAH-silenced A375 cells. (B) Quantification of SLC2A1, HK2, PFKP, PFKM, PKM, LDHA, G6PD, PGD, PRPS1, and PRPS2 mRNAs by real-time RT-PCR in siFAH-transfected A375 cells. (C) Western blot analysis of FAH, SLC2A1, HK2, PFKM, PKM, G6PD, and PRPS1 in siFAH-transfected A375 cells. (D) Analysis of the relative abundance of 13C-labeled lactate in siFAH-transfected A375 cells. Cells were cultured with 13C-D-glucose for 24 hours and analyzed by LC-MS. (E) Determination of glucose consumption rate in siFAH-transfected A375 cells. Data are expressed as mean ± SEM; n = 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Student's t-test).
Next, 13C tracing experiments were done in A375 cells to functionally validate these results. Here, glycolysis intermediates should incorporate three or six 13C atoms from 13C-D-glucose, hence the relative abundance of lactic acid containing three (m+3) 13C atoms represents glycolytic flow rate. In line with mRNA and protein expression results, 13C-labeled (m+3) lactate was decreased after FAH knockdown (Figure 6D) and slightly increased after FAH overexpression (Supplementary Figure 6D); these results indicated, respectively, reduced and increased glycolytic rates. In addition, we evaluated glucose consumption rate using enzymatic methods. Results showed that the glucose consumption rate of A375 cells was decreased after FAH knockdown (Figure 6E) and slightly increased after FAH overexpression (Supplementary Figure 6E).
These results demonstrated that FAH effectively stimulates glycolysis, the PPP pathway, and nucleotide synthesis, and increases glucose consumption in melanoma cells.
CDC5L drives FAH expression in melanoma
Mammalian cell division cycle 5-like protein (CDC5L), a functional homolog of the Schizosaccharomyces pombe cdc5 gene, has been shown to act as a positive regulator of G2/M progression and is a basic component of the non-snRNA spliceosome [27, 28]. In addition, CDC5L shares significant homology with the transcription factor c-Myb, indicating a potential role in regulating gene transcription [29, 30]. Upon searching the SABiosciences proprietary database (using the SABiosciences Text Mining Application and the UCSC Genome Browser) we found that CDC5L is one of the major predicted transcriptional factors for the FAH gene. The binding site of CDC5L on the FAH gene is located at chr15: 80430676-80430688 and the binding sequence is as follows: TTTATGTTTATTC (Figure 7A). To test the hypothesis that CDC5L is an important transcription factor for the FAH gene [31], chromatin immunoprecipitation (ChIP) and real-time PCR analysis were performed in melanoma cells. Because specific immunoprecipitation human CDC5L antibodies are not available, we constructed recombinant plasmids containing hemagglutinin- (HA) tagged CDC5L (pCMV-C-HA-CDC5L-CDS) and validated them by direct DNA sequencing (data not shown). Then we transfected this vector into A375 cells and conducted ChIP-qPCR using primers for the predicted binding sequence of the FAH gene (Supplementary Table 3). Clear amplification of a 120-bp DNA region including the predicted binding site was obtained from anti-HA precipitates, whereas anti-IgG antibody precipitates yielded negligible amplification of this DNA region (Figure 7B). These data indicated that CDC5L protein specifically binds to the FAH gene in vitro. To further confirm that CDC5L increases FAH expression, we knocked down the Cdc5l gene in melanoma cells using two siRNAs (Figure 7C and 7E), and analyzed FAH expression by real-time RT-PCR and western blot. Results showed that the expression of FAH was significantly decreased at both the mRNA (Figure 7D) and protein (Figure 7E) levels at different time points, suggesting that CDC5L effectively drives the expression of the FAH gene.
Figure 7: CDC5L drives FAH expression in melanoma cells. (A) CDC5L binding sequence and location on the FAH gene, determined using the SABiosciences database. (B) ChIP-qPCR assay. DNA fragments interacting with CDC5L protein were pulled down by an anti-HA antibody and amplified by real-time PCR. Total chromatin was used as input. Normal human IgG served as negative control. (C–E) Silencing efficiency of two CDC5L siRNAs assessed by real-time RT-PCR (C) and western blot (E) at 48 and 72 h, respectively. These samples were then used to detect FAH expression using real-time RT-PCR (D) and western blot (E). Data are expressed as the mean ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 (Student's t-test).
Anaplerotic reactions are essential for replenishing TCA cycle intermediates; they maintain the homeostasis of multiple metabolic pathways and are crucial for the survival and proliferation of tumor cells. The production of oxaloacetate from pyruvate by PC is considered the most physiologically important event within these pathways. Although multiple anaplerotic reactions have been identified in tumor cells, many of the enzymes involved remain to be identified.
In this study, we found that FAH is highly expressed in clinical specimens of melanoma, where it correlates with shortened survival times and may thus serve as an independent prognostic biomarker. Subsequently, our in vitro studies using three human melanoma cell lines revealed that FAH regulates important biological processes such as proliferation, migration, and apoptosis. Furthermore, we show that disrupting FAH in melanoma cells effectively prolongs survival in tumor-bearing mice. We attribute the pro-tumoral actions of FAH to its ability to promote anaplerotic reactions that underlie metabolic reprogramming in melanoma cells.
Little is known about the global metabolic impact of FAH on tumor cells. FAH catalyzes the conversion of 4-fumarylacetoacetate to acetoacetate and fumarate from L-phenylalanine and tyrosine catabolism. Through FAH knockdown and overexpression experiments combined with real time RT-PCR, western blot, and 13C metabolic flux analyses, the present study reveals that a remarkable number of enzymes within the glycolytic, TCA cycle, PPP, and nucleotide biosynthetic pathways are affected by fluctuations in cellular FAH levels in melanoma cells.
Based on sequence interaction evidence from genome data analyses, our ChIP-qPCR analysis confirmed that CDC5L binds to the FAH gene and drives FAH expression in A375 cells. Accordingly, disrupting CDC5L using siRNA significantly downregulated FAH levels. A transcription factor function for CDC5L has been previously suggested, but not clearly proved, based on sequence homology with c-Myb [29, 30]. Interestingly, since the binding site of CDC5L on the FAH gene is at a relatively distant position upstream of the transcription initiation point, i.e. at an unusual region of the gene's promoter, we speculate that CDC5L might not bind to FAH's promoter, but to other cis-regulatory elements (CREs) such as enhancers or silencers [32–34]. Thus, although the present data indicate that CDC5L is a putative transcription factor for FAH, detailed confirmatory experiments are warranted. Our next studies will also address the potential role of FAH in the regulation of hypoxia-inducible factor (HIF) target genes, as the elevated level of fumarate derived from increased FAH expression are likely to inhibit prolyl hydroxylase (PHD), preventing HIF degradation [35–38]. Stabilized HIF-1α is translocated to the nucleus and activates transcription of target genes including genes of glycolysis, glucose uptake and other metabolic pathways [39–41].
In summary, consistent with clinical evidence linking FAH overexpression with shortened melanoma patient survival, our study revealed that FAH plays a critical role in melanoma cell growth and survival by stimulating anaplerotic reactions that lead to increased energy metabolism and enhanced biosynthesis of fatty acids and nucleotides. The present study offers novel insights into melanoma's metabolic reprogramming, and suggests that FAH is both a useful independent prognostic biomarker and a relevant target for melanoma therapy.
Cell lines and animals
Human melanoma A375 cells were a kind gift of the Department of Oncology, Union Hospital (Wuhan, China). A875 and SK-MEL-1 cells were purchased from the Cell Resource Center, Institute of Basic Medical Sciences, CAMS/PUMC (Beijing, China). All cells were checked for mycoplasma and interspecies cross-contamination, and authenticated by short tandem repeat (STR) profiling and isoenzyme analyses at the Cell Resource Center and at the China Center for Type Culture Collection (Wuhan, China) before the study started, and occasionally during the course of this research. A375, A875, and SK-MEL-1 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco). All cell lines were maintained in culture for a maximum of 20 passages (two months). Female BALB/c-nude mice (5 to 7 weeks old) were purchased from HFK Bioscience (Beijing, China). Animal studies were approved by the Animal Care and Use Committee of Tongji Medical College (Wuhan, China).
A375 and A875 cells stably expressing short hairpin RNAs (shRNAs) against FAH and GFP were generated through lentiviral infection and puromycin selection. The shRNA-expressing virus was constructed by GeneChem (Shanghai, China), and direct sequence analysis was used to verify the inserted sequences. Melanoma cells were stably transduced with viral supernatant (MOI = 10, diluted to 107 TU/ml with 10% FBS/DMEM; 8 μg/ml polybrene) and fresh 10% FBS/DMEM was added after 12 h. Thereafter, selection with 2 μg/ml of puromycin was initiated at 48 h and allowed to continue for 3 to 5 days. Stably transduced cells were maintained in 10% FBS/DMEM with 1 μg/ml puromycin. Silencing efficiency was verified by GFP detection, western blot, and real-time RT-PCR.
For FAH and CDC5L knockdown, siRNAs against these two genes and negative control siRNAs were purchased from RiboBio (Guangzhou, China). The corresponding sequences are shown in Supplementary Table 3. Cells were transfected with siRNAs using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) at a final concentration of 50 nmol/L. Silencing efficiency was verified by western blot and real-time RT-PCR.
Microarray assay
Total RNA from A375 cells transfected with FAH-targeted siRNA (siFAH) or negative control siRNA was extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) and further purified with an RNase-Free DNase Set. RNA quality was assessed by agarose gel electrophoresis. Subsequently, biotinylated cDNA was prepared according to the standard Affymetrix (Santa Clara, CA, USA) protocol from 250 ng of total RNA using a GeneChip® WT PLUS Reagent Kit. Following labeling, 5.5 μg of cDNA was hybridized for 16 h at 45°C on a GeneChip Human Gene 1.0 ST Array in a GeneChip® Hybridization Oven 645. GeneChips were washed and stained in an Affymetrix Fluidics Station 450. We assayed three biological replicates for each sample. All arrays were then scanned using a GeneChip® Scanner 3000 (Affymetrix). Data were analyzed with the Robust Multichip Analysis (RMA) algorithm using Affymetrix default analysis settings and global scaling as the normalization method.
Bioinformatic analysis
DEGs were identified based on SAM (Significance Analysis of Microarrays). Afterwards, we selected DEGs based on ≥ 1.2-fold-change in either direction, and DEGs for which p < 0.05 were considered downregulated or upregulated.
Gene Ontology (GO) analysis was applied for DEG functional analysis. Statistical significance (defined as p < 0.05) was assessed using Fisher's exact test and multiple comparison tests, with a false discovery rate (FDR) < 0.05 as a cut-off. We calculated the enrichment (Re) score to determine the enrichment level per GO term using the following formula: Re = (nf/n)/(Nf/N), where nf is the number of target genes within a particular category, n is the total number of genes within the same category, Nf is the number of target genes in the entire microarray, and N is the total number of genes in the microarray. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to identify significant pathways related to the DEGs. Again, we used Fisher's exact test and multiple comparison test to select significant pathways with p < 0.05. Re was calculated using the equation above. GO and KEGG pathway analyses were conducted using the online analysis tool Gene Cloud of Biomedical information (GCBI; https://www.gcbi.com.cn). The microarray data presented in this study have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE86870 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE86870).
Human melanoma tissue samples' mRNA values were obtained from the Oncomine database. Survival data and related gene expression values from melanoma patients were obtained from The Cancer Genome Atlas (TCGA) database. The binding position and sequence of CDC5L in the FAH gene were obtained from the SABiosciences proprietary database.
Plasmid constructs and transfection
FAH overexpression vectors were constructed by Goodbio Company (Wuhan, China) by inserting human FAH cDNA into pcDNA3.1 plasmids. The human CDC5L (NM_001253.3) CDS without the stop codon was amplified, double-digested with SmaI and XbaI, and then cloned into the pCMV-C-HA vector (Beyotime, Shanghai, China) to construct the pCMV-C-HA-CDC5L-CDS vector. Lipofectamine 3000 (Invitrogen) was used to transfect the plasmids into melanoma cells. All constructs were subjected to direct sequence analysis to verify the identities of the inserted sequences.
In vitro migration assay
Complete medium (DMEM containing 10% FBS; 600 μl) was added to the lower chamber of 24-well transwell plates (Corning, NY, USA). Then, 5 × 104 melanoma cells resuspended in 100 μl of serum-free DMEM were seeded into the upper chambers in triplicate and incubated at 37°C. A375 cells were incubated for 24 h, whereas A875 cells were incubated for 48 h because of their lower migration capacity. After being washed with phosphate-buffered saline (PBS), the cells were fixed using 4% neutral paraformaldehyde for 30 min and stained with 0.1% crystal violet for 20 min. Tumor cells migrating into the lower surface of the membranes were photographed at 100x magnification. Cells in ten random fields were counted to quantify migration.
Cell cycle analysis
Cells were alternatively transfected with two siFAH or FAH-overexpressing plasmids for 60 h, trypsinized, washed with PBS, and fixed with cold (–20°C) 70% ethanol. After 20 h, cells were labeled using a Cell Cycle Detection Kit (KeyGen Biotech, Nanjing, China) according to the manufacturer's instructions. Cell DNA contents were measured on an Accuri C6 flow cytometer (BD Biosciences, Ann Arbor, MI, USA). The percentages of cells in G1, S, and G2 phases were determined using FlowJo 7.6 software.
Apoptosis analysis
Cells were alternatively transfected with two siFAH or FAH-overexpressing plasmids for 60 h and labeled using an Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech) according to the manufacturer's protocol. Apoptosis levels were analyzed on an Accuri C6 flow cytometer.
Semi-quantitative PCR and real-time RT-PCR
Cells were lysed in TRIzol reagent (Invitrogen), and total RNA was extracted following the manufacturer's instructions. Real-time PCR analyses were performed with SYBR Green PCR master mix (Toyobo, Osaka, Japan) using a Bio-Rad CFX ConnectTM Real-Time PCR Detection System (Bio-Rad, Mississauga, ON, Canada). Comparative quantitative mRNA levels were normalized to the housekeeping gene β-actin. Primer sequences are shown in Supplementary Table 3.
Cellular fumarate detection
Total cellular fumarate levels were determined using the Fumarate Detection Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer's protocol. Briefly, cells were homogenized in Fumarate Assay Buffer and then centrifuged to remove insoluble material. Then we set up the Master Reaction Mix which including Fumarate Developer, Enzyme Mix, and Assay Buffer. The samples and the Master Reaction Mix were mixed, incubated at 37°C for 60 min, after which absorbance was measured at 450 nm.
Glucose consumption assay
Melanoma cells were alternatively transfected with two siFAH or FAH-overexpressing plasmids for 24 h. After complete medium exchange, a cell-free control group was set. Following a 24 h incubation, the supernatants were collected and glucose concentration was measured using a Glucose Assay Kit (Sigma) according to the manufacturer's instructions. Cells were lysed for protein quantification using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Glucose consumption rate was determined as the glucose concentration in the supernatants minus that of the cell-free control group, and normalized to total protein levels.
Tissue microarray and immunohistochemistry
A human melanoma tissue microarray was purchased from Alenabio Biotechnology Co. (Xi'an, China). Immunohistochemistry using an anti-human FAH antibody (Abcam, Cambridge, MA, USA) was conducted by Goodbio Co. (Wuhan, China). IOD values of microarray samples were determined with IPP 6.0 software. Detailed information on the patient samples included in the microarray and corresponding IOD values is provided in Supplementary Table 1.
Western blot analysis
Cytosolic and mitochondrial fractions were isolated from melanoma cells using the Cell Mitochondria Isolation Kit (Beyotime, Shanghai, China). Western blot was performed as described previously [42]. Proteins were extracted and analyzed using antibodies targeting the following human proteins: FAH (Abcam), COX-IV (Beyotime), FH, ME1, PC, PDHB, CS, OGDH, IDH3A, ACACA, ACLY, HK2, PFKM, PKM, SLC2A1, G6PD, PRPS1, CDC5L (Proteintech, Wuhan, China), N-Cadherin, E-Cadherin, TCF8/ZEB1 (Cell Signaling Technology, Beverly, MA, USA), and β-actin (Abcam) at the recommended dilutions.
Two-photon confocal microscopy
A375 cells were stained with anti-human FAH (Abcam) and anti-human COX-IV (Beyotime) antibodies according to the manufacturer's instructions and visualized by two-photon confocal microscopy (Leica, Wetzlar, Germany).
13C tracing by Liquid Chromatography Q-Exactive Mass Spectrometry (LC-QE-MS)
For 13C tracing experiments, A375 cells were alternatively transfected with two siFAHs or FAH-overexpressing plasmids for 48 h. After washing, cells were cultured with D-glucose (U-13C6, 99%; Cambridge Isotope Laboratories, Inc., Andover, MA, USA) for 24 h. Cells were trypsinized, washed twice with PBS and lysed in extraction solvent (80% methanol/water) for 30 min at –80°C. After centrifugation at 13,000 g (10 min at 4°C), supernatant extracts were analyzed using LC-QE-MS. Briefly, liquid chromatography was performed using a HPLC system (Ultimate 3000 UHPLC) (Thermo Fisher Scientific, San Jose, CA, USA) equipped with an XBridge amide column (100 × 2.1 mm i.d., 3.5 μm; Waters). The column temperature was maintained at 10 °C. The mobile phase A was 20 mM ammonium acetate and 15 mM ammonium hydroxide in water with 3% acetonitrile, pH 9.0, and the mobile phase B was acetonitrile. The linear gradient was as follows: 0 min, 85% B, 1.5 min, 85% B, 5.5 min, 30% B, 8 min, 30% B, 10 min, 85% B, and 12 min, 85% B. The flow rate was 0.2 mL/min. Sample volumes of 5 μl were injected for LC-MS analysis on a Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a HESI probe; the relevant parameters were: heater temperature, 120 °C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; and spray voltage, 2.5 kV for the negative mode. A full scan ranging from 80 to 350 (m/z) was used. Resolution was set at 70,000. Data were quantified by integrating the area under the curve of each compound using Xcalibur Qual Browser (Thermo Fisher). Each metabolite's accurate mass ion and subsequent isotopic ions were extracted (EIC) using a 10 ppm window.
Chromatin immunoprecipitation (ChIP) and qPCR analysis
As specific anti-human CDC5L antibodies for immunoprecipitation studies are not available, we constructed pCMV-C-HA-CDC5L-CDS plasmids and transfected them into A375 cells using Lipofectamine 3000. ChIP was performed using the EZ-ChIP Kit (Millipore, Billerica, MA, USA). The AVCX130 system (Sonics & Materials, Newtown, CT, USA) was used for cell sonication. Anti-HA (Abcam) and normal anti-human-IgG (Millipore) antibodies were used for immunoprecipitation reactions. The DNA fragments recovered from immunoprecipitated complexes were amplified via real-time PCR (qPCR). The primer sequences are listed in Supplementary Table 3.
Statistical analysis was performed using SPSS 17.0 or GraphPad Prism 6.0 statistical software. All data are presented as mean ± standard error of the mean (SEM). Differences in mRNA expression between groups were calculated using a two-tailed Student's t-test. Survival curves were plotted using the Kaplan-Meier method and compared by the log-rank test. For all tests, statistical significance was set at p < 0.05.
FAH: fumarylacetoacetate hydrolase; FH: fumarase; acetyl-CoA: acetyl coenzyme A; SLC25A1: solute carrier family 25 (mitochondrial carrier; citrate transporter), member 1; SLC25A10: solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10; SLC25A11: solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), member 11; ME: malic enzyme; PC: pyruvate carboxylase; PDHX: pyruvate dehydrogenase complex, component X; PDPR: pyruvate dehydrogenase phosphatase regulatory subunit; PDHB: pyruvate dehydrogenase beta; PDHA1: pyruvate dehydrogenase alpha 1; CS: citrate synthase; ACO2: aconitase 2; IDH: isocitrate dehydrogenase; OGDH: oxoglutarate dehydrogenase; DLST: dihydrolipoamide S-succinyltransferase; DLD: dihydrolipoamide dehydrogenase; SUCL: succinate-CoA ligase; SDH: succinate dehydrogenase complex; MDH: malate dehydrogenase; ACLY: ATP citrate lyase; ACACA: acetyl-CoA carboxylase alpha; ACACB: acetyl-CoA carboxylase beta; SLC2A1: solute carrier family 2 (facilitated glucose transporter), member 1; HK: hexokinase; GPI: glucose-6-phosphate isomerase; PFK: phosphofructokinase; ALDO: aldolase; TPI: triosephosphate isomerase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PGK: phosphoglycerate kinase; PGAM: phosphoglycerate mutase; ENO: enolase; PKM: pyruvate kinase; LDH: lactate dehydrogenase; G6PD: glucose-6-phosphate dehydrogenase; PGLS: 6-phosphogluconolactonase; PGD: phosphogluconate dehydrogenase; TKT: transketolase; PRPS: phosphoribosyl pyrophosphate synthetase; PPAT: phosphoribosyl pyrophosphate amidotransferase; GART: phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase; PFAS: phosphoribosylformylglycinamidine synthase; PAICS: phosphoribosylaminoimidazole succinocarboxamide synthetase; ADSL: adenylosuccinate lyase; ATIC: 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase; DEGs: differentially expressed genes; TCGA: The Cancer Genome Atlas; OS: overall survival; DFS: disease-free survival; GFP: green fluorescent protein; siRNA: small interfering RNA; shRNA: short hairpin RNA; TCA cycle: tricarboxylic acid cycle; real-time RT-PCR: quantitative reverse transcription-PCR; PPP: pentose phosphate pathway; CDC5L: Cell division cycle 5-like protein; CDS: coding DNA sequence; HA: hemagglutinin; CREs: cis-regulatory elements; HIF: hypoxia-inducible factor; PHD: prolyl hydroxylase; STR: short tandem repeat; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; cDNA: complementary DNA; IOD: integrated optical density;
Conception and design: Yuchun Cao, Zhichao Gu, Bo Huang, Yong Li
Development of methodology: Yuchun Cao, Zhichao Gu, Huafeng Zhang
Acquisition of data (provided animals, recruited and managed patients, provided facilities, etc.): Zhichao Gu, Susu Shen, Wei Zhang, Xiaonan Yin, Ruimin Cheng, Yong Zhang, Xiaoyan Zhang, Hui Chen, Yuchun Cao, Bo Huang
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analyses): Zhichao Gu, Huafeng Zhang, Yong Li, Yuchun Cao
Writing, review, and/or revision of the manuscript: Zhichao Gu, Yong Li, Huafeng Zhang, Yuchun Cao
Administrative, technical, or material support: Bo Huang, Yuchun Cao, Zhichao Gu
Study supervision: Bo Huang, Yuchun Cao
We thank the specimen donors and research groups that contributed to this paper. Gene microarray detections were performed by Genminix Informatics Co. (Shanghai, China) and we thank Zhenhua Yang and Anbang Wu from this Company for bioinformatic analysis.
The authors declare no financial conflicts of interest.
This work was supported by the National Natural Science Foundation of China (Grant No. 81472000).
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PII: 23107


Nothing said about the clear foul on Candeias in the lead up to their goal. I think it was Haring practically piggy backing him on the touchline. Just before the ball broke to Ejaria who should have cleared it.
Glenroy or The Waterhouse (Dawson's) in Balloch. You won't get any bother in either I shouldn't think. Waterhouse much tidier shop right enough.


text:
First minister joins tributes to Stoke City boss
Michael O'Neill is hugely popular after his spell in charge of Northern Ireland
Northern Ireland First Minister Arlene Foster is a big fan of Michael O'Neill. (Image: PAUL FAITH/AFP via Getty Images)
If Michael O'Neill is as popular in the Potteries in eight years' time as he is in Belfast after eight-and-a-half years in charge of Northern Ireland... the trophy cabinet will be creaking.
O'Neill has stood down as international boss this week as the coronavirus pandemic kept pushing back the Euro 2020 qualifying play-offs that should have been his swansong.
A match against Bosnia and Herzegovina - with the winners taking on either Republic of Ireland or Slovakia for a place in the finals - was put back from March to June and now until at least October.
O'Neill has always admitted it was impossible to juggle two jobs in the long-term and he had to give priority to Stoke.
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Northern Ireland had slid to 129th in the world rankings as he steadied the ship but leaves them in 36th place. They reached the knock-out stages of Euro 2016 in France and only narrowly missed out on making the 2018 World Cup.
Tributes have come thick and fast, from players and pundits and politicians.
Northern Ireland's First Minister Arlene Foster, who travelled to France to support the side at Euro 2016, said: "Michael was such an inspirational leader of the team and that allowed the fans to go and be proud.
"Euro 2016 in France was such a wonderful experience and Michael made that all possible. He gave us hope and allowed us to dream. It's all down to him so I just want to say thank you."
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Ex-Stoke City captain gets all clear after brain surgery
Long-serving midfielder Steven Davis told a story that will resonate to the job O'Neill has started at Stoke.
He told the BBC: "He's been fantastic for Northern Ireland as a whole country and for us as a group of players. He gave us that self belief to go and qualify for a major tournament.
"We were going through a different period (when O'Neill took over) and seriously lacked any level of consistency to give ourselves an opportunity to qualify for a major tournament. Michael came in and made the players believe in themselves.
"He found a formula that worked for us in terms of getting results, everybody bought into that and then it grew from there. Confidence came back into the team and we showed what we were capable of. Michael was at the forefront of everything we did."

redpajama_set_name: RedPajamaCommonCrawl


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text:
Remains of a 3,700-Year-Old Pyramid Discovered in Egypt
It almost seems like you can't throw a stone or a chunk of solidified camel dung in an Egyptian desert without hitting a pyramid, yet new ones continue to be found. The latest is just a portion of a 3,700-year-old pyramid in the Dahshur Necropolis south of Cairo but it may hold information as to why this and other later pyramids did not hold up as well as the earlier ones.
Mahmoud Afifi, head of the ancient Egyptian antiquities sector in the country's antiquities ministry, made the announcement of the discovery of the pyramid's site this week. The Dahshur Necropolis is located on the Nile's west bank about 40 km (25 mi.) from Cairo. It is significant to pyramid study because it contains two of the oldest and best preserved – the Bent Pyramid and the Red Pyramid – built from 2613–2589 BCE during the reign of King Sneferu, who is believed to be buried in the Red Pyramid.
The Bent Pyramid
The Bent Pyramid gets its name from the "bend" inward the sides take about halfway up. This was the first try by the 4th Dynasty Pharaoh Sneferu at building a smooth-sided pyramid and it taught him some valuable lessons. The base was built on desert gravel and clay which offered poor support. The foundation flaw added to the problem of the lower blocks being cut so that their weight pushes to the center, requiring the design change that resulted in the bend.
Sneferu fixed some of the problems in the Red Pyramid, so named for its lean to the right. Just kidding – it was made with red limestone. The 105 meter (344 ft) Red (also called the North Pyramid) was the first true smooth-sided pyramid. Sitting at Sneferu's knee during the construction was young Khufu, who took notes and used what he learned to build the 490-foot Great Pyramid of Giza.
Corridor leading into the pyramid
The newly discovered partial pyramid is from the later 13th Dynasty which lasted from 1803-1649 BCE. What has been excavated so far is a corridor into the inside, a hall leading to a southern ramp and a room on the west side. Pieces include a granite beam and stone blocks which give some idea of the interior design. Most interesting is a 15 cm by 17 cm (6 in by 6.7 in) block engraved with hieroglyphics that are currently being deciphered. They may help identify this pyramid's builder.
Other pyramids from the 13th Dynasty in the Dahshur Necropolis include the badly damaged pyramid of Amenemhat II and the badly damaged (is there a pattern here?) Black Pyramid. Perhaps the hieroglyphics found in the new dig can explain their downfalls as well.
Why didn't these newer structures last as along as the earlier Dahshur pyramids? Did the pharaohs lose patience in waiting for quality construction? Did the workers finally figure out that they were in dead-end jobs?
Or perhaps someone spilled Egyptian coffee on the blueprints left by the aliens who helped build the first ones.
Tags ancient mysteries archeology Dahshur Necropolis egypt egyptian history Egyptology Great Pyramid pharaoh pyramid
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text:
Gram Parsons: Fallen Angel
Gram Parsons: November 5, 1946 – September 19, 1973
Gram Parsons, former member of the Byrds, the Flying Burrito Brothers, and solo artist, would have been 60 years old today.
Gram in his Nudie suit
Gram died September 19, 1973 in Joshua Tree, California, a part of the Mojave and Sonoran Deserts, at the age of 26.
During the funeral ceremony for Gram's close friend Clarence White, Gram was overheard stating that when he died, rather than being buried in the ground, he would like to be taken out to The Joshua Tree desert of southern California and burned. After Gram died in The Joshua Tree Inn, his body was taken to the Los Angeles International Airport in preparation for being flown to Louisiana for burial. Gram's road manager Phil Kaufman and a friend, Michael Martin, got very intoxicated, borrowed a broken down hearse and drove to LAX to retrieve the body. When they arrived, they told the shipping clerk that Gram's remains were to be sent out of another airport, flashed some bogus paperwork and falsely signed for the body. After crashing into a wall and almost being arrested, Phil, Michael and Gram drove back to The Joshua Tree Desert, stopping only to buy more beer and a container of gasoline. They took Gram's remains into the desert, poured gasoline inside the coffin and set him ablaze. The two were arrested several days later and fined $700.00 for stealing and burning the COFFIN (it was is not against the law to steal a dead body). Gram's partially burned remains were finally laid to rest in a modest cemetery near New Orleans, LA.
-- gramparsons dot com
Photo © MJ, Infomaniac
Gram's voice lives on in this heartbreaking duet with Emmylou Harris, "Love Hurts" (scene from Brokeback Mountain)…
Emmylou's loving, longing tribute song to Gram, "Boulder to Birmingham"…
Baby do you know what I mean
Read all about Gram in Phil Kaufman's book, Road Mangler Deluxe.
Still miss you, beautiful, poetic, harmonic angel.
Labels: Gram Parsons, hot dead guys
Prunella Jones 10:56 AM, November 05, 2006
Nice tribute. I didn't know all this about Gram. Kurt Cobain is my favorite dead rock star.
Anonymous 11:32 AM, November 05, 2006
What a lovely tribute. Though not my type of music but I listen to it and it's lovely
Geoff 11:55 AM, November 05, 2006
Brass Buttons always makes me cry.
Fancy crying over some bloody buttons!
The Mistress 12:32 PM, November 05, 2006
Pru: I had to restrain myself from writing his life story!
*raises glass*
To dead rock stars.
Charles: The Queer Chef, the busy blogger, took time to listen. Ta for that Charles.
Geoff:
Her words still dance inside my head
Her comb still lies beside my bed
And the sun comes up without her, it just doesn't know she's gone
Oh, but I remember everything she said
Nice one, Geoff. Of course it makes you cry.
However 2:21 PM, November 05, 2006
Lovely tribute MJ.
So nice to have you back! Yay! :)
allison moorer, daughter of loretta lynn - famous country singer - wrote and sang a song called send me an angel. when she made the video, it was a tribute to gram parsons. had the joshua tree, the noony suit, and everything.
FirstNations 10:49 PM, November 05, 2006
man, that voice. how did one guy hold all that voice?
nice ta see ya back chickie!
Mone 1:59 AM, November 06, 2006
What a great story. It makes me aware one more time that I was born to late. I would have been rigth there in the dessert, smoking high, feeling fine and fullfilling a friends last wish.
The Mistress 2:25 AM, November 06, 2006
Chelly: Thanks gal!
Pink: I'm crazy 'bout a Nudie suit. And have you noticed that the word "angel" follows him around everywhere? Oh yeah.
FN: Voice of an angel. Oh jeez. There I go again.
Mone: Hi Mone! It's not too late.
*passes Mone a j*
"Then we'll sweep out the ashes in the morning"
I hope "Pink" doesn't really think Allison Moorer is the daughter of "famous country singer" Loretta Lynn, 'cause discovering otherwise is only a quick Google search away. Allison Moorer and sister Shelby Lynne Moorer's parents were academics who died in a tragic murder-suicide when both girls were very young. I don't know much about Loretta Lynn's many children, but I suspect she has few, if any, as young as Moorer.
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Sexual Revolution Tuesday
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Ed Bradley: 1941 – 2006
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Male Sex Survey
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Build a 1/5 Scale Sherman Tank
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Chemicals and Drugs16
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SulfoglycosphingolipidsLipid MetabolismLipidsMarketingPoly AUser-Computer InterfaceAdvertising as TopicMarketing of Health ServicesGangliosidesMyelin Basic ProteinSialic AcidsMyelin SheathN-Acetylneuraminic AcidG(M1) GangliosideMyelin ProteinsLactosylceramidesN-Acylsphingosine GalactosyltransferaseOvarian NeoplasmsGlycolipidsCarcinomaGlycosphingolipidsMuscidaeForensic SciencesStevens-Johnson SyndromeBibliometricsFur SealsCupressusPublicationsProtein DeficiencyChromatography, Thin LayerNeutral GlycosphingolipidsTissue FixationCerebrosidesFormaldehyde
Sulfoglycosphingolipids
GLYCOSPHINGOLIPIDS with a sulfate group esterified to one of the sugar groups.
Physiological processes in biosynthesis (anabolism) and degradation (catabolism) of LIPIDS.
A generic term for fats and lipoids, the alcohol-ether-soluble constituents of protoplasm, which are insoluble in water. They comprise the fats, fatty oils, essential oils, waxes, phospholipids, glycolipids, sulfolipids, aminolipids, chromolipids (lipochromes), and fatty acids. (Grant & Hackh's Chemical Dictionary, 5th ed)
Activity involved in transfer of goods from producer to consumer or in the exchange of services.
Poly A
A group of adenine ribonucleotides in which the phosphate residues of each adenine ribonucleotide act as bridges in forming diester linkages between the ribose moieties.
User-Computer Interface
The portion of an interactive computer program that issues messages to and receives commands from a user.
Advertising as Topic
The act or practice of calling public attention to a product, service, need, etc., especially by paid announcements in newspapers, magazines, on radio, or on television. (Random House Unabridged Dictionary, 2d ed)
Application of marketing principles and techniques to maximize the use of health care resources.
A subclass of ACIDIC GLYCOSPHINGOLIPIDS. They contain one or more sialic acid (N-ACETYLNEURAMINIC ACID) residues. Using the Svennerholm system of abbrevations, gangliosides are designated G for ganglioside, plus subscript M, D, or T for mono-, di-, or trisialo, respectively, the subscript letter being followed by a subscript arabic numeral to indicated sequence of migration in thin-layer chromatograms. (From Oxford Dictionary of Biochemistry and Molecular Biology, 1997)
An abundant cytosolic protein that plays a critical role in the structure of multilamellar myelin. Myelin basic protein binds to the cytosolic sides of myelin cell membranes and causes a tight adhesion between opposing cell membranes.
Sialic Acids
A group of naturally occurring N-and O-acyl derivatives of the deoxyamino sugar neuraminic acid. They are ubiquitously distributed in many tissues.
The lipid-rich sheath surrounding AXONS in both the CENTRAL NERVOUS SYSTEMS and PERIPHERAL NERVOUS SYSTEM. The myelin sheath is an electrical insulator and allows faster and more energetically efficient conduction of impulses. The sheath is formed by the cell membranes of glial cells (SCHWANN CELLS in the peripheral and OLIGODENDROGLIA in the central nervous system). Deterioration of the sheath in DEMYELINATING DISEASES is a serious clinical problem.
N-Acetylneuraminic Acid
An N-acyl derivative of neuraminic acid. N-acetylneuraminic acid occurs in many polysaccharides, glycoproteins, and glycolipids in animals and bacteria. (From Dorland, 28th ed, p1518)
G(M1) Ganglioside
A specific monosialoganglioside that accumulates abnormally within the nervous system due to a deficiency of GM1-b-galactosidase, resulting in GM1 gangliosidosis.
Myelin Proteins
MYELIN-specific proteins that play a structural or regulatory role in the genesis and maintenance of the lamellar MYELIN SHEATH structure.
Lactosylceramides
Glycosphingolipids which contain as their polar head group a lactose moiety bound in glycosidic linkage to the hydroxyl group of ceramide. Their accumulation in tissue, due to a defect in lactosylceramide beta-galactosidase, is the cause of lactosylceramidosis.
N-Acylsphingosine Galactosyltransferase
An enzyme that catalyzes the conversion of UDP-galactose and N-acylsphingosine to D-galactosylceramide and UDP.
Ovarian Neoplasms
Tumors or cancer of the OVARY. These neoplasms can be benign or malignant. They are classified according to the tissue of origin, such as the surface EPITHELIUM, the stromal endocrine cells, and the totipotent GERM CELLS.
Glycolipids
Any compound containing one or more monosaccharide residues bound by a glycosidic linkage to a hydrophobic moiety such as an acylglycerol (see GLYCERIDES), a sphingoid, a ceramide (CERAMIDES) (N-acylsphingoid) or a prenyl phosphate. (From IUPAC's webpage)
A malignant neoplasm made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. It is a histological type of neoplasm but is often wrongly used as a synonym for "cancer." (From Dorland, 27th ed)
Lipids containing at least one monosaccharide residue and either a sphingoid or a ceramide (CERAMIDES). They are subdivided into NEUTRAL GLYCOSPHINGOLIPIDS comprising monoglycosyl- and oligoglycosylsphingoids and monoglycosyl- and oligoglycosylceramides; and ACIDIC GLYCOSPHINGOLIPIDS which comprises sialosylglycosylsphingolipids (GANGLIOSIDES); SULFOGLYCOSPHINGOLIPIDS (formerly known as sulfatides), glycuronoglycosphingolipids, and phospho- and phosphonoglycosphingolipids. (From IUPAC's webpage)
Muscidae
A family of the order DIPTERA with over 700 species. Important species that may be mechanical vectors of disease include Musca domesticus (HOUSEFLIES), Musca autumnalis (face fly), Stomoxys calcitrans (stable fly), Haematobia irritans (horn fly) and Fannia spp.
Disciplines that apply sciences to law. Forensic sciences include a wide range of disciplines, such as FORENSIC TOXICOLOGY; FORENSIC ANTHROPOLOGY; FORENSIC MEDICINE; FORENSIC DENTISTRY; and others.
Rare cutaneous eruption characterized by extensive KERATINOCYTE apoptosis resulting in skin detachment with mucosal involvement. It is often provoked by the use of drugs (e.g., antibiotics and anticonvulsants) or associated with PNEUMONIA, MYCOPLASMA. It is considered a continuum of Toxic Epidermal Necrolysis.
The use of statistical methods in the analysis of a body of literature to reveal the historical development of subject fields and patterns of authorship, publication, and use. Formerly called statistical bibliography. (from The ALA Glossary of Library and Information Science, 1983)
Fur Seals
A group comprised of several species of eared seals found in two genera, in the family Otariidae. In comparison to SEA LIONS, they have an especially dense wooly undercoat.
A plant genus of the family CUPRESSACEAE. Cypress ordinarily refers to this but also forms part of the name of plants in other genera.
Copies of a work or document distributed to the public by sale, rental, lease, or lending. (From ALA Glossary of Library and Information Science, 1983, p181)
A nutritional condition produced by a deficiency of proteins in the diet, characterized by adaptive enzyme changes in the liver, increase in amino acid synthetases, and diminution of urea formation, thus conserving nitrogen and reducing its loss in the urine. Growth, immune response, repair, and production of enzymes and hormones are all impaired in severe protein deficiency. Protein deficiency may also arise in the face of adequate protein intake if the protein is of poor quality (i.e., the content of one or more amino acids is inadequate and thus becomes the limiting factor in protein utilization). (From Merck Manual, 16th ed; Harrison's Principles of Internal Medicine, 12th ed, p406)
Chromatography, Thin Layer
Chromatography on thin layers of adsorbents rather than in columns. The adsorbent can be alumina, silica gel, silicates, charcoals, or cellulose. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)
Neutral Glycosphingolipids
A subclass of GLYCOSPHINGOLIPIDS containing one or more sugars within their head group connected directly to a ceramide moiety. They consist of monoglycosyl-, and oligoglycosylsphingoids and monoglycosyl- and oligoglycosylceramides.
Tissue Fixation
The technique of using FIXATIVES in the preparation of cytologic, histologic, or pathologic specimens for the purpose of maintaining the existing form and structure of all the constituent elements.
Cerebrosides
Neutral glycosphingolipids that contain a monosaccharide, normally glucose or galactose, in 1-ortho-beta-glycosidic linkage with the primary alcohol of an N-acyl sphingoid (ceramide). In plants the monosaccharide is normally glucose and the sphingoid usually phytosphingosine. In animals, the monosaccharide is usually galactose, though this may vary with the tissue and the sphingoid is usually sphingosine or dihydrosphingosine. (From Oxford Dictionary of Biochemistry and Molecular Biology, 1st ed)
A highly reactive aldehyde gas formed by oxidation or incomplete combustion of hydrocarbons. In solution, it has a wide range of uses: in the manufacture of resins and textiles, as a disinfectant, and as a laboratory fixative or preservative. Formaldehyde solution (formalin) is considered a hazardous compound, and its vapor toxic. (From Reynolds, Martindale The Extra Pharmacopoeia, 30th ed, p717)


Thank You for your Mitten Tree Donations!
Throughout the frosty cold month of December, Library patrons generously decorated the Mitten Tree with over 260 sixty hats, scarves and gloves!
The warm and toasty items were delivered to the Good Neighbor Center just before the last winter snow and ice storm.
Many thanks to everyone who participated in the 20th annual Mitten Tree donation program!
Mo Mo the bookworm clown is trying to learn how to lead a happy and healthy life! Join us for this hilarious physical comedy show that combines juggling, illusion, and acrobatics performed by Motion Magic! All ages are welcome to attend on Tuesday January 10, 2017 at 6:30 p.m. in the Burgess Community Room.


ARE you ready for kick off at 7.45 pm this evening?
Have you got your green, white and gold scarf draped around your neck? Have you had the dinner and boiled the kettle for the tea?
Ireland are set to kick off against Denmark at the Aviva Stadium, Dublin at 7.45pm.
Then take a minute to sit yourself down and watch this brilliant promo video for the game by RTE which features Irish actor Brendan Gleeson.
Wow. Martin O' Neill should show this to the lads before they head out to the pitch for kick off.
No big deal. It's only qualification for what would be our first World Cup in 16 years that's at stake after all!
Not long to go to kick off. Ireland are set to take to the pitch against Denmark at the Aviva Stadium, Dublin at 7.45pm.


text:
Villeperdue est une commune française située dans le département d'Indre-et-Loire en région Centre-Val de Loire.

Ses habitants sont appelés les Villeperdusiens, Villeperdusiennes, ou encore, selon le folklore local, les Introuvables.

Géographie

Paysages
Le territoire de Villeperdue constitue indéniablement une transition entre les pays de l'Indre et ceux de la Vienne. Sa géographie administrative est au demeurant là pour l'illustrer : en effet, bien que située dans le canton de Montbazon et dans l'arrondissement de Tours, Villeperdue fait partie de la communauté de communes de Sainte-Maure-de-Touraine. Ceci témoigne de ses liens, nombreux et forts, avec la Touraine du Sud.

Elle est par ailleurs traversée par d'importants axes de communication, à savoir la ligne de chemin de fer de Tours à Poitiers d'une part, et l'autoroute A10 de l'autre.

Hydrographie

Le réseau hydrographique communal, d'une longueur totale de , comprend un cours d'eau notable, le Montison (), qui constitue la limite communale est, et six petits cours d'eau pour certains temporaires.

Le Montison, d'une longueur totale de , prend sa source sur le territoire communal et se jette dans l'Indre à Artannes-sur-Indre, après avoir traversé .
Sur le plan piscicole, le Montison est classé en deuxième catégorie piscicole. Le groupe biologique dominant est constitué essentiellement de poissons blancs (cyprinidés) et de carnassiers (brochet, sandre et perche).

En 2019, la commune est membre de la communauté de communes Touraine Vallée de l'Indre qui est elle-même adhérente au syndicat d'aménagement de la vallée de l'Indre. Créé par arrêté préfectoral du  à la suite des crues historiques de  et , ce syndicat a pour vocation d'une part l'atteinte du bon état écologique des cours d'eau par des actions de restauration de zones humides et des cours d'eau, et d'autre part de participer à la lutte contre les inondations par des opérations de sensibilisation de la population ou de restauration et d'entretien sur le lit mineur, et sur les fossés situés dans le lit majeur de l'Indre appelés localement « boires », et de l'ensemble des cours d'eau du bassin versant de l'Indre.

Urbanisme

Typologie
Villeperdue est une commune rurale, car elle fait partie des communes peu ou très peu denses, au sens de la grille communale de densité de l'Insee.

Par ailleurs la commune fait partie de l'aire d'attraction de Tours, dont elle est une commune de la couronne. Cette aire, qui regroupe , est catégorisée dans les aires de  à moins de .

Occupation des sols

L'occupation des sols de la commune, telle qu'elle ressort de la base de données européenne d'occupation biophysique des sols Corine Land Cover (CLC), est marquée par l'importance des territoires agricoles (87,5 % en 2018), une proportion identique à celle de 1990 (88 %). La répartition détaillée en 2018 est la suivante :
terres arables (58,9 %), prairies (17,5 %), zones agricoles hétérogènes (11,1 %), zones urbanisées (7 %), forêts (5,5 %).

L'IGN met par ailleurs à disposition un outil en ligne permettant de comparer l'évolution dans le temps de l'occupation des sols de la commune (ou de territoires à des échelles différentes). Plusieurs époques sont accessibles sous forme de cartes ou photos aériennes : la carte de Cassini (), la carte d'état-major (1820-1866) et la période actuelle (1950 à aujourd'hui).

Un bourg rural attractif

La croissance démographique retrouvée
Néanmoins, le territoire communal n'est pas un simple espace franchi : la fréquentation de la modeste gare le prouve et, bien loin de restreindre son service, comme elle l'avait en un temps envisagé, la SNCF tend à l'élargir. Depuis le début des années 1990, Villeperdue attire de nouveau des habitants : proche de Tours, elle a joui d'un coût des terrains avantageux. Une politique avisée d'embellissement du bourg a permis de mettre plus en valeur le potentiel de ses aménités. En 2008, le conseil général d'Indre-et-Loire a financé la réalisation de logements sociaux en plein centre-bourg.

Des commerces et des services bien diversifiés
Aussi, avec ses habitants, Villeperdue tend-elle à retrouver ses commerces : à la boulangerie s'adjoint un bar-tabac, une graineterie et même un salon de coiffure. L'importante maison de matériaux Léger, vient étoffer les activités de la commune ; il en est de même de l'importante entreprise de récupération en tous genres, créée par M. Maurice Dufresne, au lendemain de la Libération.

Risques majeurs
Le territoire de la commune de Villeperdue est vulnérable à différents aléas naturels : météorologiques (tempête, orage, neige, grand froid, canicule ou sécheresse) et séisme (sismicité faible). Un site publié par le BRGM permet d'évaluer simplement et rapidement les risques d'un bien localisé soit par son adresse soit par le numéro de sa parcelle.

Le retrait-gonflement des sols argileux est susceptible d'engendrer des dommages importants aux bâtiments en cas d'alternance de périodes de sécheresse et de pluie. La totalité de la commune est en aléa moyen ou fort (90,2 % au niveau départemental et 48,5 % au niveau national). Sur les  dénombrés sur la commune en 2019, 387 sont en en aléa moyen ou fort, soit 100 %, à comparer aux 91 % au niveau départemental et 54 % au niveau national. Une cartographie de l'exposition du territoire national au retrait gonflement des sols argileux est disponible sur le site du BRGM.

Concernant les mouvements de terrains, la commune a été reconnue en état de catastrophe naturelle au titre des dommages causés par la sécheresse en 1991, 1992, 1993, 1997 et 2003 et par des mouvements de terrain en 1999.

Toponymie
Bas latin Villa Perdita. Villa = domaine rural, et perdita, participe passé adjectivé du verbe perdre = perdue, d'abord personne menacée dans sa vie physique, et au sens figuré, chemin, lieu situé à l'écart.

Census de Villa Perdita, XIIIe s. (Cartulaire de l'archevêché de Tours, charte 108, ) ; Ville Perdue,  (Archives Nationales-JJ 154, n° 181, fol. 111 v°) ; Villeperdue, XVIIIe s. (Carte de Cassini).

Délimitation réalisée de Thilouze d'avec Villeperdue par Ordonnance Royale du  : la commune de Villeperdue cède à celle de Thilouze la Faroire, que le géomètre délimitateur avait attribué par erreur à Villeperdue, dans son Procès-verbal du  ; Thilouze cède à Villeperdue 15 ha environ aux Prés Gravoux (A.N.-F 2 II Indre-et-Loire 3).

Selon une légende populaire, Villeperdue doit son nom aux Arabes qui sont montés jusqu'à la commune en 732. Prise, la ville était donc une ville perdue, bien matériel dont une personne est privé ; sens attesté vers 1260, donc compatible avec les attestations les plus anciennes de ce nom.

Histoire
Il y a beaucoup de flou sur l'origine de cette commune et son nom.

L'appellation VILLA PERDITA semblerait être issue de l'effondrement de l'empire romain et des ravages causés par les Alain et d'autres peuplades guerrières qui ont déferlé sur notre région et de la présence d'une "Villa" probablement carolingienne ; « villa » qui a bénéficié, au départ, de l'appui des moines de Saint Florent de Saumur.
D'après DOM HOUSSEAU, le château et le bourg de Villeperdue se nommaient « LA VILLA PEURERA » et, en 982, cette villa appartenait à Amalbert, abbé de Saint Florent de Saumur. Ce dernier la donna à cens au Comte d'Anjou, FOULQUES NERA, dont les descendants donnèrent cette seigneurie à cens aux archevêques de Tours au XIe siècle. Elle s'est trouvée protégée par une maison forte aux profondes douves restant en eau toute l'année. C'est au cours du XIe siècle que la localité a pris le nom de VILLA PERDITA, dont on ignore la raison et qui est à l'origine du nom actuel. Le domaine est donc devenu tourangeau et son changement de nom pourrait traduire ce changement de mouvance.
Ce nom pourrait aussi provenir du fait que le village a dû être pillé, détruit, voire brûlé, d'une part lors de l'effondrement de l'empire Romain, d'autre part par les Sarrasins vaincus ou en fuite après la célèbre bataille « de POITIERS » en 732. Il a été transformé au cours des siècles en PERDITA, peut être en témoignage de cette destruction. Elle forme alors un fief relevant de l'archevêque de TOURS. La prévôté de VILLEPERDUE constituait un autre fief relevant également de l'archevêché.

A la fin du XIIIe siècle un nommé Pierre BONNARD vint de CANDES administrer la VILLAPERDITA au nom des archevêques de Tours. Ce fut à cette période que le bois du château fut planté et, dès le début du XIVe siècle, le château ne portera plus le même nom que le village mais portera celui de Boisbonnard.
Ce Pierre BONNARD eut pour successeur JEHAN de CHAMBRE. Les seigneurs de Boisbonnard, qui constituait alors un fief relevant de Sainte MAURE, sont inconnus jusqu'au .
La famille de CORBIN le posséda au XVIe siècle puis celles de TOURS et de LANCELOT du BOUCHET au cours du XVIIe siècle. En 1670, il fut acquis par Henri PARIS, chanoine de Saint Martin qui eut son neveu pour successeur.
De 1730 à 1791, il fut aux mains des MILON de MESNES. En 1757, les habitants de Villeperdue assurent la construction d'une route allant de Boisbonnard à la Voie Royale reliant Paris à Bordeaux. La famille MILON de MESNES, propriétaire du château, a contribué à cette réalisation et obtenu du roi que, pour cette année, les habitants soient déchargés de la totalité de l'impôt.
En témoignage de cela une borne avait été placée au carrefour de ces 2 routes avec la mention : « route de Villeperdue et Boisbonnard de 1600 T (toises) à la charge des habitants de la paroisse de VILLEPERDUE 1757 ». Borne sauvée de la destruction en 1967 et installée à proximité de l'entrée du cimetière communal ainsi qu'une autre borne semblable mais ne comportant pas d'inscription.
Pendant la révolution le Procureur de la commune se serait approprié la plupart des biens de la Cure et aurait obligé le curé du village à se marier, l'église aurait été pillée et le Chartrier du château aurait peut-être été brûlé.En 1791 le château devint propriété de Mme de SAINT WAST, née CREUZE. Les CREUZE de LESSER l'héritèrent ensuite. À partir de 1839 et par héritage il devient propriété de la famille d'ESPOUS qui le possède encore aujourd'hui.

Politique et administration

Population et société

Démographie

Enseignement
Villeperdue se situe dans l'Académie d'Orléans-Tours (Zone B) et dans la circonscription de Saint Avertin.

L'école primaire accueille les élèves de la commune.

Lieux et monuments
Situé à la périphérie de la ville, le château de Boisbonnard a été construit entre le  et le , il est inscrit au titre des monuments historiques par arrêté du .

Personnalités liées à la commune
Fortunat Milon de Mesne (1767 - 1847), qui fut l'un des plus fervents préfets royalistes de la Restauration, naquit à Villeperdue.

Pierre Benoit, de l'Académie française, situa dans cette commune son roman Villeperdue (Albin Michel, 1954).

Maurice Dufresne, entrepreneur de Villeperdue, y a fondé le musée qui porte son nom. Les visiteurs en apprécieront le charmant éclectisme, ainsi que l'authenticité.

Voir aussi

Article connexe
 Liste des communes d'Indre-et-Loire

Liens externes

Notes et références

Notes

Références

Villeperdue
Aire urbaine de Tours
Aire d'attraction de Tours

redpajama_set_name: RedPajamaWikipedia


text:
Harvey J. Kaye
Harvey J. Kaye is the Ben & Joyce Rosenberg Professor of Democracy and Justice Studies at the University of Wisconsin-Green Bay and author of Thomas Paine and the Promise of America (Farrar, Straus and Giroux) and The Fight for the Four Freedoms: What Made FDR and the Greatest Generation Truly Great (Simon & Schuster). He is currently writing Radicals at Heart: Why Americans Should Embrace their Radical History (The New Press). Drawn from work on his new book project, these words were delivered as the Keynote Address at the 2017 Annual Meeting of the Wisconsin League of Women Voters. Follow him on Twitter: @harveyjkaye
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Do Democrats Really Need Wall Street?
| Harvey J. Kaye
A former Democratic operative writes in The New York Times that the party needs to drop FDR-style progressive politics and strengthen ties to Wall Street. He's dead wrong.
We Are Radicals at Heart — Don't Forget It
A historian reminds us that to truly "make America great again" we must enhance freedom, equality and democracy, not diminish them.
Time to Recall a Progressive 'Truly Great' First 100 Days
The Resistance needs to develop a memory of how past generations confronted reactionary threats to American democracy.
Democracy & Government
Who Says It Can't Happen Here?
We have endured 40 years of creeping authoritarianism and it now appears that it may run right over democracy. We must resist and act in solidarity.
Let Them Call Us Rebels
We are the heirs of Thomas Paine — let's remember his words and spirit of resistance against injustice at the dawn of the Trump era.
Progressives, Take Hold of American History
The election results may have been different had Democrats embraced America's past and its radical imperative of freedom, equality and democracy.
The Year FDR Sought to Make America 'Fairly Radical'
At this year's Democratic convention, we're not likely to hear a version of FDR's 1936 acceptance speech, but even 80 years later we still can act on his revolutionary words.
Time for Radical Action, Not National Therapy
We don't need to bridge the class divide — we need to end it.
Social Democracy Is 100-Percent American
Anyone who thinks social democracy is un-American doesn't know their history, writes historian Harvey J. Kaye.
'Life, Liberty, and the Pursuit of Happiness' Demands $15 an Hour
President Obama and aspiring Democratic candidates need to "take a note" from FDR and set a progressive-populist political agenda for the upcoming campaigns.