Background {#Sec1}
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Human gastric cancer (GC) is one of the most aggressive malignancies and the third most common cause of cancer-related death worldwide because of its rapid progression to advanced stages and highly metastatic properties \[[@CR1], [@CR2]\]. Despite advances in diagnosis and systemic therapy, the prognosis is still worse for patients diagnosed with GC, especially metastatic GC \[[@CR3]\]. It is known that epithelial-mesenchymal transition (EMT) is one of the key molecular steps in the process of distant metastasis. As the initial stage of metastatic progression, EMT is a complex process that includes not only dissolution of cell--cell junctions, but also loss of apicobasolateral polarity \[[@CR4]\]. During the EMT process, GC cells lose their expression of cellular adhesion proteins such as E-cadherin and, tight junction proteins and, concomitantly, express abundant mesenchymal markers such as N-cadherin and Vimentin. Generally, reduction of E-cadherin expression is considered as a hallmark of the EMT process \[[@CR5], [@CR6]\]. Clinically, EMT is associated with a poor prognosis \[[@CR7]\]. Consequently, a better understanding of the mechanisms underlying the EMT process involved in GC metastasis is required for facilitating the development of specific therapeutic strategies.

As the most prevalent internal chemical modification of RNAs in eukaryotes, N6-methyladenosine (m6A) modification is a reversible process which is mediated by the m6A methyltransferases methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms tumor 1 associated protein (WTAP) and eliminated by fat-mass and obesity-associated protein (FTO) or alkylation repair homolog protein 5 (ALKBH5) \[[@CR8]--[@CR11]\]. In mammals, this modification influences different aspects of RNA metabolism, resulting in mRNA stability and splicing \[[@CR12], [@CR13]\], translation efficiency \[[@CR14]\], nuclear export \[[@CR15]\], alternative polyadenylation \[[@CR16]\], as well as microRNA processing \[[@CR17]\]. Recently, the effects of m6A modification on many biological processes have been demonstrated, including fertility \[[@CR18]\], immunomodulation \[[@CR19]\], metabolism \[[@CR20]\], stemness maintenance and differentiation \[[@CR21]\]. Importantly, RNA m6A modification has been reported to play regulatory roles in human cancers. For instance, ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells \[[@CR22]\]. FTO regulates the chemo-radiotherapy resistance of cervical squamous cell carcinoma \[[@CR23]\]. METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells \[[@CR24]\]. A growing number of studies confirmed the functions of m6A in a variety of malignancies. Nevertheless, the definite role of m6A in GC remains unclear, and dysregulation of m6A in the EMT process and metastasis has never been studied.

In the present study, we demonstrate METTL3, the critical methyltransferase of RNA m6A modification, as a crucial promoter of EMT and metastasis in GC. Based on these findings, we provide several new insights into METTL3-mediated m6A modification. We also explore the molecular mechanism underlying GC metastasis through identifying the downstream target genes and signals.

Methods {#Sec2}
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Specimens and cell culture {#Sec3}
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All specimens were histopathologically confirmed by the pathologists and obtained with informed consent. This study was approved by the Ethics Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University. Human gastric epithelial cell line GES-1 and gastric cancer cell lines BGC823, AGS, MGC803, SGC7901 and MKN-28 were purchases from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Invitrogen, Camarillo, CA) at 37 °C in an atmosphere of 5% CO~2~.

Quantitative real-time PCR (qRT-PCR) and plasmid transfection {#Sec4}
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RNA extraction from all tissues and cells and quantitative real-time PCR (qRT-PCR) procedures were carried out as previously described \[[@CR25]\]. The relative gene expression of mRNAs were calculated by using 2^-ΔΔCt^ method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control to normalize the data. Stable overexpression of METTL3 or ZMYM1 was achieved by construction of lentiviral vector (Obio Technology, Shanghai). The shRNAs and siRNAs specifically targeting genes were also synthesized by Obio Technology (Shanghai, China). Transfections of expression plasmids in GC cells were performed using the Lipofectamine 2000 kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. All the primer sequences can be found in Additional file [1](#MOESM1){ref-type="media"}: Table S1.

Luciferase reporter assay {#Sec5}
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BGC823 and MKN-28 cells in 24-well plates were transfected with luciferase reporter and indicated expression constructs. All cells were harvested 48 h after transfection and analyzed using the dual-luciferase reporter gene assay system (Promega, Madison, WI, USA). The relative ratio of firefly luciferase activity to Renilla luciferase activity was determined. Each experiment was performed in triplicate.

Immunohistochemistry {#Sec6}
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Immunohistochemistry (IHC) analysis was performed using a GT Vision III Kit (Genetech, Shanghai, China) according to the manufacturer's instructions. The final stainings were scored as follows: staining intensity score, 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong); staining area score, 0 (≤10% positive staining), 1 (11--25% positive staining), 2 (26--50% positive staining), 3 (51--75% positive staining), and 4 (≥75% positive staining). Staining intensity and staining area were summed up to give a final score.

Co-immunoprecipitation (co-IP), western blot analysis and RNA immunoprecipitation (RIP) {#Sec7}
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Transfected cells were incubated in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 15 mM MgCl~2~, 5 mM EDTA, 0.1% NP-40) containing protease inhibitor Cocktail (Roche, Mennheim, Germany) for subsequent co-IP. The cell lysates were incubated with specific antibodies targeting Flag (Sigma Aldrich), CtBP1/2 (Santa Cruz), LSD1(Santa Cruz) and CoREST (Santa Cruz) at 4 °C for 2 h, then incubated with 20 μl A/G PLUSAgarose beads (Santa Cruz, USA) at 4 °C overnight. The beads were separated and washed by using cold phosphate-buffered saline, and then subjected to western blotting analysis. For the western blotting, equivalent amounts of protein were separated by SDS-PAGE at 80 V for 2.5 h and transfected to PVDF membranes for 2 h. The membranes were washed using 1% TBST for 30 min after incubation with specific antibodies targeting Flag (Sigma Aldrich), METTL3 (Abcam), HuR (Abcam), ZMYM1 (Invitrogen), E-cadherin (Cell Signaling Technology), N-cadherin (Cell Signaling Technology), Vimentin (Cell Signaling Technology), CtBP1/2 (Santa Cruz), LSD1 (Santa Cruz), CoREST (Santa Cruz), GAPDH (Abcam) and Tubulin (Abcam) at 4 °C overnight, then incubated with secondary antibodies (1:5000, BioTNT, China) for 2 h. Finally, they were washed using 1% TBST and detected by a chemiluminescence system (Amersham Biosciences, Piscataway, NJ). RIP for anti-HuR was performed as described earlier \[[@CR26]\].

ChIP and re-ChIP assays {#Sec8}
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The ChIP Assay Kit (Millipore, Bedford, MA) was utilized according to the manufacturer's instructions. Cross-linked chromatin was sonicated into fragments, and then the fragments were immunoprecipitated using different antibodies. For re-ChIP, bead eluates from the first immunoprecipitation were incubated with 10 mM DTT at 37 °C for 30 min and diluted 1:50 in dilution buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, 2 mM EDTA, pH 8.1) followed by reimmunoprecipitation with the second antibodies. The sequences of the primers used for ChIP-PCR were also listed in Additional file [1](#MOESM1){ref-type="media"}: Table S1.

mRNA-sequencing, m6A-sequencing and m6A-RNA immunoprecipitation (MeRIP) assays {#Sec9}
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Total RNAs from the transfected GC cells were extracted with TRIzol (Invitrogen). Then, mRNA sequencing and m6A sequencing were simultaneously performed (CloudSeq Biotech, Shanghai, China). For mRNA-sequencing, mRNAs were single-end sequenced on Illumina HiSeq 2000 machines. Transcript assembly and differential expression was examined by using Cufflinks with Refseq mRNAs to guide assembly. For m6A-sequencing, mRNA was fragmented and then incubated with m6A antibody (NEB, USA) for immunoprecipitation. Immunoprecipitated RNA was analyzed through qRT-PCR or high-throughput sequencing. Data analyses were performed as previously described.

LC-MS/MS analysis of m6A level {#Sec10}
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Total RNAs of GC samples were isolated by using TRIzol (Invitrogen). Then, mRNA was purified from total RNA through using NEBNext® Poly(A) mRNA Magnetic Isolation Module, and then digested and centrifuged. The supernatant after centrifugation was injected into UPLC-MS/MS. The nucleosides were separated and detected in positive ion multiple reaction-monitoring (MRM) mode. Quantitation of modifications was determined by nucleoside-to-base ion mass transitions (m6A: 282 to 150; A: 268 to 136). The m6A/A ratios were calculated.

Confocal immunofluorescent assay {#Sec11}
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Different stable GC cells and tissue sections were used. After first incubated with antibodies specific for METTL3 (Abcam), ZMYM1 (Abcam), E-cadherin (Cell Signaling Technology), N-cadherin (Cell Signaling Technology) and Vimentin (Cell Signaling Technology), and then with goat anti-rabbit IgG (Alexa Fluor 647, Invitrogen) or goat anti-mouse immunoglobulin G (IgG) (Alexa Fluor 488, Invitrogen), respectively, GC cells and tissue sections were mounted by adding DAPI (Sigma), and imaged by using a confocal microscope (Olympus).

Animal studies {#Sec12}
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6-week-old male athymic BALB/c nude mice were used. For the in vivo lung metastases model, different stable BGC823 or MKN-28 cells were injected into the tail vein of representative mice (*n* = 5 per group). The luciferase signal intensity from days 7 to 42 is on equivalent scales in the models. Bioluminescent flux (photons/s/cm2/steradian) was determined for the lung metastases. Metastatic progression was monitored and imaged using an IVIS-100 system (Caliper Life Sciences, MA, USA) 10 min after intraperitoneal injection of luciferin (300 mg/kg i.v.) in 80 μl of saline. All the mice were killed after 6 weeks, then the immunohistochemical analysis and H&E staining were performed. For the orthotopic gastric cancer model, 1 × 10^7^ stable MKN-28 cells were injected into the subserosa of mouse stomach (*n* = 5 per group), then the mouse stomach and liver were carefully examined for tumor formation and metastasis 5 weeks later. The organs were sectioned for H&E staining. All animal experiments were approved by the Institutional Animal Care and Use Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University (approval No. RA-2017-114).

Statistical analysis {#Sec13}
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For continuous variables, Student's t test and one-way ANOVA were used for comparing differences. For categorical variables, Fisher's exact test and Chi-square test were used. GC patient survival curves were plotted by the Kaplan-Meier method with log-rank test. Cox regression was utilized to estimate the hazard ratio and 95% confidence intervals for survival. All statistical analyses were performed by using SPSS version 19.0 software (SPSS, Chicago, IL). Statistical significance was considered at a value of *P* \< 0.05.

Results {#Sec14}
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METTL3 overexpression and its prognostic value in GC {#Sec15}
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To explore the expression of the major m6A-modifying enzymes in GC, we first queried the published clinical data sets TCGA (The Cancer Genome Atlas) and GSE66229, and found that METTL3 mRNA expression was significantly elevated in GC tissues compared to that in normal tissues. In contrast, the expression of METTL14 in GC tissues was reduced, and other m6A "writers" and "erasers" levels in the two data sets were inconsistent (Fig. [1](#Fig1){ref-type="fig"}a, b). Further analysis of GSE66229 survival data suggested that patients with high METTL3 expression had a shorter median survival time (Additional file [5](#MOESM5){ref-type="media"}: Figure S1a). Next, we validated the bioinformatics data in a sample cohort consisting of 60 pairs of GC tissues (Cohort 1) by qRT-PCR. The mRNA level of METTL3 was significantly overexpressed in GC tissues versus adjacent normal tissues (Fig. [1](#Fig1){ref-type="fig"}c). Moreover, in comparison to that in GC tissues at TNM low stage (I-II), METTL3 level was significantly higher in advanced-stage (III-IV) GC tissues (Fig. [1](#Fig1){ref-type="fig"}d). After excluding the samples without clinical information, similar result was also found in GSE66229 data set (Additional file [5](#MOESM5){ref-type="media"}: Figure S1b), which further supported our initial observation. Importantly, METTL3 was more highly expressed in the diffuse-type GC tissues compared with the intestinal-type samples according to the Lauren classification (Additional file [5](#MOESM5){ref-type="media"}: Figure S1c, d). In line with these findings, elevated levels of m6A mRNA were observed in GC tissues compared to that in their corresponding normal tissues (Fig. [1](#Fig1){ref-type="fig"}e). We further examined the protein level of METTL3 in the GC tissue microarray samples that included another 120 cases (Cohort 2) using immunohistochemistry. As shown in Fig. [1](#Fig1){ref-type="fig"}f, METTL3 was prominently localized in the nuclei of GC cells. In addition, METTL3 immunoreactive was predominantly positive in the majority of GC tissues. Among these specimens, 44 (36.7%) tissues displayed weak staining and 40 (33.3%) showed strong staining. Furthermore, elevated expression of METTL3 was significantly associated with pN stage (*p* = 0.003), pM stage (*p* = 0.001), TNM stage (*p* \< 0.001) and vessel invasion (*p* = 0.014) (Additional file [2](#MOESM2){ref-type="media"}: Table S2). Kaplan-Meier curves with a log-rank test revealed that patients with high METTL3 expression exhibited a worse overall survival (OS) and disease-free survival (DFS) (Fig. [1](#Fig1){ref-type="fig"}g, h). Univariate and multivariate analyses indicated that METTL3 expression was an independent prognostic indicator for both OS (Additional file [3](#MOESM3){ref-type="media"}: Table S3) and DFS (Additional file [4](#MOESM4){ref-type="media"}: Table S4) in GC patients. Taken together, these data suggested that METTL3 was significantly overexpressed in GC and might be associated with GC progression. Fig. 1METTL3 is overexpressed and correlated with tumor survival and recurrence of GC patients. **a**-**b** The expression of m6A modifying enzymes in TCGA and GSE66229 GC cohorts. **c** The mRNA levels of METTL3 in 60 GC tissues and paired normal tissues (Cohort 1) were determined by qRT-PCR. **d** The mRNA levels of METTL3 in low clinical stage (I-II) and advanced-stage (III-IV) GC patients were analyzed. **e** mRNA m6A levels in 5 GC samples determined by LCMS/MS analysis. **f** Representative images of IHC staining for METTL3 protein on a TMA constructed from 120 GC tissues. Scale bars: 200 μm. **g**-**h** Upregulation of METTL3 was significantly associated with shorter OS and DFS in GC patients

METTL3 was required for EMT {#Sec16}
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Interestingly, in our preliminary exploration through immunohistochemical serial section staining, the expression level of METTL3 in GC tissues seemed to be negatively correlated with E-cadherin and positively correlated with N-cadherin and Vimentin (Fig. [2](#Fig2){ref-type="fig"}a). This prompted us to ascertain the possible effects of METTL3 on the GC EMT process. We first determined the EMT phenotype of the human gastric epithelial cell line GES-1 and five GC cell lines. Western blotting analysis revealed that BGC823 and AGS cell lines primarily express N-cadherin and Vimentin, with a concurrent low E-cadherin level, which was consistent with their mesenchymal character. Conversely, the MKN-28 cell line showed high expression of E-cadherin and low levels of N-cadherin and Vimentin, which was representative of its epithelial phenotype (Fig. [2](#Fig2){ref-type="fig"}b). Importantly, the endogenous levels of METTL3 and mesenchymal markers were comparable, which indicated a positive correlation between METTL3 and the EMT phenotype in GC (Additional file [5](#MOESM5){ref-type="media"}: Figure S1e). Next, to validate whether METTL3 was required for the EMT program, loss- and gain-of-function studies were carried out (Fig. [2](#Fig2){ref-type="fig"}c, d). We found that knockdown of METTL3 led to a significant downregulation of N-cadherin and Vimentin, accompanied by a prominent upregulation of E-cadherin at the protein levels in BGC823 and AGS cells. On the other hand, METTL3 overexpression caused an opposite result in MKN-28 cells (Fig. [2](#Fig2){ref-type="fig"}e). Furthermore, altered METTL3 expression resulted in significant changes in the EMT markers' levels as confirmed by confocal immunofluorescent microscopy assay (Additional file [5](#MOESM5){ref-type="media"}: Figure S1f). As expected, knockdown or overexpression of METTL3, respectively, also decreased or increased the global m6A modification level in GC cells (Fig. [2](#Fig2){ref-type="fig"}f, g). Collectively, these findings strongly suggested that METTL3 promoted the EMT program in GC cells. Fig. 2Gain of mesenchymal features and simultaneous loss of epithelial characteristics correlate with METTL3 expression in GC. **a** Representative IHC staining of EMT markers in pathologic serial sections of GC tissues with low or high METTL3 expression. Scale bars: 200 μm. **b** Western blot analysis of METTL3 and EMT markers in different cell lines. **c** The shRNA-mediated METTL3 repression was confirmed by qRT-PCR after lentivirus infection in BGC823 and AGS cells. **d** Overexpression of METTL3 was confirmed by qRT-PCR after lentivirus infection in MKN-28 cells. **e** METTL3 knockdown or overexpression influenced the expression levels of EMT markers in the western blot assay. **f**-**g** The global m6A modification levels in METTL3 knockdown and overexpression GC cells determined by LC-MS/MS analysis. \*\**p* \< 0.01, \*\*\**p* \< 0.001

METTL3 promoted GC cell invasion and metastasis in vitro and in vivo {#Sec17}
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To assess whether METTL3-mediated EMT was responsible for GC metastasis, we explored the effects of METTL3 on cell motility. Wound healing assay showed that attenuation of METTL3 expression significantly impeded the migratory ability of BGC823 cells (Fig. [3](#Fig3){ref-type="fig"}a). Additionally, forced expression of METTL3 apparently increased the migration speed of MKN-28 cells (Fig. [3](#Fig3){ref-type="fig"}b). Correspondingly, transwell matrigel invasion assay confirmed that the invasive ability of GC cells was markedly suppressed in response to METTL3 knockdown (Fig. [3](#Fig3){ref-type="fig"}c), while it was dramatically enhanced by ectopic expression of METTL3 (Fig. [3](#Fig3){ref-type="fig"}d). Next, we evaluated the physiological relevance of METTL3 to GC metastasis in vivo. Stable cells with modified METTL3 expression were injected into the tail vein of BABL/c nude mice, and luciferase signals were monitored at different time points to observe the location and growth of tumor xenografts in the lung. Notably, we discovered that BGC823-shRNA cells metastasized to the lungs of nude mice less effectively compared to those in the control group (Fig. [3](#Fig3){ref-type="fig"}e, f). Conversely, overexpression of METTL3 significantly enhanced the lung metastases burden of MKN-28 cells in contrast to that in the control group (Fig. [3](#Fig3){ref-type="fig"}g, h). An orthotopic mouse model assay was also performed to further analyze the effect of METTL3 on metastasis. Stable MKN-28 cells were injected beneath the serosa of the stomach of nude mice. Tumors infiltrating muscularis and mucosa were observed 5 weeks after GC cells implantation. We found that the tumors in METTL3-overexpressing group were larger than those in the control. Moreover, metastatic foci in the liver could be observed in METTL3-overexpressing group, but not in the control (Fig. [3](#Fig3){ref-type="fig"}i-k). All these findings indicated a crucial role of METTL3 in facilitating GC cell invasion and metastasis. Fig. 3METTL3 promotes GC cell invasion and metastasis in vitro and in vivo. **a**-**d** Knockdown or overexpression of METTL3, decreased or increased, respectively, GC cells' wound healing and invasion abilities in vitro. **e**-**f** Representative mice injected with modified METTL3 expressing BGC823 cells, downregulation of METTL3 decreased GC lung metastases. **g**-**h** Representative mice injected with modified METTL3 expressing MKN-28 cells, upregulation of METTL3 increased GC lung metastases. **i**-**k** Stomachs and livers from the orthotopic gastric cancer model. The stomach tumors were larger in METTL3-overexpressing group than those in the control. Overexpression of METTL3 facilitated the formation of liver metastasis. Scale bars: 100 μm. \**p* \< 0.05, \*\**p* \< 0.01, \*\*\**p* \< 0.001

Transcriptome-sequencing and m6A-sequencing identified ZMYM1 as a direct target of METTL3 {#Sec18}
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In order to investigate the functional implications of METTL3 and identify its potential targets in GC, we performed transcriptome-sequencing to compare the gene expression profile following METTL3 knockdown in BGC823 cells. We found that 798 genes were significantly downregulated, while 662 genes were significantly upregulated (Fig. [4](#Fig4){ref-type="fig"}a, b). To test whether the altered gene expression could be a consequence of METTL3-mediated m6A modification, we also performed m6A-sequencing to map the m6A modification in control or METTL3 knockdown BGC823 cells. In total, m6A-sequencing identified 9465 and 8794 m6A peaks in control and METTL3-deficient cells, respectively (Fig. [4](#Fig4){ref-type="fig"}c). In the BGC823 cells with METTL3 stable knockdown by shRNA, 850 peaks were found to be diminished (Fig. [4](#Fig4){ref-type="fig"}d). Gene ontology (GO) analysis revealed that differentially expressed m6A-modified transcripts were significantly enriched in gene sets involved in hemidesmosome assembly, centrosome localization and regulation of attachment of spindle in sh-METTL3 cells, suggesting that m6A might have profound impacts on chromosome (Additional file [5](#MOESM5){ref-type="media"}: Figure S2a). Pathway analysis showed that multiple cancers and related signaling pathways (such as renal cell carcinoma, thyroid cancer and HIF-signaling pathway) correlated with METTL3-mediated m6A modification (Additional file [5](#MOESM5){ref-type="media"}: Figure S2b). When mapped the m6A methylomes in BGC823 cells, the m6A consensus sequence GGAC (RRACH) motif was identified to be highly enriched within m6A sites in the immunopurified RNA (Fig. [4](#Fig4){ref-type="fig"}e). Consistently with previous studies, we demonstrated that m6A signal was enriched around the stop codon of mRNAs (Fig. [4](#Fig4){ref-type="fig"}f). Next, we asked whether the altered m6A peaks were associated with differentially expressed genes. Importantly, filtering the 850 diminished m6A peaks with the 798 downregulated genes resulted in the identification of 15 peaks harbored by 13 genes (Fig. [4](#Fig4){ref-type="fig"}g). Among these 13 potential regulators, our focus was on ZMYM1 as two m6A peaks were detected around the stope codon of its mRNA in sh-NC BGC823 cells and were all diminished upon METTL3 knockdown (Fig. [4](#Fig4){ref-type="fig"}h). Nowadays, the biological function of ZMYM1 remains unclear. Thus, we selected ZMYM1 as a candidate target of METTL3 mediated m6A modification for further investigation. Fig. 4ZMYM1 is a direct target of METTL3-mediated m6A modification. **a**-**b** RNA-sequencing identified differentially expressed genes in METTL3 knockdown GC cells when compared to the sh-NC control group. **c**-**d** m6A-sequencing identified the diminished m6A peaks in METTL3 knockdown GC cells when compared to the sh-NC control group. **e** The m6A consensus sequence motif was identified in BGC823 cells. **f** Distribution of m6A IP signal in mRNA transcripts in BGC823 cells. The m6A signal was enriched around the stop codon of mRNAs. **g** Filtering the diminished m6A peaks with the downregulated genes in sh-METTL3 BGC823 cells identified ZMYM1 as a direct target of METTL3. **h** Attenuation of METTL3 diminishes m6A modification of ZMYM1mRNA in BGC823 cells. A HuR binding site locates within the m6A modification region of ZMYM1

METTL3 maintained ZMYM11 expression in GC {#Sec19}
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To investigate the regulation of ZMYM11 expression by METTL3, qRT-PCR and western blot analysis were performed. Consistent with the gene expression data, knockdown or overexpression of METTL3 downregulated or upregulated, respectively, ZMYM1 at both the mRNA and protein levels (Fig. [5](#Fig5){ref-type="fig"}a-d). Immunofluorescence staining indicated the nuclear localization of ZMYM1 and revealed that depletion of METTL3 induced loss of ZMYM1 expression in BGC823 cells, whereas overexpression of METTL3 increased ZMYM1 levels in MKN-28 cells (Fig. [5](#Fig5){ref-type="fig"}e). Next, we validated ZMYM1 expression in cohort 1 using qRT-PCR. In this sample cohort, ZMYM1 expression was frequently elevated in GC tissues and positively correlated with METTL3 expression (Fig. [5](#Fig5){ref-type="fig"}f, g). The nuclear co-localization of METTL3 and ZMYM1 in GC tissues was also confirmed via confocal imaging (Fig. [5](#Fig5){ref-type="fig"}h). These data indicated that METTL3 positively regulated ZMYM1 expression in GC. Fig. 5METTL3 maintains ZMYM1 expression in GC. **a**-**b** ZMYM1 transcripts upon METTL3 knockdown or overexpression as measured by qRT-PCR. **c**-**d** ZMYM1 protein levels upon METTL3 knockdown or overexpression as measured by western blotting analysis. **e** Co-expression of METTL3 and ZMYM1 in GC cells was determined by confocal immunofluorescent assay. Scale bars: 50 μm. **f** The mRNA levels of ZMYM1 in 60 GC tissues and paired normal tissues (Cohort 1) were determined by qRT-PCR. **g** METTL3 was positively correlated with ZMYM1 expression in 60 GC tissues (Cohort 1). **h** The nuclear colocalization of METTL3 and ZMYM1 in GC tissues were confirmed by confocal immunofluorescent assay. Scale bars: 100 μm. \*\**p* \< 0.01

METTL3 enhanced ZMYM1 mRNA expression through the m6A-HuR-dependent pathway {#Sec20}
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To further support the notion that METTL3 targeted ZMYM1 mRNA for m6A modification, we validated the m6A-sequencing data set using MeRIP qRT-PCR. As a result, anti-m6A antibody significantly enriched ZMYM1 mRNA level in GC cells. Moreover, knockdown or overexpression of METTL3, dramatically reduced or increased the m6A level of ZMYM1 mRNA (Fig. [6](#Fig6){ref-type="fig"}a, b). Bcl2 \[[@CR24]\] was used as a positive control in this assay (Additional file [5](#MOESM5){ref-type="media"}: Figure S2c, d). Then, we constructed luciferase reporters containing either the wild-type or mutant ZMYM1 to address the effect of m6A modification on ZMYM1 expression. For the mutant ZMYM1, m6A modification was abrogated because of the replacement of adenosine base by cytosine in m6A consensus sequences (RRACH) (Fig. [6](#Fig6){ref-type="fig"}c). Neither knocking down nor overexpressing METTL3 exerts significant effect on the protein expression of mutant ZMYM1 (Additional file [5](#MOESM5){ref-type="media"}: Figure S2e). Luciferase reporter assay showed that the transcriptional level of wild-type ZMYM1, but not the mutation, significantly decreased in the absence of METTL3 (Fig. [6](#Fig6){ref-type="fig"}d). Reciprocally, METTL3 overexpression augmented the expression of wild-type ZMYM1-fused reporter, but failed to influence the expression of the mutated ZMYM1 constructs, revealing the regulation of ZMYM1 level was the under control of METTL3 associated m6A modification (Fig. [6](#Fig6){ref-type="fig"}e). Notably, inhibiting m6A activity by 3-deazaadenosine (DAA), the global methylation inhibitor, substantially decreased the expression of ZMYM1 (Additional file [5](#MOESM5){ref-type="media"}: Figure S2f). The above results conformed the m6A modification of ZMYM1 by METTL3. Then, we chose to investigate the potential role of RNA-bound protein HuR (also known as ELAVL1) in METTL3-mediated m6A modification of ZMYM1. Unlike other readers located in the cytoplasm, HuR is one of the few known readers located in the nucleus, which is consistent with METTL3. Moreover, HuR was considered as a m6A "reader" protein recruited by METTL3 in the nucleus \[[@CR27]\]. As expected, the HuR binding site was located in the m6A modification region of ZMYM1 as revealed by our m6A-sequencing (Fig. [4](#Fig4){ref-type="fig"}f). RIP-qPCR using anti-HuR antibody showed dramatically reduced affinity of HuR to ZMYM1 mRNA in METTL3-silenced BGC823 cells, whereas overexpression of METTL3 in MKN-28 cells displayed a reverse result (Fig. [6](#Fig6){ref-type="fig"}f, g). SOX2 \[[@CR27]\] was used as a positive control to prove that the HuR-RIP assay were successfully performed (Additional file [5](#MOESM5){ref-type="media"}: Figure S2 g, h). Consistently, we found that knockdown of HuR could partially counteract the effects of elevated METTL3 on the expression of ZMYM1. Interestingly, the status of HuR expression showed no obvious changes in all GC cells with modified METTL3 expression used in our study (Fig. [6](#Fig6){ref-type="fig"}h-j). Together, our findings revealed that METTL3-mediated m6A modification enhanced ZMYM1 expression via m6A-HuR-dependent pathway. Fig. 6METTL3 enhanced ZMYM1 mRNA expression through m6A-HuR dependent pathway. **a**-**b** RIP with anti-m6A antibody was performed in GC cells. m6A modification on ZMYM1 was depleted upon METTL3 knockdown, whereas enhanced by METTL3 overexpression. **c** Wild-type or mutant m6A consensus sequence was fused with firefly luciferase reporter, respectively. **d** The transcriptional level of wild-type ZMYM1, but not the mutation, significantly decreased in METTL3 knockdown cells. **e** Elevated expression of METTL3 increased the luciferase activity of wild-type ZMYM1-fused reporter, but failed to interact with mutated ZMYM1 constructs. **f**-**g** RIP-qPCR using anti-HuR antibody showed the affinity of ZMYM1 RNA to HuR in different modified METTL3 expressing cells. **h**-**i** Relative ZMYM1 and HuR mRNA levels were examined by qRT-PCR in GC cells transfected with lentiviruses carrying METTL3 and/or sh-HuR. **j** ZMYM1 and HuR protein levels were examined by western blot analysis in GC cells transfected with lentiviruses carrying METTL3 and/or sh-HuR. \**p* \< 0.05, \*\**p* \< 0.01

ZMYM1 was physically associated with the CtBP/LSD1/CoREST complex in the nucleus {#Sec21}
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The zinc-finger family proteins have been implicated in transcriptional regulation \[[@CR28], [@CR29]\]. Thus, we fused the full length ZMYM1 to the DNA-binding domain of Gal4 and tested its transcriptional activity in GC cells. The results showed that Flag-tagged ZMYM1 significantly inhibited the reporter activity in a dose-dependent manner in both BGC823 and MKN-28 cells (Fig. [7](#Fig7){ref-type="fig"}a, b). Overexpression of ZMYM1 simply did not influence the activity of Gal4-driven reporter (Fig. [7](#Fig7){ref-type="fig"}c), suggesting that ZMYM1 physically bound to DNA to exert its transcriptional repressive activity. As a member of the MYM (myeloproliferative and mental retardation) gene family, the function of ZMYM1 remains unclear. Some other MYM members have been shown to physically associate with the CtBP/LSD1/CoREST complex via the MYM-type zinc fingers, thereby enhancing the transcriptional repression of target genes \[[@CR30], [@CR31]\], implying that ZMYM1 may play a similar role. To ascertain whether ZMYM1 regulated metastasis-related gene expression in such a manner, we performed co-immunoprecipitation (co-IP) experiments with specific antibodies in GC cells. IP with antibodies against ZMYM1 followed by immunoblotting with antibodies against components of the CtBP/LSD1/CoREST complex demonstrated the interaction of ZMYM1 with all the tested proteins (Fig. [7](#Fig7){ref-type="fig"}d, e). Reciprocally, IP with antibodies against CtBP1/2, LSD1, or CoREST followed by immunoblotting with antibodies against ZMYM1 also confirmed that ZMYM1 was efficiently co-immunoprecipitated by all the components of this complex (Fig. [7](#Fig7){ref-type="fig"}f). All these results showed that ZMYM1 was physically associated with the CtBP/LSD1/CoREST complex. Fig. 7Transcription repression of E-cadherin by ZMYM1-CtBP/LSD1/CoREST complex. **a**-**b** BGC823 and MKN-28 cells were transfected with different amounts of Gal4-ZMYM1 (Flag-tagged) expression plasmids, together with the Gal4-luciferase reporter. Relative luciferase activity was measured after 48 h. **c** MKN-28 cells were transfected with Flag-ZMYM1 plasmids together with the luciferase reporter. Relative luciferase activity was measured after 48 h. **d**-**e** Whole-cell lysates from BGC823 and MKN-28 cells were immunoprecipitated with antibodies against ZMYM1 or IgG, followed by immunoblotting with the antibodies against CtBP1/2, LSD1 and CoREST. **f** Whole-cell lysates from GC cells were immunoprecipitated with antibodies against CtBP1/2, LSD1 and CoREST followed by immunoblotting with the antibodies against ZMYM1. **g** ZMYM1 associated complex bound specifically to the E-cadherin proximal promoter in MKN-28 cells. **h** Co-occupancy of the E-cadherin promoter by ZMYM1 and the components of CtBP/LSD1/CoREST complex. **i** The protein level of E-cadherin was determined by western blot analysis after transfection of si-CtBP1/si-LSD1/si-CoREST, together with ZMYM1 expression construct. \**p* \< 0.05, \*\**p* \< 0.01

Transcription repression of E-cadherin by the ZMYM1-associated complex {#Sec22}
----------------------------------------------------------------------

The CtBP/LSD1/CoREST complex is known to be recruited by a panel of transcription repressors to modulate the expression of a cohort of genes \[[@CR32], [@CR33]\]. It has been shown that a consensus recognition sequence (CRS) present in the E-cadherin promoter (− 221/+ 30), specifically binding the zinc-finger protein/HDAC/CtBP/LSD1/CoREST co-repressors \[[@CR34]\]. We therefore speculated that ZMYM1-CtBP/LSD1/CoREST complex might be capable of binding this promoter region. ChIP/Re-ChIP experiments were carried out in MKN-28 cells using primers specific to the CRS site, or using negative control primers located 3 kb upstream of the E-cadherin promoter. In these experiments, the enrichments of all complex components on CRS were validated (Fig. [7](#Fig7){ref-type="fig"}g). Moreover, the co-occupancy of ZMYM1, CtBP1, LSD1, and CoREST on the E-cadherin promoter was identified via Re-ChIP assay (Fig. [7](#Fig7){ref-type="fig"}h). To further confirm the transcription repression of E-cadherin by the ZMYM1-associated complex, we performed western blotting analysis and found that gain-of-function of ZMYM1 was accompanied by a reduced expression of E-cadherin. Significantly, the attenuation of E-cadherin caused by ZMYM1 overexpression was abolished when CtBP1, LSD1, or CoREST was knocked down (Fig. [7](#Fig7){ref-type="fig"}i). Collectively, these data suggested that ZMYM1 targeted and repressed the transcription of E-cadherin by associating with the CtBP/LSD1/CoREST complex.

METTL3-mediated epigenetic activation of ZMYM1 was responsible for EMT and metastasis of GC {#Sec23}
-------------------------------------------------------------------------------------------

Despite establishing the critical role of METTL3 in EMT and metastasis, whether this impact is specifically attributed to the METTL3/ZMYM1 axis needs to be further explored. Firstly, BGC823 cells were transduced with lentiviruses carrying METTL3 and/or sh-ZMYM1, and MKN-28 cells were transduced with lentiviruses carrying sh-METTL3 and/or ZMYM1. These cells were utilized in the next functional assays. As shown in Fig. [8](#Fig8){ref-type="fig"}a, elevation of ZMYM1 expression recapitulated the levels of N-cadherin and Vimentin, and reduced the expression of E-cadherin in METTL3-knockdown cells. Conversely, inhibition of ZMYM1, at least in part, counteracted the acceleration of the EMT process caused by METTL3 overexpression (Fig. [8](#Fig8){ref-type="fig"}b). Transwell invasion assay showed that knockdown of METTL3 decreased the invasive ability of GC cells, and ectopic expression of ZMYM1 rescued this ability (Fig. [8](#Fig8){ref-type="fig"}c, e). Reciprocally, overexpression of METTL3 enhanced cell invasion, which was partially attenuated by co-transfection with sh-ZMYM1 plasmid (Fig. [8](#Fig8){ref-type="fig"}d, f). For the lung metastatic model, a similar result was observed. The inhibitory effect of METTL3 knockdown on metastasis could be, at least partially, offset by introduction of ZMYM1, whereas the increase in the metastatic potential associated with METTL3 overexpression could be partially attenuated by depletion of ZMYM1 (Fig. [8](#Fig8){ref-type="fig"}g, h). Furthermore, the protein level of ZMYM1 was reduced with decreased lung metastasis burden and upregulated with more lung colonization caused by modified METTL3 expression according to the immunohistochemistry results (Fig. [8](#Fig8){ref-type="fig"}i, j). These findings revealed that overexpression of ZMYM1 was responsible for METTL3-mediated EMT and metastasis. Fig. 8METTL3-mediated epigenetic activation of ZMYM1 is responsible for EMT and metastasis of GC. **a** The protein levels of EMT markers were measured by western blot analysis in BGC823 cells transfected with lentiviruses carrying sh-METTL3 and/or ZMYM1. **b** The protein levels of EMT markers were measured by western blot analysis in MKN-28 cells transfected with lentiviruses carrying METTL3 and/or sh-ZMYM1. **c**-**f** The invasive abilities were explored by transwell assay in GC cells transfected with lentiviruses carrying sh-METTL3 and/or ZMYM1, or transfected with lentiviruses carrying METTL3 and/or sh-ZMYM1. **g**-**h** Lung metastatic assay was performed to determine whether ZMYM1 enhanced or inhibited the metastatic abilities of BGC823 cells or MKN-28 cells by repressing or overexpressing METTL3 expression in nude mice, respectively. **i**-**j** ZMYM1 expression was reduced with decreased lung metastasis burden and upregulated with more lung colonization caused by modified METTL3 expression according to IHC analysis. Scale bars: 100 μm. \**p* \< 0.05, \*\**p* \< 0.01, \*\*\**p* \< 0.001

Discussion {#Sec24}
==========

As one of the most prominent features of malignant tumors, distant metastasis is a complex process affected by genetic and epigenetic modifications, and accounts for more than 90% of cancer-related deaths \[[@CR35], [@CR36]\]. EMT exerts critical impacts on the early events of tumor cell metastatic dissemination which has been demonstrated to be the dominant process of human GC \[[@CR37]\]. Recently, m6A modulators, including "writers" (METTL3, METTL14, WTAP and RBM15), "easers" (ALKBH5 and FTO), and "readers" (YTHDF1/2/3, HuR and HNRNPA2B1), have been considered to be essential for regulation of cancer biology, thus, m6A modification in cancers is gradually becoming the focus of research. However, the involvement of m6A in regulation of EMT and the metastatic cascade remains largely unclear. In this study, we reported for the first time that the major m6A writer METTL3 was required for the EMT program and metastasis of GC both in vitro and in vivo, suggesting that METTL3 might be a candidate inducer of EMT through endowing GC cells with more motile, invasive abilities. Optimal prognostic biomarkers for GC have not been established until now. Here, we confirmed the upregulation of METTL3 in GC samples and uncovered its clinical significance, indicating its potential value in GC prognosis.

The latest findings regarding m6A functions in cancer progression are controversial. For instance, the m6A demethylase ALKBH5 was reported to induce the breast cancer stem cell phenotype by demethylating NANOG mRNA \[[@CR38]\], whereas a later study revealed an important role of ALKBH5 in inhibiting the progression of pancreatic cancer \[[@CR39]\]. On the other hand, METTL14 plays an oncogenic role in acute myeloid leukemia \[[@CR40]\], but suppresses the metastatic potential of hepatocellular carcinoma by modulating m6A dependent primary microRNA processing \[[@CR41]\]. The reasonable explanations for these contradictory phenomena could be attributed to the different reader proteins responding to these m6A modifications, different cellular functions regulated by the target genes and different mRNA regions that m6A is distributed on. In the current study, we found that METTL3 epigenetically activated ZMYM1 via the m6A-HuR dependent pathway. The binding between ZMYM1 mRNA and METTL3 was validated by MeRIP and luciferase reporter assays. Through m6A-sequencing, we showed that the HuR-binding sequence was located within the m6A enrichment region of ZMYM1 around the stop codon. Interestingly, the HuR-binding site does not seem to exist in the other decreased m6A region in METTL3-knockdown cells, suggesting that METTL3 may also rely on additional "reader" proteins to exert its m6A modification effects. Clearly, the enrichment of some m6A-associated biological processes is beyond what we know according to the GO analysis. However, we cannot exclude the involvement and importance of other molecular mechanisms in GC.

ZMYM1 is a member of the MYM family of proteins with a molecular weight of \~ 129 kD. As it is a novel molecule, its functions in human tumors have not yet been reported. Given that zinc-finger proteins are known to play transcription activating or repressive roles \[[@CR42], [@CR43]\], investigated the transcriptional regulatory roles of ZMYM1. In the present study, elevated expression of ZMYM1 was a frequent event in GC. The transcriptional repressive activity of ZMYM1 was also measured in our system. Indeed, biochemical studies suggested that ZMYM1 was associated with the CtBP/LSD1/CoREST co-repressor complex. These findings are consistent with those of previous studies on other MYM family members. It is believed that the CtBP/LSD1/CoREST complex is required for transcriptional repression of a number of genes. For example, ZNF516 recruits the CtBP/LSD1/CoREST complex to the EGFR promoter and requires the activity of this complex to repress EGFR expression \[[@CR33]\]. MCRIP1 recruits the CtBP co-repressor complex, and silences the E-cadherin promoter by inducing chromatin modifications \[[@CR44]\]. Moreover, some zinc-finger family members function as sequence-specific DNA-binding proteins through identifying CRS site \[[@CR45], [@CR46]\]. This CRS is highly conserved within the proximal promoters of several human genes, including that encoding E-cadherin \[[@CR34]\]. Therefore, based on the above evidences, we thought that ZMYM1 might bind E-cadherin in a sequence-specific manner by recruiting the CtBP/LSD1/CoREST complex. In fact, our results demonstrated that ZMYM1 interacted with the CtBP/LSD1/CoREST complex and that this association resulted in E-cadherin downregulation. ChIP/Re-ChIP assays indicated that all the core components of the ZMYM1-associated complex were enriched on the specific region of the endogenous E-cadherin promoter containing the identified CRS site. Based on the fact that attenuation of E-cadherin expression is generally accepted as a hallmark of the EMT process and has often been observed in cancer metastasis, in this study, we showed that METTL3-mediated m6A modification in epigenetic activation of ZMYM1 represses E-cadherin by targeting the CtBP/LSD1/CoREST complex to chromatin, thus facilitating EMT and metastasis of GC.

Chromosomal level-based transcription repression of E-cadherin has been found to be linked to the EMT progress in many malignancies \[[@CR6], [@CR47], [@CR48]\]. The mechanism of altered EMT markers levels induced by decreased E-cadherin expression is worth exploring. On the other hand, further investigations that whether other genes play important roles in METTL3-mediated EMT and metastasis via a m6A-dependent or -independent manner will enhance our understanding of METTL3-associated regulatory network.

Conclusions {#Sec25}
===========

Collectively, we elucidated the critical role of METTL3-mediated m6A modification in human GC progression, wherein it promotes the EMT process and metastasis. The discovery of the METTL3/ZMYM1/E-cadherin axis and its impacts on metastasis will aid in further GC investigation and in developing therapeutic strategies against GC.

Additional files
================

 {#Sec26}

Additional file 1:**Table S1.** shRNA, siRNA and primer sequences. (PDF 11 kb) Additional file 2:**Table S2.** Association between METTL3 expression and clinicopathological characteristics in gastric cancer. (PDF 74 kb) Additional file 3:**Table S3.** Univariate and multivariate analysis of overall survival (OS) after surgery. (PDF 116 kb) Additional file 4:**Table S4.** Univariate and multivariate analysis of disease-free survival (DFS) after surgery. (PDF 116 kb) Additional file 5:**Figure S1.** a GC patients with high METTL3 expression had a shorter overall survival time in GSE66229 data set. b After excluding the samples without clinical information, analysis of GSE66229 data set showed that METTL3 level was significantly higher in advanced-stage GC tissues. c-d METTL3 was more highly expressed in the diffuse-type GC tissues compared with the intestinal-type samples in both GSE66229 (excluding the samples without clinical information) and Cohort 1. e The mRNA levels of METTL3 and EMT markers were evaluated by qRT-PCR in three GC cells, gastric epithelial cell line GES-1 was used as control. f Confocal immunofluorescent analysis of the expression of EMT markers in indicated GC cell clones. \**p* \< 0.05. (PDF 322 kb)

ALKBH5

:   Alkylation repair homolog protein 5

Co-IP

:   Co-immunoprecipitation

DFS

:   Disease-free survival

EMT

:   Epithelial-Mesenchymal Transition

FTO

:   Fat-mass and obesity-associated protein

GC

:   Gastric cancer

IHC

:   Immunohistochemistry

m6A

:   N6-methyladenosine

MeRIP

:   m6A-RNA Immunoprecipitation

METTL14

:   methyltransferase-like 14

METTL3

:   Methyltransferase-like 3

OS

:   Overall survival

qRT-PCR

:   Quantitative real-time PCR

RIP

:   RNA immunoprecipitation

TCGA

:   The Cancer Genome Atlas

ZMYM1

:   Zinc finger MYM-type containing 1

**Publisher's Note**

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ben Yue, Chenlong Song and Linxi Yang contributed equally to this work.

Not applicable.

ZZ and GZ conceived and designed the study. BY and CS contributed to carry out the experiments. LY and RC provided clinical samples and clinical information. BY wrote the manuscript. XC supervised the research. All authors read and approved the final manuscript.

This study was supported by funds from the National Natural Science Foundation of China (31872740, 81802314), Excellent Academic Leader Training Program of Shanghai Municipal Commission of Health and Family Planning (2017BR043), Cultivation Foundation of Renji Hospital, School of Medicine, Shanghai Jiao Tong University (PY-17-015).

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The m6A-sequencing and RNA-sequencing datasets have been submitted to the GEO database under the accession number GSE133132.

This research was approved by the Ethics Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University, and informed consent was obtained from all patients before enrolling in the research program. The in vivo assay using nude mice was approved by the Institutional Animal Care and Use Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University.

All subjects have written informed consent.

The authors declare that they have no competing interests.


Differential activities of plant polyphenols on the binding and internalization of cholera toxin in vero cells.
Plant polyphenols, RG-tannin, and applephenon had been reported to inhibit cholera toxin (CT) ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. We report here the effect of those polyphenols on the binding and entry of CT into Vero cells. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon but not RG-tannin. These observations were confirmed by fluorescence microscopy using Cy3-labeled CT. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CTA, and CTB from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization.


Del Potro sets up quarter-final showdown with Nadal

12/07/2018

It was a case of down but definitely not out as a Rafael Nadal kept picking himself off a slippery and dusty Centre Court surface to storm into the Wimbledon semi-finals with a pulsating 7-5 6-7 (7) 4-6 6-4 6-4 over Juan Martin del Potro.

Del Potro refused to surrender, breaking back in game 10 to level the score at 5-5 and he saved four set points in the tie break, climbing back from 3-6 down to steal the set 9-7 and make a huge roar of relief, ready to fight until the very last point.

"Sometimes you play your best tennis and it's not enough to beat him", said the teary-eyed Argentine, who has had the misfortune of running into Nadal at the French Open and Wimbledon championships over the past five weeks.

That came at a key moment in the third set, which was a tougher examination of Nadal than the initial two.

The Spaniard hit back immediately though and sealed victory on his third match point when Vesely served at 4-5.

Nadal carved out the crucial break in the opening set for a 6-5 lead and claimed the opener off a Del Potro backhand error.

Cardi B Announces The Birth Of Her Baby Girl On Instagram
While on iHeartMedia's Power 105.1 The Breakfast Club she declared: 'I'm a grown woman, I'm 25 years ago, I'm a schmillionaire. In a now-deleted post, she wrote, "I'm going to be able to hold baby you and give her all the kisses and hugs".

Next up for the Spaniard is a blockbuster last four showdowns with Serb Novak Djokovic. Nadal took him to deuce but couldn't nail the break and they went to the shootout, where, against all likelihood, Nadal double-faulted on set point.

Just 90 minutes after defending champion Roger Federer was sensationally knocked out by South African Kevin Anderson, it seemed as if the grasscourt major would lose its top two seeds on the same day as Del Potro took a two-sets-to-one lead.

Nadal broke for 3-2 but had to save break points in the sixth and eighth games before securing a memorable victory.

"Rafa is a fighter". Eventually, things went to a tiebreak Del Potro won to capture the set. A blistering, nearly joyous whipped forehand sealed the set for Nadal and they went to a fifth.

"I think it was great quality tennis and in the final set there were some wonderful points", said 2008 and 2010 champion Nadal.

Crocodiles are farmed for their meat and hides, but large and battle-scarred crocs are usually unsuitable for the handbag market. Authorities had been hunting the giant crocodile for a decade, ranger John Burke said.

The 13-time Champions League winners believe the Belgian striker is the one to fill Ronaldo's boots in Madrid next season. Manchester United chose not to sign Ronaldo due to their interest in his Real Madrid team-mate Gareth Bale.

The world's biggest entertainment company and owner of NBC and Universal Pictures gate-crashed Murdoch's bid for Sky in February . Fox, which already owns 39 percent of Sky, said it had secured the agreement of the independent committee of Sky for the deal.

Brad Keselowski won the battle off of pit road and would lead the field at the start of stage 2 in the Coe Zero 400 at Daytona. With 36 laps to go Larson had a tire go down and Stenhouse Jr. drove into the rear of the No. 42 Chevrolet.

Lorna Wilkinson, the hospital's director of nursing, said: "While this is welcome news, clearly we are not out of the woods yet". Britain and its allies blamed Russia for the attack on the Skripals, prompting a major Western expulsion of Russian diplomats.

In a statement Tuesday, White House press secretary Sarah Sanders called that decision to resentence the Hammonds "unjust". The announcement came as President Trump set out for North Atlantic Treaty Organisation meetings in Brussels, Belgium.

TS Chris begins NE path in Atlantic Ocean
She added: "The weather we will be seeing after Chris is not particularly windy, but we could see rain moving in from the west". Hurricane Chris is expected to bring heavy rainfall and strong winds to the southeastern half of Newfoundland.

England fans pack pub
Despite a valiant effort, the team will come home without the Jules Rimet trophy after losing the semi final 2-1 to Croatia . The game will kick off in Saint Petersburg on Saturday, before Croatia play France in the final in Moscow on Sunday.


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TEL AVIV, Israel — Father Tesfayohanns Tesfamariam has always prayed his way through the darkest days. Growing up in Eritrea — a small East African country run by one of the world’s most brutal dictatorships — he prayed to God to find freedom.

When he fled Eritrea, as tens of thousands of others have to escape the slavery-like military conscription there, he prayed for God’s protection. When he was then trapped by human traffickers and tortured by smugglers in Libya and Egypt’s Sinai Peninsula — the physical and psychological wounds from which are still raw today — he prayed for the strength to survive.

When the priest made it across the border into Israel in 2010, he prayed that he would finally be safe.

And Tesfamariam, 44, was relatively safe, for eight years. Many people don’t know it, but Israel — home to the world’s largest Jewish community — also houses an estimated 40,000 African refugees who started arriving in the country en masse in the mid-2000s to escape war, economic hardship, and persecution.

In the southern part of Tel Aviv, Israel’s vibrant commercial capital, African food stands are a common sight. The streets echo with many languages, including Tigrinya, which many people speak in Eritrea, and Arabic, which is spoken in Sudan.

But all of that may soon change because of Israel’s new, and deeply controversial, push to rid the country of its African asylum seekers. Israel says they are economic migrants that the Jewish state can’t and shouldn’t have to care for; critics say Israel’s moves violate international law by denying legitimate asylum claims and deporting people to countries where they’ll be unsafe again.

This month, Israel started issuing deportation orders that present a bleak choice: take $3,500 and leave — or face imprisonment. The issue has divided Israelis as well as the larger Jewish community; some argue that Israel’s identity as a refuge for persecuted Jews should extend to non-Jewish asylum seekers as well.

The problem for Tesfamariam is that a majority of Israelis seem to support the government push to deport the Africans. Sixty-six percent of Jewish Israelis (and half of Israeli Arabs, who make up 20 percent of Israel’s population) favor the deportation plans, according to a late January poll by the Israel Democracy Institute.

It’s part of a worldwide wave of anti-immigrant fervor that is playing out in dozens of countries ranging from smaller places like Hungary to larger powers like the US.

The stakes extend well beyond Tesfamariam and his community of Eritrean refugees. If Israel continues to deport Africans, it will be another sign of how the Jewish state is solidifying a more right-wing nationalist identity and an increasingly closed conception of who belongs and deserves rights.

But if asylum seekers and activist groups in Israel succeed in blocking the effort, it could rejuvenate the more liberal parts of Israel’s civil society that have struggled to build broad enough coalitions for nationwide change. In the meantime, Tesfamariam — and tens of thousands of Africans — are waiting and watching to see which way the country goes.

“They have no idea what is waiting for them on the other side,” he said in a hushed voice.

A country created to take in persecuted Jews is struggling with how to take in persecuted Africans

In Europe last year, about 90 percent of the tens of thousands of Eritreans who applied for asylum were allowed in. In Israel, just 10 Eritreans and one Sudanese person have received asylum since 2009.

That reflects how Israel — a country built to be a refuge for Jews fleeing persecution — is struggling to develop its own policies toward non-Jews seeking asylum amid fears of losing its Jewish majority.

Instead, the country’s law classifies the mainly Eritreans and Sudanese who have crossed over from Egypt in recent years as “infiltrators” — a term first used in the 1950s to refer to Palestinians who would infiltrate from the then-Jordanian-controlled West Bank to attack Israel.

This wave of Africans have been trying to make their way into Israel since about 2006, and by 2012, roughly 60,000 Africans had succeeded. That influx largely ended around 2013, when Israel completed construction of a wall along its southern border with Egypt.

But the wall didn’t fix the question of what to do with the Africans who were already there. So Israel developed different policies along the way, making life harder and harder for those who stayed in an effort to coerce them into leaving, according to Human Rights Watch.

“These [methods] include indefinite detention, obstacles to accessing Israel’s asylum system, the rejection of 99.9 percent of Eritrean and Sudanese asylum claims, ambiguous policies on being allowed to work, and severely restricted access to healthcare,” a 2014 HRW report found.

Over the years, new laws have meant more paperwork and rules that people have to follow to avoid being deported. In 2013, Israel built Holot, an open-air detention center in the south for men; those who didn’t report when summoned could be imprisoned or deported. (In 2015, Israel’s supreme court ruled that African refugees and migrants could only be held there for 12 months.)

Last year, Israel enacted new legislation adding an extra tax on the salaries of asylum seekers, most of whom were already working menial and low-paying jobs. (Asylum seekers aren’t technically supposed to work, but the government allows it in some circumstances.) The law made it more expensive for employers to hire asylum seekers and created a new fund where 20 percent of each person’s monthly salary is set aside — to be accessed only once they’ve left Israel.

This last part is crucial: The government is using money to pressure people to leave — a process that human rights groups say violates international law because they’re being sent to countries that can’t ensure their safety.

A few years ago, Israel started offering asylum seekers $5,000 and a plane ticket to undisclosed countries in Africa, widely known to be Rwanda and Uganda. The Israeli government denies that it is deporting anyone against their will and insists that Rwanda and Uganda, with which Israel has warming ties, are safe.

But Africans who have taken the money and left Israel, and human rights groups that have monitored what happens next, warn that the reality is quite different. Once in Rwanda or Uganda, the asylum seekers have had their money and paperwork stolen and have often become ensnared in human trafficking.

In one particularly brutal case, at least three Eritreans who left Israel and then tried their luck on the migrant trail to Europe were beheaded by ISIS in Libya in 2015; relatives recognized their faces from pictures and videos ISIS posted online.

About 20,000 Africans have left Israel in recent years, according to the Israeli government.

In February, Israel began issuing deportation orders to some people renewing their visas, giving them 60 days to take the money and leave or be imprisoned. The government says that for now it is only deporting single men who had open asylum applications as of the start of 2018.

But African refugees of all nationalities, including the estimated 6,000 children who have been born in Israel, are scared for their future.

Israel is the latest country to adopt harshly anti-immigrant policies

The debate over what to do about Israel’s asylum seekers has divided the country and Jewish communities in America and raised larger questions about Israel’s identity and Jewish values.

On one side is Israeli Prime Minister Benjamin Netanyahu — Israel’s veteran kingpin currently battling several corruption cases — who has blamed Africans for crime in Israel and stirred up his voter base by using racially charged language and promising to deport the asylum seekers. He’s backed by members of his far-right ruling coalition, including culture and sports minister Miri Regev, who has likened the Africans to “a cancer.”

This summer, Netanyahu toured the southern part of Tel Aviv, where the government initially sent Africans to live, and promised its Israeli residents that the government would “give back” the area. Some of southern Tel Aviv’s Jewish residents, many of whose families were immigrants to Israel only decades ago, have been organizing against the growing African community in the neighborhood. (Men who have served time in Holot are legally banned from living in Tel Aviv, though many do anyway, as it’s easier to find work there.)

Israeli politicians may not see much to gain in today’s coalitions by speaking out against the deportations. But a determined sector of Israelis in Jerusalem and Tel Aviv is organizing against it by drawing on the Jewish people’s own history of repression and the Holocaust that preceded Israel’s creation.

Rabbis both in Israel and abroad have signed petitions opposing the plan and pledging to hide Africans in their homes to prevent their deportation — citing Anne Frank’s story as precedent. Pilots from Israel’s national airline, El Al, have called for a boycott of flights with deportees (a gesture activists lauded, though they then pointed out that El Al isn’t actually chartering those flights). On February 24, an estimated 20,000 Israelis joined Eritreans for a solidarity march through southern Tel Aviv.

At a protest in Jerusalem in February, one of the organizers, 18-year-old Omer Leven, told me he’d been moved to stand up against the deportations because “we have to do something about it.” As in similar events, Israelis at the rally chanted in defense of human and refugee rights and accused the government of racism.

Some of the slogans, like “human rights for all” or “racist government, don’t deport the refugees” were reminiscent of chants at protests against the Israeli occupation of the Palestinian territories — an issue that’s all but taboo to talk about in mainstream Israel circles today.

Leven, who wants to be a combat soldier during his mandatory military service, said he sees the two issues as very different. “We decided that what we are doing here has nothing to do with politics,” he said. “It’s about basic human rights.”

African refugees are fighting to preserve their new lives in Israel

African refugees in Israel have been living for years with talk of deportations. Humor sometimes helps people cope with those fears: Some Eritreans now joke when making plans with friends that they should hang out “before they deport us.”

Others are buckling under the pressure. Israel has never been an easy place for single Eritrean men, who, after surviving the perilous trek to Israel, have struggled to get by with low-paying work and without social and familial ties.

Fed up and without hope, some have taken the money to leave, and others say they’d rather leave than face jail. Earlier this month, hundreds of African asylum seekers in Holot started a hunger strike after seven men there were transferred to the nearby Saharonim Prison for refusing to be deported.

But now a new generation of Eritrean activists across Israel is working to organize coalitions against the deportations, educate community members about their rights and what awaits them, and make sure people’s paperwork is up to date.

One of those activists is 29-year-old Teklit Michael, who was once one of the fastest runners in Eritrea until he fled to avoid military and religious persecution. Today he’s constantly fielding interview requests amid his full-time job as a coordinator at a center for embattled Eritreans.

“The people deported to Rwanda and Uganda have no protection,” Michael told me. “They could face torture and slavery.”

Tesfamariam, the priest, understands why many in the community are now angry and scared after years trying to rebuild their lives amid all the uncertainty in Israel.

“Nobody hears their cries,” he said.

Tesfamariam works at a nondescript Eritrean church in Tel Aviv without drawing a salary; he moonlights as a plumber to earn enough money to pay his rent. He told me that he’s willing to go to jail rather than leave the country, but he worries about what will happen to others who choose to take the money to leave Israel.

In Libya, his torturers targeted him for being a Christian; he’s worried that the Eritreans in his community, too, will face further danger if they fall back into the hands of human traffickers.

In the meantime, he’s keeping the faith that God will provide. His source of strength is his church, known for its opposition to the Eritrean government. A decade ago, the Eritrean government arrested the head of the Eritrean Orthodox Church, put him under house arrest, and targeted church members who didn’t accept his replacement. Tesfamariam and Michael were among those who never did. Now they can worship freely here, in exile in southern Tel Aviv. It’s all that Tesfamariam has to depend on after everything that’s passed.

“This church serves the people who have become victims,” said Tesfamariam, taking a break from early morning Saturday prayer to speak to me. “We have to stand with our people. We have to stand with the victims.”

Miriam Berger is a freelance journalist with a focus on people and politics in the Middle East. She is currently based in Jerusalem.


China has changed the way it talks about its actions in the South China Sea, signalling a shift in the way it thinks.

The Philippines released photos of China’s construction and land reclamation activities in the South China Sea, on May 15, 2014, a day after Manila accused Beijing of violating the 2002 Declaration on the Conduct of Parties in the South China Sea by carrying out such construction. Chinese Foreign Ministry Spokesperson Hua Chunying was asked about the photographs in her regular press conference on May 15. Here’s her response, in full:

China has indisputable sovereignty over Nansha [Spratly] Islands including Chigua Reef [Johnson South Reef] and the contiguous waters. Whatever construction China carries out in the Chigua Reef is completely within China’s sovereignty.

That response did not evolve much over the next ten months. In March 2015, Hua was still defending China’s construction with terse proclamations: “China’s normal construction activities on our own islands and in our own waters are lawful, reasonable and justifiable.”

Over the last four months, however, we’ve seen a major change in the way China talks about its activities in the South China Sea – first a clarification of the purposes of the construction, then an announcement that land reclamation will soon be coming to a halt. As others have pointed out, China’s change in rhetoric does not accompany a change in behavior – it still plans to finish all its previously-started construction, for example. Nonetheless, the shift in tone is important.

This shift signals that China realizes the damage being done to its soft power in the region by sticking to a because-I-said-so attitude (ie, our construction is “lawful, reasonable, and justifiable” simply because we said so). In its new statements, China is actively trying to convince listeners (most prominently, the governments of ASEAN countries and the United States) that it does not pose a threat – clear proof that Beijing does, indeed, care about its image.

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The change began on April 9, when, for the first time, Hua (speaking for China’s Foreign Ministry) provided a detailed explanation of the purposes for China’s construction on disputed islands. In her remarks, Hua framed the construction as an example of China upholding its “international responsibility and obligation” by providing new facilities to assist with “maritime search and rescue, disaster prevention and mitigation, marine science and research, meteorological observation, environmental protection, navigation safety, fishery production service and other areas.” Beijing wants to flip accusations of irresponsibility on their head, arguing that China (as the region’s major power) has not only the right but the responsibility to construct facilities in the South China Sea.

In later comments, Beijing expanded on this by suggesting that other countries would also be welcome to use the new outposts. The National Development and Reform Commission (NDRC) released a statement outlining its plan for the South China Sea reefs, including the construction of “large-scale lighthouses”; wireless navigation facilities; base stations for maritime security and communications; emergency rescue stations; moorings for fishermen to shelter from storms or replenish supplies; and weather stations, oceanic observation platforms, and scientific research centers.

Beijing also announced on June 16 that its land reclamation activities would finish “in the upcoming days,” although Chinese officials have declined to provide a concrete timeline. Construction on the reclaimed land will continue, however, as China builds up facilities on newly-created islands. Foreign Ministry Lu Kang made that clear in the initial announcement: “After the land reclamation, we will start the building of facilities to meet relevant functional requirements.”

Experts at the Center for Strategic and International Studies note that, prior to the announcement, China had already finished land reclamation on Johnson South Reef and Fiery Cross Reef and is nearly done in other places. “[T]his recent announcement appears only to confirm what analysts already knew: Beijing has almost finished its planned land reclamation activities in the Spratlys,” CSIS experts concluded. They categorize the announcement as “a change in message, not in policy.”

Very true, but the change in message is important nonetheless. Compared to a year – or even four months – ago, China’s rhetoric on the South China Sea issue is almost unrecognizable. Instead of beginning and ending the conversation with proclamations of China’s “indisputable” sovereign rights, we’re seeing a genuine attempt at putting some China-friendly ‘spin’on these construction projects. That these new proclamations are unlikely to convince China’s critics is beside the point. The attempt itself is new, and noteworthy in the sense that it signals a level of recognition of how China’s actions appear through other’s eyes.

As Chinese Academy of Social Sciences scholar Xue Li recently pointed out in a piece for The Diplomat, the South China Sea disputes could jeopardize the success of Beijing’s top foreign policy priority: implementing the “Belt and Road” projects. The Maritime Silk Road in particular will require cooperation and buy-in from China’s neighbors in ASEAN. If the South China Sea disputes cause problems for the “Belt and Road” (as Xue argues is likely to happen), Xue suggest that China “adjust its South China Sea strategy and policies.”

The shift in messaging may be the first stage in this adjustment. As my colleague Prashanth has noted before, China’s South China Sea strategy involves both asserting sovereignty in the region and maintaining adequately friendly ties with rival claimants, largely based on economic relations. As China itself put it in its military strategy white paper, Beijing must strike a balance between “rights protection” (defending China’s claims) and “stability maintenance” (making sure tensions in the region do not get out of hand).

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China’s “Belt and Road” project was announced in fall of 2013, during a larger ‘charm offensive’ that accompanied Xi Jinping’s tour of Southeast Asia. But the fruit of that labor (including the Belt and Road) withered away during the tensions of 2014 and 2015. The policy fell out of balance, tipping too far toward the side of “right protection” at the expense of “stability maintenance.” China seems to have woken up to that fact and is adjusting its messaging accordingly.

There are limits to this shift, of course — most notably that China has changed rhetoric but not behavior. Beijing has repeatedly stated it will not give up an inch of its territory, including the nine-dash line in the South China Sea; that policy will remain the bedrock of its maneuvering in the disputed area. In addition, though Beijing has provided a lengthy list of civilian functions for the island outposts, it has not been similarly upfront about its plans for militarizing the islands, although officials have confirmed that the new facilities will be used for defensive purposes, including “better safeguarding territorial sovereignty.” That means the area that most concerns other claimants – newly militarized Chinese outposts in the South China Sea – remains a black box.


Q:

Return Title Record With Cast using Netflix OData Service

Is there a way to return a result set made up of a Title record and the associated Cast collection if I filter on Title.Id?
I.E., give me all Title fields where Id = "ApUFq" and associated Cast (Person) collection.

A:

I think this is the query you're describing: http://odata.netflix.com/v1/Catalog/Titles('ApUFq')?$expand=Cast
Or in LINQ:
from t in Titles where t.Id == "ApUFq" select new {t, t.Cast}


The most crushing aspect of Andy Murray’s Australian Open was not the first press conference, when he wept after two words and then announced his possible retirement at Wimbledon, if not before, due to a persistent hip injury. Nor was it the instant classic first-round match, which Murray lost to Roberto Bautista Agut in five sets. After the eulogies and the public mourning and the widespread consensus on the significance of his glittering career, the most dispiriting moment came when, shattered by his regrets, he rebutted them all.

“Some people might say: ‘It’s a positive thing that Andy worked really, really hard, such and such, trained hard,’” said Murray in his post-match press conference. “But I also often didn’t stop myself when I was being told to do things. I should have sometimes said, ‘No, I’m not doing that today.’ Or: ‘No, I don’t want to train today. I’m sore. I need a day off.’ I didn’t do that.”

Murray’s doubts and his ravaged cartilage are the most significant news from the Australian Open this year, but that news is also a larger reflection of the grind that tennis inflicts on its subjects. Governed by points, prize money, and appearance fees, the athletes circle the globe for 11 months each year, chasing the sun and success. Tennis players must meet specific tournament quotas to collect bonuses and maintain their rankings. For most, this means a constant cycle of traveling, playing matches, or training for their next event, with the threat of burnout and injury looming endlessly.

“You don’t know how long you’re going to be successful,” says Roger Federer, one of the most successful players in the history of the sport. “You just don’t know. That’s why I think a tennis player’s life, it’s very short-term planning. It makes it kind of difficult because we don’t have a five-year deal in some club, like team sports.” It’s often only when players meet people from outside of their bubble that they consider the strangeness of their professional lives. “I speak to French handball player Daniel Narcisse and he says: ‘Fuck! It’s crazy for you guys. One week you’re in Australia. One week you’re in France,’” says young Frenchman Lucas Pouille. “We travel all the time, we play, and we never rest.”

Related Andy Murray Deserves a Better Farewell

The grueling schedule grinds players mentally and robs them of their free time, but the physical cost is often insurmountable. In the past two years, almost every male top player has suffered a serious, career-threatening injury. Roger Federer tore his meniscus, Novak Djokovic sobbed for three days after he was forced undergo surgery, and Rafael Nadal has spent his career at war with his own body. Murray, meanwhile, has struggled with his hip since 2017. “The men have the longest season,” says Andrea Petkovic. “Plus, they have the best of five at the slams. Especially the best players, I think they will start to rethink everything.”

Still, female players face similar demands in a physical sport with immense depth. “It’s a very taxing profession,” says Johanna Konta, laughing. “There’s no other way to say it. We consciously put our bodies and our mental and physical health on the line to be able to be in the position to get a chance to achieve something.”

Murray’s injury, and his emotional press conference, have recently highlighted the grueling nature of the sport, but it has been a constant refrain from the players for decades. At the ATP Finals in mid-November last year, just six weeks before the beginning of the new year, Alexander Zverev spoke out about the length of the season, branding it “ridiculous”: “We don’t have time to prepare. We finish here, we go for a 10-day holiday, then we train for two weeks, then we go to Australia,” he said.

“I think every sport has somewhat of an offseason. We have, too, but you can’t count it like that,” Federer says. Nadal in particular takes issue with the dominance of the concrete surface that pounds the joint and his knees. At the Australian Open, he shrugged. “It’s true that I’ve had so many injuries in my career,” he says. “I’ve said it many times but in the end, I think that one has to consider if tennis wants to go down this route on this surface.”

Though most players can’t relate to the potentially life-altering aspects of Murray’s injuries, they understand that it’s a possibility for all. “You’re not asking yourself, ‘Hey, am I going to be able to play tennis next week, or next match, or the next Grand Slam down the road,” Milos Raonic says. “You’re sort of asking yourself: ‘Can I live a normal life after? Could I go out for a run, can I stay in shape? Or am I going to be confined to very limited activities in life?”

The greatest measure of tennis’s dysfunction is in the way it contradicts regular athletic norms. In most other sports, injuries are the greatest enemy: They sideline competitors from their livelihoods and overwhelm an individual with doubt. But recent tennis seasons have been filled with players who suffered serious injuries, departed the tour for a long period, and then returned with a refreshed perspective. In tennis, injuries can inadvertently drive players to their greatest success.

U.S. no. 1 Sloane Stephens achieved one of the great comebacks in 2017 by winning the U.S. Open in her fifth tournament back from foot surgery after only 11 months away. For Stephens, her break from the sport offered her a “reset” and the chance to live like a real person. “I felt like I was missing something at home,” she says. “I was able to be with my grandparents and really experience life.”

The latest wave of players to return to the sport after career-threatening injuries included former slam finalists Tomas Berdych and Jo-Wilfried Tsonga. For Berdych, it wasn’t until he finally paused for six months in 2018 that he was able to sit back and reflect on the absurdity of his existence in the sport. He likened the tour to being trapped: “For me, it was like getting out of a box which is just spinning around, and you can’t really get out of it, and it‘s melting you down. When you took the time off, you get out of it and you’re just watching the box from the outside, and you see all what’s happening.”

Berdych saw his back injury as a simple opportunity to let go. He used the time to refresh mentally and decided to return only when he felt like it. “If I needed one year, then I [would have] taken one more year,” he says. “I wanna say that [this break is] the best thing I’ve done in really long years. It just opens up my eyes and gives me a great perspective on tennis.”

Tsonga, who missed seven months after knee surgery, underlined the joy he discovered in mundanity. “You feel light!” he says, chuckling. “Because, of course, to travel every week, never sleep in your bed, always in hotel every week, it’s not something common. We’ve lived this life since we were kids. For us it’s also refreshing to stay at home, do the same things every day.” U.S. no. 3 Madison Keys concurs: “I was really excited to go to the grocery store, like, every day,” she says. Only in tennis can physical ruin promote mental self-care.

Personal crisis has also proved an avenue for players to find themselves away from the tour. Timea Bacsinszky effectively retired from tennis in 2013, and she worked in a hotel until spontaneously deciding to return to the sport. In a sport where players are often thrust into competition from a young age, flanked by older coaches and instructed to focus only on maximizing their chances of success, autonomy can be an alien concept for younger players. Bacsinszky returned determined to play tennis only for herself, breezing past her previous career high of 37 and into the top 10.

“I had to step aside and do what I wanted to do. Not what I was good at and what people were telling me to do because ‘It would have been a shame if I was not playing tennis,’” she says, her voice rising. “I’m the owner of my life. I have the right to do whatever I wanted to do. And even with my career now, I have the right to choose to come back or not to come back or to pick the persons around me.”

“It’s like a washing machine,” says Pouille, who reached the top 10 in March before considering a break from the sport by the end of the year. “You stop, you go in the wash, and then you come back and you’re clean, you’re ready to go mentally and physically. I think we’re going to see it more and more on the tour.”

In many ways, tennis players function far more like celebrities than athletes: Their “teams” consist of no teammates, just an entourage of employed coaches, advisers, and specialists. Meanwhile, players are disproportionately responsible for their own management and health. When soccer players are injured during a match, it’s usually the coach that decides on a substitution; in tennis, the players make decisions that affect their future, often while under the burden of extreme financial and competitive pressure. As Murray noted in his second press conference, the athlete’s instinct is always to attempt to endure the pain. When combined with a professional’s need to keep a career going, the average tennis player’s instinct to persevere can be catastrophic.

It’s no coincidence that the players who have attained unprecedented longevity are the Williams sisters and Roger Federer, three athletes with historic success and the financial freedom that comes with it. Venus and Serena Williams spent much of the first half of their careers savaged by the media for making their own standards, pursuing their businesses, and playing limited schedules. Their lives beyond the court are what have sustained their interest in the sport for so long. Unlike most of their rivals, they have always remained on the periphery, playing the barest minimum.

For Serena, the demands of the tour weren’t compatible, so she simply didn’t comply with them. “I just literally couldn’t try to do all the traveling,” she says, shrugging. “I was always doing something. I was either in school or working on some company. It was always something I was doing, so it didn’t actually allow me to travel as much. I always feel when I play tournaments, I just try to do the best I can in them, and then maybe I wouldn’t have to play as many.”

On the other hand, Federer’s longevity is the consequence of long-term planning. After winning his first Australian Open in 2004, Federer sat down with his fitness coach, who implored him not to be drawn by the appearance fees offered by lower events. “He told me, ‘Just do me a favor, please, and don’t chase appearance fees and play every tournament.’ I was like, ‘No, I won’t. I will try to play the best schedule possible.’ … I think I can be very happy that I didn’t do that.”

For many players, however, the examples of both the Williams sisters and Federer are unattainable. They stress that their ability allows them to pick and choose the events they play and the amount of time they spend competing. “Roger is really strong because he accepts that even if he loses somewhere, he’s gonna take three weeks, then rest, practice and come back,” Pouille says. “Of course, he’s no. 1, it’s easy! But still, he plays 13 tournaments a year when some players are playing 25, 28, and 30 tournaments, and that’s a lot.”

The great tragedy of Andy Murray’s career is that, like so many tennis players, he never got healthy enough to plan for longevity. He announced his first surgery at the end of 2013 with the reassuring yet oxymoronic phrase “minor back operation.” He ended the following season desperately chasing his ranking, exhausting himself through grueling matches and countless tournaments in succession. In 2016 again, he pursued the no. 1 ranking with a brutally demanding schedule; six months after finishing the year as no. 1, he faced Stan Wawrinka in the five-set French Open semifinal. And then, give or take a goodbye match this summer, his hip was done.

For the rest of the players, what struck them most was the cruelty of Murray’s fate, dictated not by his own choices but by an injury that proved insurmountable. “This is really sad for all of us,” Novak Djokovic says. “We talked about it. It’s sad to see him go that way, that is not on his terms. Injuries are the greatest obstacle that an athlete can have.”

Bacsinszky says: “This is the thing that I would fear the most in the future. This is the only wish I have. It’s really—the day I decide I wanna stop, let it be my own decision. Mine. It belongs to me. Not to an injury.”


Gufo radar

The Gufo radar was an Italian naval search radar developed during World War II by the Regio Istituto Elettrotecnico e delle Comunicazioni della Marina (RIEC). Also known as the EC-3 ter, its name is the Italian word for owl.

Description
The first prototypes were designed by navy technicians Ugo Tiberio, Nello Carrara and Alfeo Brandimarte in the period 1936–1937. The project was stalled due to budget cuts until 1941, when interest was revived soon after the Italian navy suffered a series of heavy setbacks in night actions against the radar-equipped units of the Royal Navy, especially that of the Battle of Cape Matapan.

The first tests were conducted on board the ageing torpedo boat Giacinto Carini in April 1941. The radar sets were produced by the Italian company SAFAR. Only 12 devices had been installed on board Italian warships by 8 September 1943, the day Italy signed an armistice with the Allies. Beginning in the spring of 1943, the recommendation of the Italian high command was to switch the radar on only in proximity of enemy forces, after an incorrect German advisory that the British had radar warning receivers similar to the Metox. The Allies, however, did not develop such technology until 1944. In spite of this, it has been reported that the crews made a wide use of the Gufo as a search radar, omitting to mention it on the ship's logbook to avoid sanctions.

The radar was used in combat by the light cruiser Scipione Africano on the night of 17 July 1943, while on passage from La Spezia to Taranto, when she detected a flotilla of four British Elco motor torpedo boats five miles ahead in the strait of Messina. One of the motor boats, MTB 316, was destroyed by the cruiser's guns, and another one was seriously damaged. Twelve British seamen lost their lives.

See also
 Operation Scylla

References

Bibliography

Category:Naval radars
Category:World War II radars
Category:Military radars of Italy
Category:Regia Marina
Category:World War II military equipment of Italy
Category:World War II Axis electronics


Gadwall

Mareca strepera

In many of our dabbling ducks, the males have bright ornate patterns, while the females are plainly marked with brown and gray. In the Gadwall, even the male looks plain at a distance; only a close view reveals subtle but beautiful colors. Although it is widespread in North America (and in Europe and Asia as well), the Gadwall is most common on inland waters west of the Mississippi River.

Conservation status

Settlement of the northern Great Plains may have reduced Gadwall numbers more than those of most ducks. Current populations vary substantially from year to year, but not in serious decline.

Lakes, ponds, marshes. In summer mainly around fresh or alkaline lakes in prairie regions or western intermountain valleys where land is open, not forested; also locally in coastal marshes. In migration and winter on marshes, lakes, estuaries, but generally not on salt water.

In many of our dabbling ducks, the males have bright ornate patterns, while the females are plainly marked with brown and gray. In the Gadwall, even the male looks plain at a distance; only a close view reveals subtle but beautiful colors. Although it is widespread in North America (and in Europe and Asia as well), the Gadwall is most common on inland waters west of the Mississippi River.

Photo Gallery

Feeding Behavior

Forages mainly while swimming by taking items from surface or by dabbling with head submerged, sometimes by up-ending, occasionally by diving. Rather seldom forages on land.

Eggs

8-11, sometimes 5-13. White. 2 or more females sometimes lay in same nest. Incubation is by female only, 24-27 days. Young: leave nest shortly after hatching. Female leads young to water, where they find their own food; often seen on more open water than young of other dabbling ducks. Young are capable of flight 48-59 days after hatching.

Young

Leave nest shortly after hatching. Female leads young to water, where they find their own food; often seen on more open water than young of other dabbling ducks. Young are capable of flight 48-59 days after hatching.

Diet

Mostly plant material. Feeds mainly on aquatic plants. Compared to other dabbling ducks, eats more leaves and stems of these plants, fewer seeds. Also eats small numbers of mollusks, insects, crustaceans, rarely small fish. Very young ducklings eat many insects at first before shifting to more vegetarian diet.

Nesting

In one courtship display, male pulls head far back on shoulders and raises rear part of body out of water, with wingtips lifted to show off white patch in wing. Compared to most ducks, nesting begins rather late. Nest: female, accompanied by male, makes prospecting flights to seek site for nest. Site is usually near water, on dry land, surrounded by dense weeds or grass. Nest (built by female) is in a shallow depression, built of grasses, weeds, lined with down.

How climate change could affect this bird's range

In the broadest and most detailed study of its kind, Audubon scientists have used hundreds of thousands of citizen-science observations and sophisticated climate models to predict how birds in the U.S. and Canada will react to climate change.

Gadwall

Each map is a visual guide to where a particular bird species may find the climate conditions it needs to survive in the future. We call this the bird’s “climatic range.”

The colors indicate the season in which the bird may find suitable conditions— blue for winter, yellow for summer (breeding), and green for where they overlap (indicating their presence year-round).

The darker the shaded area, the more likely it is the bird species will find suitable climate conditions to survive there.

The outline of the approximate current range for each season remains fixed in each frame, allowing you to compare how the range will expand, contract, or shift in the future.

The first frame of the animation shows where the bird can find a suitable climate today (based on data from 2000). The next three frames predict where this bird’s suitable climate may shift in the future—one frame each for 2020, 2050, and 2080.

You can play or pause the animation with the orange button in the lower left, or select an individual frame to study by clicking on its year.

The darker the color, the more favorable the climate conditions are for survival. The outlined areas represent approximate current range for each season. More on reading these maps.


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