BUSCADOR RECOLECTA

Additional file 3: Supplementary Figure S3. tRNAs are methylated at guanine-7 by METTL1. A) Western blot showing the expression of doxycycline-inducible HA-METTL1 in PC3 cells. B, C) Density distribution of reads per million (RPM) identified after PAR-CLIP analysis of METTL1-bound tRNAs (B) and other RNA species (C) in PC3 (control) and HA-METTL1 expressing PC3 cells. The red line indicates the third lower quartile of total RPMs. D) tRNAs are the most common RNA species that bind Flag-METTL1 in HEK293T cells. Density distribution of reads per million (RPM) identified after PAR-CLIP analysis of METTL1-bound RNAs: The red line indicates the third lower quartile of total RPMs. Data were retrieved from Bao et al. study. E) tRNAs comprise half of the reads of all RNAs bound to METTL1 after PAR-CLIP analysis in HEK293 cells. F) Boxplot representing the median with interquartile range of reads per million (RPM) per transcript bound to METTL1 in HEK293T cells. G) Schematic representation of genomic editing introduced by CRISPR-Cas9 technology in the PC3 METTL1 KO clones used in this study. H) METTL1 mRNA expression levels in METTL1 KO, and WT control clones used in this study. Mean ± SD, n = 3. I) Graphics representing the experimental workflow followed to generate AlkAniline-seq libraries. J) Normalised cleavage signals for METTL1-methylated tRNAs Cys and Ile, and METTL1 non-methylated tRNAs His and Glu in PC3 WT and METTL1 KO cells., Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

Figure S1: microscopic and spectroscopic characterization of ultrasound-assisted liquid-phase exfoliated MoS2 colloidal dispersions. Figure S2: AFM characterization of cathodically exfoliated MoS2 materials obtained using ammonium salts other than HTMABr as electrolyte. Figure S3: X-ray diffraction patterns of bulk and exfoliated MoS2 materials. Figure S4: cyclic voltammograms and electrochemical impedance spectroscopy (EIS) of the exfoliated MoS2 materials after long-term cycling. Figure S5: microscopic characterization of the ee-MoS2 electrodes. Figure S6: post-mortem microscopic characterization of the ee-MoS2 electrodes cycled at 0.2 A g−1. Figure S7: post-mortem microscopic characterization of the ee-MoS2 electrodes cycled at 0.5 A g−1. Figure S8: post-mortem microscopic characterization of the lpe-MoS2 electrodes cycled at 0.2 A g−1. Supplementary Movies S1–S3: electrochemical expansion of MoS2 over 30 min using HTMABr, TEACl and THACl as electrolytes, respectively., Peer reviewed

Additional file 4: Supplementary Figure S4. Accumulation of 5'tRNA fragments in METTL1 KO cells. A) The most common sequences of 5' terminal guanines (5G) containing 5'TOGs and 4G 5'TOGs are shown. B, C) Northern blot showing the absence of 5'tRNA fragments derived from Lys and Pro tRNA in PC3 WT METTL1 KO cells under normal conditions. D) qPCR showing reduced expression of METTL1 in 22Rv1 cells upon METTL1 silencing using siRNA. Mean ± SD, n = 3. E) Dot blot against m7G showing reduced methylation levels in tRNAs extracted from METTL1-silenced 22Rv1 cells. Mean ± SD, n = 3. F) Northern blot detection of Cys-derived 5'TOGs in METTL1-silenced 22Rv1 cells. Mean ± SD, n = 3. G) Northern blot detection of Cys-derived 5'TOGs in PC3 METTL1 KO cells (second biological replicate), unexposed (0 h) or after 2 and 8 h of oxidative stress exposure. H) Northern blot detection of Lys-derived 5'TOGs in PC3 METTL1 KO cells unexposed (0 h) or after 2 and 8 h of oxidative stress exposure. I) Western blotting of DU145 METTL1 KO and WT cell clones generated using CRISPR-Cas9 technology and parental DU145 (DU) cells. J) Northern blot detection of Cys-derived 5'TOGs in DU145 METTL1 KO cells (second biological replicate), unexposed (0 h) or after 2 and 8 h of oxidative stress exposure. The loading control by red-safe staining (tRNA) is shown in the bottom panels (B, C, F–H, J). Bands corresponding to full-length tRNAs are indicated with stars, and 5’tRNA fragments are indicated with arrows. Statistical tests: One-tailed Student’s t-test (C-F). *p < 0.05., Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

1 file, The current trend of climatic alterations will accelerate the modification of the ocean system by, among other aspects, changing the metal speciation and its bioavailability which may have an impact in their accumulation by marine organisms. Understanding the impact of these potential changes is essential for future risk assessment of metal contamination. In the present study, we selected the species Mediterranean mussel (Mytilus galloprovincialis) as the main European aquaculture production bivalve and due to its widespread use for biomonitoring purposes. A long-term test (2 months) was carried out to explore the impact that global change in the marine environment (warming and CO2 increase) may exert on the accumulation of dissolved trace metals (Cu, Co, Pb, Cd, Cr, As and Ni) in different body parts of mussels (foot and soft tissue). Studied mussels were collected at two different climatic locations (Atlantic and Mediterranean Sea) and exposed to unspiked, unpolluted seawater from the Vigo Ria (NW Iberian Peninsula). Results showed that under the global change conditions proposed in this study (1100 pCO2 and 25 °C), the increase in temperature resulted in a lower condition index and byssus strength for mussels from Atlantic Sea, while Mediterranean sea mussels, adapted to higher temperatures, did not show remarkable variations. According to trace metals accumulation in different body parts of the studied mussels, it was observed that the effect of increasing CO2 alone did not show to have an impact in the bioaccumulation, but the combined stressors (increase in CO2 and temperature) may lead to an increase in the bioaccumulation for some elements. The increase in temperature resulted in a decrease of the Cu content of foot tissue (byssus gland) in mussels from Atlantic Sea, which is in accordance with the lower byssus strength observed under such conditions. Our results indicate that the expected seawater temperature increase, which will be produced gradually during next decades, should be further study to ensure the species adaptability and aquaculture production, Peer reviewed

Additional TEM studies; irradiation effects, and videos of a series of high-resolution TEM images recorder under different conditions. Complementary and extra HRSTEM images, EEL spectra, and DFT calculations are also included in the Supporting Information., Peer reviewed

Additional file 5: Supplementary Figure S5. 5’TOGs bind to translation initiation factors. A) No differences in protein expression of translation initiation complex factors were detected by western blotting in PC3 parental, WT, and METTL1 KO cells. The bottom boxplot shows the normalised protein level quantification, and the dotted lines indicate the mean protein levels in WT cells. B) Global protein synthesis rate measured by OP-puromycin (OP-puro) incorporation in PC3 METTL1 KO cells compared to the control (WT) after NaAsO2 exposure at the indicated time points. Fluorescence was normalised to cell size (FSC). n = 3; mean ± SD. C) Representative western blot showing translation initiation factors pulled down by m7G-cap sepharose beads in PC3 METTL1 KO vs. WT cell extracts. D) Representative western blot showing translation initiation factors pulled with synthetic biotinylated-5'TOG in PC3 WT cells transfected with biotinylated-5'TOGs (5'TOG), anti-TOG (ANT) or scramble RNA (-). E) Representative western blot showing mRNA-cap-bound translation initiation factors after 5'TOG (TOG), anti-TOG (ANT), or scramble control (Ctrl) transfection in PC3 WT and METTL1 KO cells. Quantification of western blots are shown in Fig. 5 (C, D, E). Statistical tests: One-tailed Student’s t-test (A), and multiple t-test (B); ***p < 0.001,, Consejo Superior de Investigaciones Científicas (España), Peer reviewed

Additional file 6: Supplementary Figure S6. METTL1 mediated methylation promotes cell proliferation, and tumour growth in vivo. A) Proliferation of DU145 METTL1 KO and WT cell clones. Mean ± SD, n = 6. The thick dotted line represents the average growth of the WT and KO cells. B) Proliferation of METTL1-silenced 22Rv1 cells. Mean ± SD, n = 3. C) Cell cycle analysis of PC3 METTL1 KO, WT, and parental cells showing decreased cell cycle progression in METTL1 KO cells. Mean ± SD, n = 3. The dotted lines indicate the mean of three biological replicates. D) Representative images of BrdU staining of three clones of PC3 METTL1 KO and WT cells. Quantification is shown in Fig. 5. E) Schematic representation of generation of single-cell-derived spheroids, and representative images of single-cell-derived spheroids (lower panel). Quantification is shown in Fig. 5. F) METTL1 depletion affects tumour growth in vivo. Immunohistochemical staining for METTL1, Ki67, and cleaved caspase 3 (Cl-Casp3) in PC3 METTL1 KO and WT cell-derived xenografts. Right charts show the quantification of Ki67 + and Cl-Casp3 + cells per microscopic field. Mean ± SD, n = 5, and ten images per biological replicate. G-I) Protein expression (G) and m7G methylation levels (H, I) of a second PC3 METTL1 KO cell clone (KO2) ectopically expressing an HA-tagged wild-type (WT) or catalytic dead mutant (AFPA) version of METTL1. METTL1 KO2 cells were infected with the empty vector (eV) as a control. Methylene blue staining was used as the loading control (H, bottom panel). Mean ± SD, n = 3 (H). J, K) Proliferation (J) and spheroid formation capacity (K) of PC3 METTL1 KO2 cells re-expressing METTL1 (WT) or catalytic dead mutant (AFPA) compared with METTL1 KO2 (eV). Mean ± SD, n = 6. L) WDR4 mRNA expression and cell growth of PC3 cells expressing doxycycline-inducible SCR shRNA or two shRNA against WDR4 with or without doxycycline induction. Mean ± SD are represented (n = 6). M) WDR4 mRNA expression and tumour growth of xenografts of cells expressing doxycycline-inducible SCR shRNA or a shRNA against WDR4 with or without doxycycline induction. Mean ± SEM are represented (n = 10 for shWD2, n = 5 for SCR, for each Dox condition). Scale bar represents 100 μm (D), 50 μm (F). Statistical tests: Two-way ANOVA (A, I, J), one-way ANOVA (C), and one-tailed Student’s t-test (B, F, K, L, M). **p < 0.01, ***p < 0.001, ****p < 0.0001., Consejo Superior de Investigaciones Científicas (España), Peer reviewed

HRTEM image of a thick walled TbS-CrS2 nanotubular structure, elemental quantification of a radial EELS line highlighting the O-T alternating system and the surface oxidation of one wavy NT, EELS analyses of the surface of one nanotube highlighting the CrS termination, and Cs-HRTEM micrograph of the surface of a TbS-CrS2 nanotube showing the presence of an outer crystalline layer., Peer reviewed

Additional file 7: Supplementary Figure S7. Translational and transcriptional characterisation of METTL1 KO cells. A) Differential mRNA expression ranked in a volcano plot according to their statistical P-value (-Log10 pV) and their relative abundance ratio (Log2 FC) between PC3 WT and METTL1 KO cells. Coloured dots represent statistically significant (p < 0.05) upregulated (red) and downregulated (blue) mRNAs levels. n = 3. B) GAPDH and KIF20A were not differentially translated in PC3 METTL1 KO cells. Percentage per fraction of mRNA abundance of the indicated genes found in polysomes of WT and METTL1 KO cell lysates. n = 3, mean ± SEM. The boxplot shows the fold change (FC) of mRNA abundance in polysome fractions of METTL1 KO versus WT cells. C) Global protein expression differences ranked in a volcano plot of PC3 METTL1 KO versus WT cells. D) Gene Ontology (GO) category enrichment of biological processes in significantly (p < 0.05) upregulated (UP) or downregulated (Down) proteins identified in PC3 METTL1 KO cells compared with WT cells. E) Western blots of PC3 WT and METTL1 KO cells transfected with synthetic 5’TOG RNAs (TOG), synthetic anti-TOG RNAs (ANT), or scramble RNAs (Ct). High (h) and low (l) western blot exposures are shown for pSTAT1, total STAT1 and IRF9. The bottom bar plots indicate the expression of each protein relative to GAPDH. Mean ± SEM, n = 3. F) Immunohistochemical staining for pSTAT1 in PC3 METTL1 KO and WT cell-derived xenografts. Scale bar represents 50 μm. G, H) Increased mRNA expression levels of ISGs genes in METTL1 KO PC3 (G) and METTL1-silenced 22Rv1 cells (H) cells. Mean ± SD, n = 3. I) Western blot of IRF9 and METTL1 protein levels in METTL1-silenced 22Rv1 cells compared to control cells (SCR). Bottom bar plots show quantification of IRF9 expression normalised to GAPDH. Mean ± SD, n = 3. J) Correlation analysis between METTL1 expression and ISG genes in three human PCa expression datasets reflects an inverse expression correlation. Correlations with p-value <  = 0.05 and |R|> = 0.2 are indicated with (*). Statistical tests: One-tailed (B, G-I) and two-tailed Student’s t-test and Pearson correlation test (J) were performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001., Peer reviewed

By combining ab initio simulations with state-of-the-art electron microscopy and electron energy loss spectroscopy, we study the mechanism of electron beam damage in nitrogen-doped graphene and carbon nanotubes. Our results show that the incorporation of nitrogen atoms results in noticeable knock-on damage in these structures already at an acceleration voltage of 80 kV, at which essentially no damage is created in pristine structures at corresponding doses. Contrary to an early estimate predicting rapid destruction via sputtering of the nitrogen atoms, in the case of substitutional doping, damage is initiated by displacement of carbon atoms neighboring the nitrogen dopant, leading to the conversion of substitutional dopant sites into pyridinic ones. Although such events are relatively rare at 80 kV, they become significant at higher voltages typically used in electron energy loss spectroscopy studies. Correspondingly, we measured an energy loss spectrum time series at 100 kV that provides direct evidence for such conversions in nitrogen-doped single-walled carbon nanotubes, in excellent agreement with our theoretical prediction. Besides providing an improved understanding of the irradiation stability of these structures, we show that structural changes cannot be neglected in their characterization employing high-energy electrons., T.S. received support from the Finnish Foundation for Technology Promotion. T.S., H.J., and E.I.K. further acknowledge financial support from the CNB-E project of the Aalto MIDE program and from the LiBaCAM project of TEKES. J.K. and A.V.K. acknowledge financial support from Helsinki University Funds and Academy of Finland through several projects as well as CSC Ltd. Finland for computational resources. S.K., U.K., and J.C.M. acknowledge the financial support by the DFG (German Research Foundation) and the Ministry of Science, Research and the Arts (MWK) of Baden-Wuerttemberg in the frame of the SALVE (Sub Angstrom Low-Voltage Electron microscopy) project. V.S. acknowledges an EC Grant No. 266391 related to the project ELECTROGRAPH (FP7/2007-2013)., Peer reviewed