BUSCADOR RECOLECTA

Additional file 7: Fig. S7. Aβo neurotoxicity is worsened when microglial cells are present. (A) To characterize Aβo, a negative stain with 1% uranyl acetate was performed followed by electron microscopy analysis. Arrows indicate small oligomeric structures (scale bar, 200 nm). (B, C) Representative images and quantitative analysis of neurons treated as indicated, immunostained with anti-acetylated tubulin antibody (scale bar, 100 μm). (D, E) Representative micrographs and quantitative measurement of neurons and neuron/microglia cocultures treated as indicated, immunolabeled with anti-acetylated tubulin and anti-Iba1 antibodies as specified in the images (scale bar, 100 μm in the middle panel; 50 μm in the right panel). Each experiment was performed at least three independent times, with three replicates per condition each time. The data are presented as mean ± S.E.M. and were analyzed by one-way ANOVA followed by Bonferroni post-test. *p < 0.05; **p < 0.01; ****p < 0.0001., Fondecyt FONDAP ISCIII CIBERNED Junta de Andalucia Consejería de Economía y Conocimiento MICIN Ramon y Cajal Program National Institutes of Health., Peer reviewed

Additional file 8: Fig. S8. Full blots of pMLKL and RIPK3 western blots. (A) The membrane was first probed with an anti-hsp90 antibody and then incubated with anti-RIPK3. (B) The membrane was cut, and each piece was incubated with the indicated antibody. Left panel: colorimetric image showing protein ladder. (C) The membrane was cut and incubated with an anti-pMLKL antibody. Following stripping, and after confirming that no bands could be visualized with ECL, membranes were blocked, and detection of the IgG was done. Left panel: colorimetric image showing protein ladder. (D) The membrane was cut, and each piece was incubated with the indicated antibody. Left panel: colorimetric image showing protein ladder., Fondecyt FONDAP ISCIII CIBERNED Junta de Andalucia Consejería de Economía y Conocimiento MICIN Ramon y Cajal Program National Institutes of Health., Peer reviewed

Additional file 9: Fig. S9. TNF-α alone is not sufficient to trigger neuronal necroptosis. (G, H) Representative micrographs and quantitative measurements of neurons treated for 72 h with CMctrl, CMAβ, TNF-α, and with CMctrl/TNF-α, immunolabeled with anti-acetylated tubulin antibody (scale bar, 200 μm). The data are presented as mean ± S.E.M. and were analyzed by one-way ANOVA followed by Bonferroni post-test. *p < 0.05; **p < 0.01., Fondecyt FONDAP ISCIII CIBERNED Junta de Andalucia Consejería de Economía y Conocimiento MICIN Ramon y Cajal Program National Institutes of Health., Peer reviewed

FIGURES. Figure 1. Workflow of the SNVs for the AF-2018 and MEGASTROKE-CES datasets. NaN: Not a number. SNV: Single Nucleotide Variant. Figure 2. Manhattan plot of MTAG-CES. The X axis represents chromosome location, and the Y axis represents the minus logarithm on base 10 of p-value. Figure 3. Polygenic risk score (PRS) performance. Panel A is a bar plot of the r2 for the PRS models of eight different thresholds in the training set. Panel B represents the p-value variation along the full range of thresholds evaluated in the training set. Panel C shows ROC curves and panel D Precision-Recall curves for the PRS performance in the independent test set. SUPPLEMENTARY FIGURES. Supplementary Figure 1. GO Biological processes enriched in CES prioritized gene set. Supplementary Figure 2. GO Biological processes enriched exclusively in analysis of AF associated genes independently of CES risk. Supplementary Figure 3. Polygenic risk score (PRS) performance for the individual predictors. Panel A shows ROC curves and panel B Precision-Recall curves for the PRS performance in the independent test set., [Background] Occult atrial fibrillation (AF) is one of the major causes of embolic stroke of undetermined source (ESUS). Knowing the underlying etiology of an ESUS will reduce stroke recurrence and/or unnecessary use of anticoagulants. Understanding cardioembolic strokes (CES), whose main cause is AF, will provide tools to select patients who would benefit from anticoagulants among those with ESUS or AF. We aimed to discover novel loci associated with CES and create a polygenetic risk score (PRS) for a more efficient CES risk stratification., [Methods] Multitrait analysis of GWAS (MTAG) was performed with MEGASTROKE-CES cohort (n = 362,661) and AF cohort (n = 1,030,836). We considered significant variants and replicated those variants with MTAG p-value < 5 × 10−8 influencing both traits (GWAS-pairwise) with a p-value < 0.05 in the original GWAS and in an independent cohort (n = 9,105). The PRS was created with PRSice-2 and evaluated in the independent cohort., [Results] We found and replicated eleven loci associated with CES. Eight were novel loci. Seven of them had been previously associated with AF, namely, CAV1, ESR2, GORAB, IGF1R, NEURL1, WIPF1, and ZEB2. KIAA1755 locus had never been associated with CES/AF, leading its index variant to a missense change (R1045W). The PRS generated has been significantly associated with CES improving discrimination and patient reclassification of a model with age, sex, and hypertension., [Conclusion] The loci found significantly associated with CES in the MTAG, together with the creation of a PRS that improves the predictive clinical models of CES, might help guide future clinical trials of anticoagulant therapy in patients with ESUS or AF., Peer reviewed

Supplementary Table 2. Detailed number of participants (IS and controls) included from each project. IS: ischemic strokes patients., [Background] Occult atrial fibrillation (AF) is one of the major causes of embolic stroke of undetermined source (ESUS). Knowing the underlying etiology of an ESUS will reduce stroke recurrence and/or unnecessary use of anticoagulants. Understanding cardioembolic strokes (CES), whose main cause is AF, will provide tools to select patients who would benefit from anticoagulants among those with ESUS or AF. We aimed to discover novel loci associated with CES and create a polygenetic risk score (PRS) for a more efficient CES risk stratification., [Methods] Multitrait analysis of GWAS (MTAG) was performed with MEGASTROKE-CES cohort (n = 362,661) and AF cohort (n = 1,030,836). We considered significant variants and replicated those variants with MTAG p-value < 5 × 10−8 influencing both traits (GWAS-pairwise) with a p-value < 0.05 in the original GWAS and in an independent cohort (n = 9,105). The PRS was created with PRSice-2 and evaluated in the independent cohort., [Results] We found and replicated eleven loci associated with CES. Eight were novel loci. Seven of them had been previously associated with AF, namely, CAV1, ESR2, GORAB, IGF1R, NEURL1, WIPF1, and ZEB2. KIAA1755 locus had never been associated with CES/AF, leading its index variant to a missense change (R1045W). The PRS generated has been significantly associated with CES improving discrimination and patient reclassification of a model with age, sex, and hypertension., [Conclusion] The loci found significantly associated with CES in the MTAG, together with the creation of a PRS that improves the predictive clinical models of CES, might help guide future clinical trials of anticoagulant therapy in patients with ESUS or AF., Peer reviewed

[Background] Occult atrial fibrillation (AF) is one of the major causes of embolic stroke of undetermined source (ESUS). Knowing the underlying etiology of an ESUS will reduce stroke recurrence and/or unnecessary use of anticoagulants. Understanding cardioembolic strokes (CES), whose main cause is AF, will provide tools to select patients who would benefit from anticoagulants among those with ESUS or AF. We aimed to discover novel loci associated with CES and create a polygenetic risk score (PRS) for a more efficient CES risk stratification., [Methods] Multitrait analysis of GWAS (MTAG) was performed with MEGASTROKE-CES cohort (n = 362,661) and AF cohort (n = 1,030,836). We considered significant variants and replicated those variants with MTAG p-value < 5 × 10−8 influencing both traits (GWAS-pairwise) with a p-value < 0.05 in the original GWAS and in an independent cohort (n = 9,105). The PRS was created with PRSice-2 and evaluated in the independent cohort., [Results] We found and replicated eleven loci associated with CES. Eight were novel loci. Seven of them had been previously associated with AF, namely, CAV1, ESR2, GORAB, IGF1R, NEURL1, WIPF1, and ZEB2. KIAA1755 locus had never been associated with CES/AF, leading its index variant to a missense change (R1045W). The PRS generated has been significantly associated with CES improving discrimination and patient reclassification of a model with age, sex, and hypertension., [Conclusion] The loci found significantly associated with CES in the MTAG, together with the creation of a PRS that improves the predictive clinical models of CES, might help guide future clinical trials of anticoagulant therapy in patients with ESUS or AF., Peer reviewed

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers14010192/s1, Figure S1: Bioinformatic analysis of 353 human HCC samples based on data generated by the Cancer Genome Atlas (TCGA) Research Network, Figure S2: Representative IHC-stained sections demonstrating nuclear expression of c-MYC. (A) c-MYC nuclear expression inside tumor nodules in patients with MAFLD-related HCC. Left: the DAB-stained images. Right: Hematoxylin stained images. (B) c-MYC nuclear expression in MAFLD patients with advanced liver fibrosis. Left: the DAB-stained images. Right: Hematoxylin stained images, Figure S3: MS in alb-myctg at baseline. WT (n = 9) and alb-myctg (n = 8) mice at the age of 36 weeks. (A) Food caloric intake was stable in both groups through the duration of study. (B) Representative MRI scan images of WT and alb-myctg mice showing body fat distribution in 26 weeks old animals. The quantitative data represent fat/body ratio (%) means. (C) Representative F4/80 IF staining of WAT. Positive cells are stained in green and marked with red arrows. Nuclei are stained in blue using DAPI as counterstain. Scale bar is 100 µm (n = 4), Figure S4: Glucose intolerance. (A) The histogram represents the incremental area under the respective glucose tolerance curve (n = 3). (B) Insulin tolerance test (ITT), and the histogram represents the incremental area under the curve (n = 3). Data are expressed as the mean ± SD, * = p < 0.05; ** = p < 0.01, alb-myctg mice vs. WT controls, Figure S5: Hepatosomatic ratio at 36 weeks. WT (n = 9) and alb-myctg (n = 8) mice at the age of 36 weeks. (A) Liver weight. (B) Liver to body weight ratio, Figure S6: Hepatic phenotype in WT and alb-myctg mice at 36 weeks. (A) qPCR analysis of hepatic mRNA expression expression of Scd1. (B) Histograms showing densitometric analysis of western blots for c-MYC, CYP2E1 and CHOP. (C) qPCR analysis of hepatic mRNA expression expression of Ucp2 (n = 4). (D) Representative TEM images of hepatic endoplasmic reticulum. Representative Transmission Electro-Microscopy (TEM) pictures of WT and alb-myctg mice. Asterisks indicate fat droplets in hepatocytes. Arrows mark endoplasmic reticulum (ER). Scale= 10 µm and 1 µm, Figure S7: Spontaneous proliferation and mild inflammation in alb-myctg mice at 36 weeks. (A) Histograms showing densitometric analysis of western blots for CC3 and PCNA. (B) Ki-67 immunofluorescence of crytosections showing increased cell proliferation (green, as indicated by arrow) of hepatocytes in alb-myctg mice liver and the quantification of % Ki-67 positive cells (n = 8). (C-E) Quantification of % CD45, F4/80- positive cells and SR area respectively using ImageJ software (n = 8). Data are expressed as the mean ± SD, * =p < 0.05; ** = p < 0.01, *** = p < 0.001, alb-myctg mice vs. WT controls, Figure S8: Increased glutamine catabolism in alb-myctg mice at 36 weeks. (n = 5). (A) IHC staining of Glutamine synthetase (GS). (B) Protein expression of Glutaminase 1 (GLS1) in the livers of alb-myctg and WT mice, Figure S9: Activation of signaling pathways relevant to human MAFLD in the liver of mice. (A) Dysregulated genes involved in different pathways. (B) Heat map demonstrating deregulated genes. Red and green colors indicate high and low gene expression, respectively. (C) Top canonical pathways. (D) Molecules involved in hepatotoxicity. All data are based on statistical significance, Figure S10: Spontaneous HCC development in alb-myctg mice. WT (n = 3) and alb-myctg (n = 5) mice at the age of 52 weeks. (A) Macroscopic images of the liver. Numbers represent tumor incidence (%) means (B) Representative liver sections stained with H&E, Figure S11: Phenotypical changes in WT and transgenic mice fed with WD for 24 weeks. (A) Growth curve demonstrating body weight gain, assessed at 7 days interval at each group (B) Relevant adipocyte area of eWAT (n = 6). (C) Representative F4/80 IF staining of WAT. Positive cells are stained in green and marked with red arrows. Nuclei are stained in blue using DAPI as counterstain. Scale bar is 100 µm (n = 4). (D) Quantification of Oil Red O positive area (n = 5). (E) Histological NAFLD activity score (n = 5). (F) Quantification of % CD45 positive cells (n = 6). (G) Quantification of % SR positive area (n = 4). (H) Quantification of % αSMA positive area (n = 3). (I) qPCR analysis of hepatic tissue of fibrosis gene αSma (n = 6). (J) Quantification of % KI-67 positive cells (n = 6). (K) Histograms showing densitometric analysis of western blot for CC3. Data are expressed as the mean ± SD, * = p < 0.05; ** = p < 0.01, *** = p < 0.001, alb-myctg mice vs. WT animals fed WD, Figure S12: MS and phenotypical changes in animals fed with metformin enriched diet. (A) Growth curve demonstrating body weight gain of mice, assessed at 7 days interval at each group, and (B) Relevant histogram represents the incremental area under the respective body weight curve. (C) Level of AST in serum (n = 6–8). (D) Quantification of % Ki-67 positive cells (n = 6). (E) qPCR analysis of hepatic mRNA expression expression of c-Myc. (F) Histograms showing densitometric analysis of western blots for SREBP1. Data are expressed as the mean ± SD, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, alb-myctg mice fed metformin enriched chow diet vs. alb-myctg mice fed chow diet; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, WT mice fed metformin enriched chow diet vs. alb-myctg mice fed chow diet, Figure S13: Uncropped blots for Figure 4C, Figure S14: Uncropped blots for Figure 4E, Figure S15: Uncropped blots for Figure 5I, Figure S16: Uncropped blots for Figure 7I, Table S1: Etiology of human liver samples used for c-MYC gene expression analysis, Table S2: Clinical features of human liver samples, Table S3: Kcal% of chow diet (CD) and western diet (WD), Table S4: Diets used for the development of MAFLD, Table S5: Antibodies used for IF staining, Table S6: Antibodies used for IHC and IF stainings, Table S7: Primers for qPCR, Table S8: List of antibodies used for WB., Peer reviewed

Table 1. Summary of PROM data over the different time points., Dysferlinopathy is a muscular dystrophy with a highly variable functional disease progression in which the relationship of function to some patient reported outcome measures (PROMs) has not been previously reported. This analysis aims to identify the suitability of PROMs and their association with motor performance.Two-hundred and four patients with dysferlinopathy were identified in the Jain Foundation's Clinical Outcome Study in Dysferlinopathy from 14 sites in 8 countries. All patients completed the following PROMs: Individualized Neuromuscular Quality of Life Questionnaire (INQoL), International Physical Activity Questionnaire (IPAQ), and activity limitations for patients with upper and/or lower limb impairments (ACTIVLIMs). In addition, nonambulant patients completed the Egen Klassifikation Scale (EK). Assessments were conducted annually at baseline, years 1, 2, 3, and 4. Data were also collected on the North Star Assessment for Limb Girdle Type Muscular Dystrophies (NSAD) and Performance of Upper Limb (PUL) at these time points from year 2. Data were analyzed using descriptive statistics and Rasch analysis was conducted on ACTIVLIM, EK, INQoL. For associations, graphs (NSAD with ACTIVLIM, IPAQ and INQoL and EK with PUL) were generated from generalized estimating equations (GEE). The ACTIVLIM appeared robust psychometrically and was strongly associated with the NSAD total score (Pseudo R2 0.68). The INQoL performed less well and was poorly associated with the NSAD total score (Pseudo R2 0.18). EK scores were strongly associated with PUL (Pseudo R2 0.69). IPAQ was poorly associated with NSAD scores (Pseudo R2 0.09). This study showed that several of the chosen PROMs demonstrated change over time and a good association with functional outcomes. An alternative quality of life measure and method of collecting data on physical activity may need to be selected for assessing dysferlinopathy., Peer reviewed

Appendix B. Coinvestigators - The Jain COS Consortium., Dysferlinopathy is a muscular dystrophy with a highly variable functional disease progression in which the relationship of function to some patient reported outcome measures (PROMs) has not been previously reported. This analysis aims to identify the suitability of PROMs and their association with motor performance.Two-hundred and four patients with dysferlinopathy were identified in the Jain Foundation's Clinical Outcome Study in Dysferlinopathy from 14 sites in 8 countries. All patients completed the following PROMs: Individualized Neuromuscular Quality of Life Questionnaire (INQoL), International Physical Activity Questionnaire (IPAQ), and activity limitations for patients with upper and/or lower limb impairments (ACTIVLIMs). In addition, nonambulant patients completed the Egen Klassifikation Scale (EK). Assessments were conducted annually at baseline, years 1, 2, 3, and 4. Data were also collected on the North Star Assessment for Limb Girdle Type Muscular Dystrophies (NSAD) and Performance of Upper Limb (PUL) at these time points from year 2. Data were analyzed using descriptive statistics and Rasch analysis was conducted on ACTIVLIM, EK, INQoL. For associations, graphs (NSAD with ACTIVLIM, IPAQ and INQoL and EK with PUL) were generated from generalized estimating equations (GEE). The ACTIVLIM appeared robust psychometrically and was strongly associated with the NSAD total score (Pseudo R2 0.68). The INQoL performed less well and was poorly associated with the NSAD total score (Pseudo R2 0.18). EK scores were strongly associated with PUL (Pseudo R2 0.69). IPAQ was poorly associated with NSAD scores (Pseudo R2 0.09). This study showed that several of the chosen PROMs demonstrated change over time and a good association with functional outcomes. An alternative quality of life measure and method of collecting data on physical activity may need to be selected for assessing dysferlinopathy., Peer reviewed

The available data contains: sky images before, during, and after the flash, event file, corresponding ARF and RMF, light-curves. See README file for further information., Peer reviewed