BUSCADOR RECOLECTA

Muscle development is a multistep process that involves cell specification, myoblast fusion, myotube migration, and attachment to the tendons. In spite of great efforts trying to understand the basis of these events, little is known about the molecular mechanisms underlying myotube migration. Knowledge of the few molecular cues that guide this migration comes mainly from studies in Drosophila. The migratory process of Drosophila embryonic muscles involves a first phase of migration, where muscle progenitors migrate relative to each other, and a second phase, where myotubes migrate searching for their future attachment sites. During this phase, myotubes form extensive filopodia at their ends oriented preferentially toward their attachment sites. This myotube migration and the subsequent muscle attachment establishment are regulated by cell adhesion receptors, such as the conserved proteoglycan Kon-tiki/Perdido. Laminins have been shown to regulate the migratory behavior of many cell populations, but their role in myotube migration remains largely unexplored. Here, we show that laminins, previously implicated in muscle attachment, are indeed required for muscle migration to tendon cells. Furthermore, we find that laminins genetically interact with kon-tiki/perdido to control both myotube migration and attachment. All together, our results uncover a new role for the interaction between laminins and Kon-tiki/Perdido during Drosophila myogenesis. The identification of new players and molecular interactions underlying myotube migration broadens our understanding of muscle development and disease., Peer reviewed

Muscle development is a multistep process that involves cell specification, myoblast fusion, myotube migration, and attachment to the tendons. In spite of great efforts trying to understand the basis of these events, little is known about the molecular mechanisms underlying myotube migration. Knowledge of the few molecular cues that guide this migration comes mainly from studies in Drosophila. The migratory process of Drosophila embryonic muscles involves a first phase of migration, where muscle progenitors migrate relative to each other, and a second phase, where myotubes migrate searching for their future attachment sites. During this phase, myotubes form extensive filopodia at their ends oriented preferentially toward their attachment sites. This myotube migration and the subsequent muscle attachment establishment are regulated by cell adhesion receptors, such as the conserved proteoglycan Kon-tiki/Perdido. Laminins have been shown to regulate the migratory behavior of many cell populations, but their role in myotube migration remains largely unexplored. Here, we show that laminins, previously implicated in muscle attachment, are indeed required for muscle migration to tendon cells. Furthermore, we find that laminins genetically interact with kon-tiki/perdido to control both myotube migration and attachment. All together, our results uncover a new role for the interaction between laminins and Kon-tiki/Perdido during Drosophila myogenesis. The identification of new players and molecular interactions underlying myotube migration broadens our understanding of muscle development and disease., Peer reviewed

Muscle development is a multistep process that involves cell specification, myoblast fusion, myotube migration, and attachment to the tendons. In spite of great efforts trying to understand the basis of these events, little is known about the molecular mechanisms underlying myotube migration. Knowledge of the few molecular cues that guide this migration comes mainly from studies in Drosophila. The migratory process of Drosophila embryonic muscles involves a first phase of migration, where muscle progenitors migrate relative to each other, and a second phase, where myotubes migrate searching for their future attachment sites. During this phase, myotubes form extensive filopodia at their ends oriented preferentially toward their attachment sites. This myotube migration and the subsequent muscle attachment establishment are regulated by cell adhesion receptors, such as the conserved proteoglycan Kon-tiki/Perdido. Laminins have been shown to regulate the migratory behavior of many cell populations, but their role in myotube migration remains largely unexplored. Here, we show that laminins, previously implicated in muscle attachment, are indeed required for muscle migration to tendon cells. Furthermore, we find that laminins genetically interact with kon-tiki/perdido to control both myotube migration and attachment. All together, our results uncover a new role for the interaction between laminins and Kon-tiki/Perdido during Drosophila myogenesis. The identification of new players and molecular interactions underlying myotube migration broadens our understanding of muscle development and disease., Peer reviewed

Figure S1. Sequencing results of Orai1βE43Q-EGFP (corresponding to the E106Q mutant of the Orai1 α variant) (a) and GECO-Orai1E106Q mutants (b). Figure S2. STIM1, Orai1 variants/mutants and TRPC1 expression in HeLa cells. a HeLa cells were co-transfected with STIM1-CFP, Orai1α-GFP (or dnOrai1α mutant, as indicated), Orai1β-GFP (or dnOrai1β-GFP mutant, as indicated) and TRPC1. Forty-eight hours later cells were lysed and subjected to 10% SDS-PAGE and Western blotting with anti-STIM1 antibody, anti-Orai1 C-terminal antibody or anti-TRPC1 antibody, as described in Material and Methods. Membranes were reprobed with anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of three separate experiments. b HeLa cells were co-transfected with STIM1 and Orai1α, STIM1 and Orai1β or empty vector (mock). Fura-2-loaded cells were perfused with a Ca2+-free medium (250 μM EGTA added) and then stimulated with TG (1 μM) followed by reintroduction of external Ca2+ (final concentration 1 mM) to initiate Ca2+ entry. c Quantification of Ca2+ entry estimated as described in Material and Methods. Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn´s test). *p < 0.05 as compared to mock-treated cells. Figure S3. Histamine-induced Ca2+ oscillations in mock-treated HeLa cells. a Representative Ca2+ oscillations in response to 3 µM histamine measured using fura-2 in HeLa cells not incubated with plasmids but otherwise treated as cells in Figure 1. Cells were superfused with HBSS containing 1 mM Ca2+ and stimulated with 3 µM histamine at 1 min (indicated by arrow). Representative traces from five cells were chosen to represent the datasets. b-c Quantification of the percentage of oscillating and plateau cells (b) and total oscillations/cell in 10 min (c) for data presented in a (for b, n = 10; n-values correspond to independent experiments; for C n=24; n-values correspond to individual cells). d Quantification of Ca2+ mobilization estimated in mock-treated cells in comparison to cells expressing STIM1, Orai1α, Orai1β and TRPC1 (data from Fig. 1). Scatter plots are represented as mean ± SEM and were statistically analyzed using Mann–Whitney U test to HeLa cells expressing STIM1, Orai1α, Orai1β and TRPC1 (***p < 0.001). Figure S4. Orai1α and Orai1β, but not TRPC1, are required for histamine-induced Ca2+ oscillations. a-h Representative Ca2+ oscillations in response to 3 µM histamine measured using fura-2 in HeLa cells co-transfected with STIM1, Orai1α or Orai1β and TRPC1 or the corresponding dominant negative mutants, as described. Cells were superfused with HBSS containing 1 mM Ca2+ and stimulated with 3 µM histamine at 1 min (indicated by arrow). Representative traces from five cells/condition were chosen to represent the datasets. i-l Quantification of the percentage of oscillating cells (i), percentage of plateau cells (j), percentage of non-responding cells (k) and total oscillations/cell in 10 min (l) for data presented in a-h(for i to k, n = 4-5; n-values correspond to independent experiments; for l, from left to right, n=22, 20, 8, 6, 30, 28, 16 and 11; n-values correspond to individual cells). m-o Quantification of Ca2+ mobilization for all the conditions from a to h estimated in all the cells (m), oscillating cells (n) and plateau cells (o). Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn´s test) to HeLa cells expressing STIM1, Orai1α or Orai1β and TRPC1 (*p < 0.05 and ***p < 0.001), HeLa cells expressing STIM1, Orai1α or Orai1β and dnTRPC1 (for conditions including the expression of dnTRPC1; $p < 0.05 and $$p < 0.01) or the corresponding condition with WT TRPC1 vs dnTRPC1 (#p < 0.05). Figure S5. TRPC1 interacts exclusively with Orai1α. HeLa cells were suspended in HBS containing 1 mM Ca2+ and then stimulated for 1 min with 2 µM TG or the vehicle and lysed. Whole-cell lysates were immunoprecipitated with anti-Orai1 antibody (epitope N-terminal (NT): amino acids 2-61). The immunoprecipitates (pellet) were then subjected to 10% SDS-PAGE and Western blotting with the anti-TRPC1 antibody (a), as described in Material and Methods. Membranes were reprobed with the anti-Orai1 antibody (epitope C-terminal (CT): amino acids 288-301) for protein loading control (c). The supernatant of the immunoprecipitation with anti-Orai1 NT-antibody was further immunoprecipitated with the anti-Orai1 CT-antibody. The pellet was subjected to 10% SDS-PAGE and Western blotting with the anti-TRPC1 antibody (b) and membranes were reprobed with the anti-Orai1 CT-antibody for protein loading control (d). Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. Blots are representative of five separate experiments. e Quantification of TRPC1-Orai1 association under the different experimental conditions normalized to the Orai1 expression. Scatter plots are represented as mean ± SEM and were statistically analyzed using Mann–Whitney U test. ***p < 0.001 as compared to Control. Figure S6. TRPC1 does not alter either the plasma membrane location or serine phosphorylation of Orai1α. a-b HeLa cells were co-transfected with STIM1-CFP, Orai1α-GFP and TRPC1 (or dnTRPC1 mutant, as indicated). Forty-eight hours later cells were suspended in HBS containing 1 mM Ca2+ and then stimulated with 3 µM histamine. Samples were taken 1s before and 10 s, 1 min and 10 min after the addition of histamine and lysed. Whole-cell lysates were immunoprecipitated with anti-Orai1 C-terminal antibody. The immunoprecipitates were then subjected to 8% SDS-PAGE and Western blotting with specific anti-phosphoserine antibody (a, top panel), as described in Material and Methods. Membranes were reprobed with the anti-Orai1 C-terminal antibody for protein loading control (a, bottom panel). Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. b Quantification of Orai1α serine phosphorylation under the different experimental conditions normalized to the Orai1α expression. Scatter plots are represented as mean ± SEM, expressed as fold change (experimental/control) and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn´s test). *p < 0.05 as compared to Control. c-d HeLa cells were co-transfected with STIM1-CFP, Orai1α-GFP and either TRPC1, dnTRPC1 mutant or shTRPC1, as indicated. Forty-eight hours later cells were suspended in HBS containing 1 mM Ca2+, stimulated for 1 min with 3 µM histamine or left untreated and mixed with biotinylation buffer containing EZ-Link sulfo-NHS-LC-biotin. Cell surface proteins were labeled by biotinylation as described in Material and Methods. Labeled proteins were pulled down with streptavidin-coated agarose beads. The pellet (containing the plasma membrane fraction) was analyzed by SDS-PAGE and Western blotting using anti-Orai1α (C terminal) or anti-PMCA antibody, as indicated. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. These results are representative of 3 separate experiments. d Quantification of Orai1α plasma membrane expression under the different experimental conditions normalized to the PMCA expression. Scatter plots are represented as mean ± SEM and expressed as fold change (experimental/control (resting cells co-transfected with STIM1-CFP, Orai1α-GFP and TRPC1)). Data were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn´s test). Figure S7. Mn2+ influx in HeLa cells expressing STIM1, Orai1α, Orai1β and TRPC1. a Representative responses to 2 µM TG in HeLa cells co-transfected with STIM1, Orai1α, Orai1β and TRPC1 or mock transfected, as described. Cells were superfused with HBSS containing 0.5 mM Mn2+ and 1 mM Ca2+ and stimulated with 2 µM TG (indicated by arrow). Fura-2 fluorescence was measured at an excitation wavelength of 360 nm, the isoemissive wavelength. Representative traces were chosen to represent the datasets. b Quantification of the rate of decay of fura-2 fluorescence under the different experimental conditions (from left to right, n=28; n-values correspond to individual cells). Scatter plots are represented as mean ± SEM and were statistically analyzed using the Mann–Whitney U test. ***p < 0.001 as compared to mock transfected HeLa cells. Figure S8. Determination of Mn2+ influx in HeLa cells expressing STIM1 and Orai1α or STIM1 and Orai1β. Representative responses to 2 µM TG in HeLa cells co-transfected with STIM1 and Orai1α (a) or STIM1 and Orai1β (b), as described. Cells were superfused with HBSS containing 0.5 mM Mn2+ and 1 mM Ca2+ and stimulated with 2 µM TG (indicated by arrow). Fura-2 fluorescence was measured at an excitation wavelength of 360 nm, the isoemissive wavelength. Traces are representative of 3 independent experiments (n=28-34; n-values correspond to individual cells). Figure S9. Orai1α and Oraiβ modulate Mn2+ influx through TRPC1 in HEK293 cells. a-e Representative responses to TG in HEK293 cells co-transfected with empty vectors (mock cells; a), or expression plasmids for STIM1, TRPC1 and either EYFP-Orai1 (b), the dominant negative Orai1 mutant (c), Orai1α-EGFP (d) or Orai1β-EGFP (e), as described. Cells were superfused with HBSS containing 0.5 mM Mn2+ and 1 mM Ca2+ and stimulated with 2 µM TG (indicated by arrow). Fura-2 fluorescence was measured at an excitation wavelength of 360 nm, the isoemissive wavelength. Representative traces were chosen to represent the datasets. f Quantification of the rate of decay of fura-2 fluorescence under the different experimental conditions (from left to right, n=65, 53, 53, 78 and 70; n-values correspond to individual cells). Scatter plots are represented as mean ± SEM and were statistically analyzed using Kruskal–Wallis test with multiple comparisons (Dunn´s test). ***p < 0.001 as compared to mock cells. $$$p < 0.001 as compared to cells expressing STIM1, Orai1 and TRPC1., Peer reviewed

Supplementary Figure 1. Related to Figure 1. Characterization of the cholinergic system in bone. Supplementary Figure 2. Related to Figure 2. Interleukin-6 induces a cholinergic switch in sympathetic neurons. Supplementary Figure 3. Related to Figures 2 and 3. Interleukin-6 induces a cholinergic switch of sympathetic fibers in bone. Supplementary Figure 4. Related to Figure 4. Osteolineage cells contribute to the non-neuronal cholinergic system. Supplementary Figure 5. Related to Figure 5. GFRa2 loss causes reduced bone thickness and osteocyte degeneration. Supplementary Figure 6. Related to Figure 6. GFRa2 signaling maintains osteocyte connectivity and survival. Supplementary Figure 7. Related to Figure 7. Moderate exercise increases bone cholinergic innervation through sympathetic cholinergic fibers. Table S1. Oligonucleotide sequences used for mouse genotyping. Table S2. Oligonucleotide sequences used for quantitative real-time RT-PCR., Peer reviewed

Figure S1. Representative standard curve of the Melatonin (A) and 6-SMT (B) assays. Figure S2. Representative standard curve of the antioxidative assays used in this study (TAC, FRAP, ORAC)., Peer reviewed

Supplementary material to DESI's publication "The DESI N-body Simulation Project II: Suppressing Sample Variance with Fast Simulations" to comply with the data management plan., We collect all the data and code needed to produce the figures in the paper. The data set mainly consists of halo two-point correlation function (CF), power spectra (Pk) and bispectra (Bk) from the AbacusSummit and FastPM simulations. For Pk, we include results from the base cosmology (c000), the secondary cosmology c002 and c004, respectively. For Pk and CF, we separate results with different halo mass cut 10^11 and 10^13 Msun/h. We provide python jupyter notebooks for the plotting routines. The subfolder "output" in each directory contains all the necessary data for the plots., ZD and YY were supported by the National Key Basic Research and Development Program of China (No. 2018YFA0404504) and the National Science Foundation of China (grant Nos. 11621303, 1189069, 111773048)., Peer reviewed

1-Supplemental Materials and Methods. 2- Supplemental e-FIGURES. Figure I: Workflow for CpG-sites and sample QCs. Figure II: Batch effect evaluation with MDS and SVD plots. Figure III: Manhattan plots for DMCT. Figure IV: cg00039070 methylation correlation between blood and brain. Figure V: Blood–Brain Epigenetic Concordance (BECon) results for cg00039070. 3- Supplemental e-TABLES. Table I: Analysis of variables associated with ∆NIHSS in bivariate and regression analysis. Table II: Analysis of variables associated with mRS at 3 months in bivariate and regression analysis. Table III: Summary statistics for the discovery EWAS adjusted by batch. Table IV: Feature enrichment analysis. Table V: Differentially methylated region (DMR) results. Table VI: Differentially methylated block (DMB) results. Table VII: EWAS summary statistics in the meta-analyses for dichotomic ∆NIHSS. Table VIII: Demographic and clinical data for the subjects included in the analysis with SOMAscan. Table IX: eFORGE analysis. 4- Supplemental References., Boehringer Ingelheim España Instituto de Salud Carlos III Agència de Gestió d'Ajuts Universitaris i de Recerca FUNDACIÓ DOCÈNCIA I RECERCA MÚTUATERRASSA Bristol-Myers Squibb Eranet-Neuron Fundació la Marató de TV3., Peer reviewed

Antibiotic usage in this study (GTI1503) was within the range reported for other subcutaneous immune globulin products approved for primary immunodeficiency syndromes in Europe and North America, as shown in the table below. Days of Antibiotic Use Per Subject-Year for Subcutaneous Immune Globulins., Peer reviewed

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape., Peer reviewed