Resultados de investigación

Quantitative variables are expressing as number (percentage) or median (interquartile range). Pa value for differences between patients who were or not discharged. Pb value for differences between patients who who did and did not get wore. SpO2, peripheral capillary oxygen saturation; CRP, C-reactive protein; LDH, Lactate dehydrogenase; NLR, neutrophil/lymphocyte ratio., Peer reviewed

AUC, area under the curve; SE, sensitivity; S, specificity; PPV, positive predictive value; NPV, negative predictive value. SpO2, peripheral capillary oxygen saturation; CRP, C-reactive protein; LDH, Lactate dehydrogenase; NLR, neutrophil/lymphocyte ratio; TNF-α; tumor necrosis factor α; IL-6, interleukine-6; IL-8, interleukine-8; IL-1β, interleukine-1β; MIP-1β, macrophage inflammatory proteins 1β; sCD25, soluble receptor interleukine-2; IP-10, interferon γ-induced protein 10., Peer reviewed

AUC, area under the curve; SE, sensitivity; S, specificity; PPV, positive predictive value; NPV, negative predictive value. SpO2, peripheral capillary oxygen saturation; CRP, C-reactive protein; LDH, Lactate dehydrogenase; NLR, neutrophil/lymphocyte ratio; TNF-α; tumor necrosis factor α; IL-6, interleukine-6; IL-8, interleukine-8; IL-1β, interleukine-1β; MIP-1β, macrophage inflammatory proteins 1β; sCD25, soluble receptor interleukine-2; IP-10, interferon γ-induced protein 10., Peer reviewed

Data are expressed by percentage and interquartile range. Medians fluorescence intensitive (MFI) were calculated in those markets that have a high rate of expression., Peer reviewed

Supplementary Table 1: Antibodies for Flow Cytometry. Supplementary Figure 1. CD25 and Foxp3 expression, absolute Treg quantification and Tcon:Treg ratio. a. Representative cytometries showing CD25 and Foxp3 staining of CD4+ (blue) and CD8+ (red) cells after anti-CD3/CD28 activation and ruxolitinib treatment. b. Absolute quantification of CD4+CD25+Foxp3high cells. Average and S.E.M. of two independent experiments with three technical replicates each are shown. Cells are normalized to counting beads in the culture (1 bead/50 PBMNCs in the initial culture) c. Quantification of CD4+ or CD8+ to CD4+CD25+Foxp3high cells ratio . Average and S.E.M. of two independent experiments with three technical replicates each are shown. Supplementary Figure 2. Correlation of PD1 and Helios, cytometries of CTLA4 and CD39. a. Freshly isolated huPBMNCs were analyzed for Helios and PD1 expression in CD4+ CD25+ Foxp3high Tregs. One representative cytometry is showing the left, and the quantification of 5 independent experiments is shown in the right panel (mean +/-S.E.M.). b. Dot plot of the percentage of PD1+ cells versus Helios+ cells of CD4+ Foxp3+ gated cells after 2, 5 and 8 days of antiCD3 and antiCD8 stimulation and Ruxolitinib treatments. The linear regression of each day is shown, with the equation, Pearson correlation (R2) and p value. c. Representative cytometry of stimulated huPBMNCs, gated for CD4+, showing CD39 and CTLA4 staining after 2, 5 and 8 days of treatment with 0 or 0.3μM Ruxolitinib. Supplementary Figure 3. Suppression assay after pretreatment of Treg with Ruxolitinib. MACS sorted Treg were preincubated in the presence of ruxolitinib 0.1 and 0.3 μM for 24h and then ruxolitinib was washed. Later, CD4+ cells labeled with cell division tracking dye were activated with anti CD3 and anti CD28 antibodies and the pretreated Treg were added at different proportions. a. CD4+ proliferation was determined by flow cytometry. b. The percentage of suppression was calculated as {1-(%proliferation in the sample/%proliferation in the control)}×100%. Supplementary Figure 4. Cytometry analysis of samples from the GvHD mouse model. Mice were sacrificed 10 weeks after treatment start (14 weeks after BM transplantation) and the cells of different organs were analyzed by cytometry. a. Representative cytometries of peripheral blood from BM only transplanted mice (no GvHD), or spleen and BM transplanted cells treated with vehicle, Tregs, Ruxolitinib or both. CD45 gated cells are shown in the upper row for CD3 and CD19 staining, CD3 gated cells are further gated for CD8 and CD4 staining (middle row) and CD4 gated cells are analyzed for CD25 and GFP expression (lower row). b. Cells from bone marrow are analyzed as in A. c. Cells from BM are stained for Foxp3 expression. Cells are gated for CD4 expression (upper row), and the represented for Foxp3 versus CD25 (middle row) or Foxp3 versus GFP. GFP signal is reduced compared to figure B due to the fixation and permeabilization used for Foxp3 intracellular staining. d. Bone marrow biopsies of mice infused with GFP Tregs at the indicated times post-infusion. CD4 gated cells are analyzed for CD25 and GFP expression. Supplementary figure 5. Quantification of cell populations in the GvHD mouse model. a. Peripheral blood samples were extracted from mice at 2, 4 and 6 weeks after treatment start (6, 8 and 10 weeks post transplantation). Samples of at least 4 mice per group were analyzed following the gating strategy shown in supplemental figure 4A. Mean and S.E.M. is represented. b. Mice were sacrificed 10 weeks after treatment start (14 weeks after BM transplantation). Cell populations from peripheral blood, bone marrow (BM), large intestine (LI), Peyer ́s patches (PP), small intestine (SI), spleen and thymus were analyzed as in A. c. CD25+ Foxp3high Tregs from peripheral blood, bone marrow (BM), spleen and thymus were analyzed following the gating strategy shown in supplementary figure 4C. Mean and S.E.M. is represented. d. Blood samples from mice at 2, 4 and 6 weeks after treatment start were analyzed using an haematocytometer. LYM= lymphocytes (103 /μl), MON=monocytes (103 /μl), GRA=granulocytes (103 /μl), WBC= White blood cells (103 /μl), percent_LYM= percentage of lymphocytes, percent_MON= percentage of monocytes, percent_GRA= percentage of granulocytes. p values of a Student ́s t-test are shown. Significant values are marked with red arrows. Supplementary figure 6. Histopathological analysis of small and large intestine and skin of the GvHD mouse model. Large intestine, small intestine and skin from mice sacrificed after 10 weeks of treatment were fixed in formalin, and included in paraffin. Slices were stained with hematoxylin-eosin and Masson ́s trichrome, and were evaluated by a pathologist for GvHD signs. A histopathological score was assigned according to published scoring system56. Average plus standard error of the mean of four mice per group are shown. LI, Large Intestine. SI, Small Intestine., Peer reviewed

1 table. -- Supplementary Table 1. Primary and secondary antibodies used in this study., There exists considerable interest to unveil preclinical period and prodromal stages of Alzheimer's disease (AD). The mild cognitive impairment (MCI) is characterized by significant memory and/or other cognitive domains impairments, and is often considered the prodromal phase of AD. The cerebrospinal fluid (CSF) levels of β-amyloid (βA), total tau (t-tau), and phosphorylated tau (p-tau) have been used as biomarkers of AD albeit their significance as indicators during early stages of AD remains far from accurate. The new biomarkers are being intensively sought as to allow identification of pathological processes underlying early stages of AD. Fifty-three participants (75.4 ± 8.3 years) were classified in three groups as cognitively normal healthy controls (HC), MCI, and subjective memory complaints (SMC). The subjects were subjected to a battery of neurocognitive tests and underwent lumbar puncture for CSF extraction. The CSF levels of estrogen-receptor (ER)-signalosome proteins, βA, t-tau and p-tau, were submitted to univariate, bivariate, and multivariate statistical analyses. We have found that the components of the ER-signalosome, namely, caveolin-1, flotilin-1, and estrogen receptor alpha (ERα), insulin growth factor-1 receptor β (IGF1Rβ), prion protein (PrP), and plasmalemmal voltage dependent anion channel 1 (VDAC) could be detected in the CSF from all subjects of the HC, MCI, and SMC groups. The six proteins appeared elevated in MCI and slightly increased in SMC subjects compared to HC, suggesting that signalosome proteins undergo very early modifications in nerve cells. Using a multivariate approach, we have found that the combination of ERα, IGF-1Rβ, and VDAC are the main determinants of group segregation with resolution enough to predict the MCI stage. The analyses of bivariate relationships indicated that collinearity of ER-signalosome proteins vary depending on the stage, with some pairs displaying opposed relationships between HC and MCI groups, and the SMC stage showing either no relationships or behaviors similar to either HC or MCI stages. The multinomial logistic regression models of changes in ER-signalosome proteins provide reliable predictive criteria, particularly for the MCI. Notably, most of the statistical analyses revealed no significant relationships or interactions with classical AD biomarkers at either disease stage. Finally, the multivariate functions were highly correlated with outcomes from neurocognitive tests for episodic memory. These results demonstrate that alterations in ER-signalosome might provide useful diagnostic information on preclinical stages of AD, independently from classical biomarkers., Peer reviewed

It contains technical information provided for transparency and reproducibility of the results presented in the manuscript., Peer reviewed

13 pages. -- Table S1. Division of data for 289 pesticides against A. bahia for mortality at 96 h (mean lethal concentration, (LC50). -- Fig. S1. Linear Backward Selection Regression results for variable selection. -- Fig. S2. Correlation heat maps of selected descriptors. -- Fig. S3. Density and frequency of pLC50 (R package). -- Fig. S4. Density line (A), histogram (B) and scatter plot (C) of the molecular descriptors. (R package). -- Fig. S5. Observed values versus predicted values for the training set. -- Fig. S6. Observed values vs. predicted values for prediction set. -- Table S2. Symbols, definitions and sign value of molecular descriptors in the QSTR-MLR model. -- Table S3. RPTree. Training Sets., Peer reviewed

Fig. S1. Cln311A accumulates in the nucleus during G1 and reaches a maximum around Start Fig. S2. Cln3 boosts nuclear import of Cdc28-GFP during cell cycle entry Fig. S3. Mad3 regulates Cln3 levels in G1 but does not modulate Whi5 levels at Start Fig. S4. Mad3 protein levels oscillate during the cell cycle as a function of APC activity Fig. S5. Mad3 tilts the sizer behavior of G1 control Table S1. Yeast strains. Data S1., Peer reviewed

Archivo Excel, This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 861088., Peer reviewed