Adipose-Derived Stem Cells in Crohn's Rectovaginal Fistula

Therapeutic options for recto-vaginal fistula in the setting of Crohn's disease are limited and many data are available in the literature. The manuscript describes the history of a patient who has been the pioneer of our Clinical Trials in treating this disease in fistulizing Crohn's disease environment. We believe it is the first time that a patient with this disease has been treated by adipose-derived stem cells in allogeneic form. The conclusion of our study with Mary is that the use of mesenchymal stem cells derived from adipose tissue is secure, either in autologous or allogeneic form. Furthermore, we have proved that if we use multi-dose and multiple applications on a patient, it does not produce any adverse effect, which confirms us the safety of using these cells in patients at least in the fistulizing Crohn's disease environment

Protumorigenic effects of Snail-expression fibroblasts on colon cancer cells

et al.Snail1 is a transcriptional factor that plays an important role in epithelial–mesenchymal transition and in the acquisition of invasive properties by epithelial cells. In colon tumors, Snail1 expression in the stroma correlates with lower specific survival of cancer patients. However, the role(s) of Snail1 expression in stroma and its association with patients' survival have not been determined. We used human primary carcinoma-associated fibroblasts (CAFs) or normal fibroblasts (NFs) and fibroblast cell lines to analyze the effects of Snail1 expression on the protumorigenic capabilities in colon cancer cells. Snail1 expression was higher in CAFs than in NFs and, as well as α-SMA, a classic marker of activated CAFs. Moreover, in tumor samples from 50 colon cancer patients, SNAI1 expression was associated with expression of other CAF markers, such as α-SMA and fibroblast activation protein. Interestingly, coculture of CAFs with colon cells induced a significant increase in epithelial cell migration and proliferation, which was associated with endogenous SNAI1 expression levels. Ectopic manipulation of Snail1 in fibroblasts demonstrated that Snail1 expression controlled migration as well as proliferation of cocultured colon cancer cells in a paracrine manner. Furthermore, expression of Snail1 in fibroblasts was required for the coadjuvant effect of these cells on colon cancer cell growth and invasion when coxenografted in nude mice. Finally, cytokine profile changes, particularly MCP-3 expression, in fibroblasts are put forward as mediators of Snail1-derived effects on colon tumor cell migration. In summary, these studies demonstrate that Snail1 is necessary for the protumorigenic effects of fibroblasts on colon cancer cells.This research was supported by the PI12/02037, Fundación Científica AECC, SAF2010-20750, S2010/BMD-2344, RTICC-RD12/0036/0041 and by the Fundación Banco Santander. Antonio García de Herreros’ laboratory was supported by RTICC-RD12/0036/0005 and SAF 2010-16089. Ma Jesús Larriba’s laboratory was supported by RD12/0036/0021. Cristina Peña and José Miguel García are recipients of Miguel Servet Contracts from the Instituto de Salud Carlos III.Peer reviewe

Transcriptional Profile Associated with Clinical Outcomes in Metastatic Hormone-Sensitive Prostate Cancer Treated with Androgen Deprivation and Docetaxel

(1) Background: Androgen deprivation therapy (ADT) and docetaxel (DX) combination is a standard therapy for metastatic hormone-sensitive prostate cancer (mHSPC) patients. (2) Methods: We investigate if tumor transcriptomic analysis predicts mHSPC evolution in a multicenter retrospective biomarker study. A customized panel of 184 genes was tested in mRNA from tumor samples by the nCounter platform in 125 mHSPC patients treated with ADT+DX. Gene expression was correlated with castration-resistant prostate cancer-free survival (CRPC-FS) and overall survival (OS). (3) Results: High expression of androgen receptor (AR) signature was independently associated with longer CRPC-FS (hazard ratio (HR) 0.6, 95% confidence interval (CI) 0.3-0.9; p = 0.015), high expression of estrogen receptor (ESR) signature with longer CRPC-FS (HR 0.6, 95% CI 0.4-0.9; p = 0.019) and OS (HR 0.5, 95% CI 0.2-0.9, p = 0.024), and lower expression of tumor suppressor genes (TSG) (RB1, PTEN and TP53) with shorter OS (HR 2, 95% CI 1-3.8; p = 0.044). ARV7 expression was independently associated with shorter CRPC-FS (HR 1.5, 95% CI 1.1-2.1, p = 0.008) and OS (HR 1.8, 95% CI 1.2-2.6, p = 0.004), high ESR2 was associated with longer OS (HR 0.5, 95% CI 0.2-1, p = 0.048) and low expression of RB1 was independently associated with shorter OS (HR 1.9, 95% CI 1.1-3.2, p = 0.014). (4) Conclusions: AR, ESR, and TSG expression signatures, as well as ARV7, RB1, and ESR2 expression, have a prognostic value in mHSPC patients treated with ADT+DX

A novel gene signature unveils three distinct immune- metabolic rewiring patterns conserved across diverse tumor types and associated with outcomes

Existing immune signatures and tumor mutational burden have only modest predictive capacity for the efficacy of immune check point inhibitors. In this study, we developed an immune-metabolic signature suitable for personalized ICI therapies. A classifier using an immune-metabolic signature (IMMETCOLS) was developed on a training set of 77 metastatic colorectal cancer (mCRC) samples and validated on 4,200 tumors from the TCGA database belonging to 11 types. Here, we reveal that the IMMETCOLS signature classifies tumors into three distinct immune-metabolic clusters. Cluster 1 displays markers of enhanced glycolisis, hexosamine byosinthesis and epithelial-to-mesenchymal transition. On multivariate analysis, cluster 1 tumors were enriched in pro-immune signature but not in immunophenoscore and were associated with the poorest median survival. Its predicted tumor metabolic features suggest an acidic-lactate-rich tumor microenvironment (TME) geared to an immunosuppressive setting, enriched in fibroblasts. Cluster 2 displays features of gluconeogenesis ability, which is needed for glucose-independent survival and preferential use of alternative carbon sources, including glutamine and lipid uptake/β-oxidation. Its metabolic features suggest a hypoxic and hypoglycemic TME, associated with poor tumor-associated antigen presentation. Finally, cluster 3 is highly glycolytic but also has a solid mitochondrial function, with concomitant upregulation of glutamine and essential amino acid transporters and the pentose phosphate pathway leading to glucose exhaustion in the TME and immunosuppression. Together, these findings suggest that the IMMETCOLS signature provides a classifier of tumors from diverse origins, yielding three clusters with distinct immune-metabolic profiles, representing a new predictive tool for patient selection for specific immune-metabolic therapeutic approaches

Cryo-EM and single-particle analysis with Scipion

Cryo-electron microscopy has become one of the most important tools in biological research to reveal the structural information of macromolecules at near-atomic resolution. In single-particle analysis, the vitrified sample is imaged by an electron beam and the detectors at the end of the microscope column produce movies of that sample. These movies contain thousands of images of identical particles in random orientations. The data need to go through an image processing workflow with multiple steps to obtain the final 3D reconstructed volume. The goal of the image processing workflow is to identify the acquisition parameters to be able to reconstruct the specimen under study. Scipion provides all the tools to create this workflow using several image processing packages in an integrative framework, also allowing the traceability of the results. In this article the whole image processing workflow in Scipion is presented and discussed with data coming from a real test case, giving all the details necessary to go from the movies obtained by the microscope to a high resolution final 3D reconstruction. Also, the power of using consensus tools that allow combining methods, and confirming results along every step of the workflow, improving the accuracy of the obtained results, is discussed

Preclinical characterization of antagomiR-218 as a potential treatment for myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by expansion of unstable CTG repeats in a non-coding region of the DMPK gene. CUG expansions in mutant DMPK transcripts sequester MBNL1 proteins in ribonuclear foci. Depletion of this protein is a primary contributor to disease symptoms such as muscle weakness and atrophy and myotonia, yet upregulation of endogenous MBNL1 levels may compensate for this sequestration. Having previously demonstrated that antisense oligonucleotides against miR-218 boost MBNL1 expression and rescue phenotypes in disease models, here we provide preclinical characterization of an antagomiR-218 molecule using the HSALR mouse model and patient-derived myotubes. In HSALR, antagomiR-218 reached 40-60 pM 2weeks after injection, rescued molecular and functional phenotypes in a dose- and time-dependent manner, and showed a good toxicity profile after a single subcutaneous administration. In muscle tissue, antagomiR rescued the normal subcellular distribution of Mbnl1 and did not alter the proportion of myonuclei containing CUG foci. In patient-derived cells, antagomiR-218 improved defective fusion and differentiation and rescued up to 34% of the gene expression alterations found in the transcriptome of patient cells. Importantly, miR-218 was found to be upregulated in DM1 muscle biopsies, pinpointing this microRNA (miRNA) as a relevant therapeutic target.This work was funded by research grants from Instituto de Salud Carlos III, including funds from FEDER, to M.P.-A. and B.L. (PI17/00352) and HR17-00268 (TATAMI project) from the “la Caixa” Banking Foundation to R.A. I.G.-M. was funded by the Precipita Project titled “Desarrollo de una terapia innovadora contra la distrofia miotónica,” E.C.-H. and J.M.F.-C. were supported by the post-doctoral fellowships APOSTD/2019/142 and APOSTD/2017/088 from the Fondo Social Europeo for science and investigation, while J.E.-E. was the recipient of a Santiago Grisolia fellowship (Grisolip2018/098) from the Generalidad Valenciana. Part of the equipment employed in this work has been funded by Generalitat Valenciana and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana 2014-2020). Antibody MB1a (4A8) was provided by MDA Monoclonal Antibody Resource

Algorithmic robustness to preferred orientations in single particle analysis by CryoEM

The presence of preferred orientations in single particle analysis (SPA) by cryo-Electron Microscopy (cryoEM) is currently one of the hurdles preventing many structural analyses from yielding high-resolution structures. Although the existence of preferred orientations is mostly related to the grid preparation, in this technical note, we show that some image processing algorithms used for angular assignment and three-dimensional (3D) reconstruction are more robust than others to these detrimental conditions. We exemplify this argument with three different data sets in which the presence of preferred orientations hindered achieving a 3D reconstruction without artifacts or, even worse, a 3D reconstruction could never be achievedWe acknowledge support from “la Caixa” Foundation (Fellowship LCF/BQ/DI18/11660021. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 713673. We also thank the financial support from the Spanish Ministry of Economy and Competitiveness through Grants BIO2016-76400-R(AEI/FEDER, UE) and SEV 2017-0712, the “Comunidad Autónoma de Madrid” through Grant: S2017/BMD-3817, Instituto de Salud Carlos III, PT17/ 0009/0010 (ISCIII-SGEFI/ERDF), European Union (EU) and Horizon 2020 through grants: CORBEL (INFRADEV-1-2014-1, Proposal: 654248), INSTRUCT-ULTRA (INFRADEV-03-2016-2017, Proposal: 731005), EOSC Life (INFRAEOSC-04-2018, Proposal: 824087), High- ResCells (ERC-2018-SyG, Proposal: 810057), IMpaCT (WIDESPREAD-03-2018 – Proposal: 857203), EOSC-Synergy (EINFRA-EOSC-5, Proposal: 857647), and iNEXT-Discovery (Proposal: 871037). The authors acknowledge the support and the use of resources of Instruct, a Landmark ESFRI projec

Table_4_A novel gene signature unveils three distinct immune-metabolic rewiring patterns conserved across diverse tumor types and associated with outcomes.docx [Dataset]

Supplementary Table S4: Important features identified by One-way ANOVA and post-hoc analysis (Fisher’s LSD) comparing the expression of immune signatures in the IMMETCOLS Clusters.Existing immune signatures and tumor mutational burden have only modest predictive capacity for the efficacy of immune check point inhibitors. In this study, we developed an immune-metabolic signature suitable for personalized ICI therapies. A classifier using an immune-metabolic signature (IMMETCOLS) was developed on a training set of 77 metastatic colorectal cancer (mCRC) samples and validated on 4,200 tumors from the TCGA database belonging to 11 types. Here, we reveal that the IMMETCOLS signature classifies tumors into three distinct immune-metabolic clusters. Cluster 1 displays markers of enhanced glycolisis, hexosamine byosinthesis and epithelial-to-mesenchymal transition. On multivariate analysis, cluster 1 tumors were enriched in pro-immune signature but not in immunophenoscore and were associated with the poorest median survival. Its predicted tumor metabolic features suggest an acidic-lactate-rich tumor microenvironment (TME) geared to an immunosuppressive setting, enriched in fibroblasts. Cluster 2 displays features of gluconeogenesis ability, which is needed for glucose-independent survival and preferential use of alternative carbon sources, including glutamine and lipid uptake/β-oxidation. Its metabolic features suggest a hypoxic and hypoglycemic TME, associated with poor tumor-associated antigen presentation. Finally, cluster 3 is highly glycolytic but also has a solid mitochondrial function, with concomitant upregulation of glutamine and essential amino acid transporters and the pentose phosphate pathway leading to glucose exhaustion in the TME and immunosuppression. Together, these findings suggest that the IMMETCOLS signature provides a classifier of tumors from diverse origins, yielding three clusters with distinct immune-metabolic profiles, representing a new predictive tool for patient selection for specific immune-metabolic therapeutic approaches.Peer reviewe

Table_5_A novel gene signature unveils three distinct immune-metabolic rewiring patterns conserved across diverse tumor types and associated with outcomes.docx [Dataset]

Supplementary Table S5: Important features identified by One-way ANOVA and post-hoc analysis (Fisher’s LSD) comparing the expression of immune genes in the IMMETCOLS Clusters.Existing immune signatures and tumor mutational burden have only modest predictive capacity for the efficacy of immune check point inhibitors. In this study, we developed an immune-metabolic signature suitable for personalized ICI therapies. A classifier using an immune-metabolic signature (IMMETCOLS) was developed on a training set of 77 metastatic colorectal cancer (mCRC) samples and validated on 4,200 tumors from the TCGA database belonging to 11 types. Here, we reveal that the IMMETCOLS signature classifies tumors into three distinct immune-metabolic clusters. Cluster 1 displays markers of enhanced glycolisis, hexosamine byosinthesis and epithelial-to-mesenchymal transition. On multivariate analysis, cluster 1 tumors were enriched in pro-immune signature but not in immunophenoscore and were associated with the poorest median survival. Its predicted tumor metabolic features suggest an acidic-lactate-rich tumor microenvironment (TME) geared to an immunosuppressive setting, enriched in fibroblasts. Cluster 2 displays features of gluconeogenesis ability, which is needed for glucose-independent survival and preferential use of alternative carbon sources, including glutamine and lipid uptake/β-oxidation. Its metabolic features suggest a hypoxic and hypoglycemic TME, associated with poor tumor-associated antigen presentation. Finally, cluster 3 is highly glycolytic but also has a solid mitochondrial function, with concomitant upregulation of glutamine and essential amino acid transporters and the pentose phosphate pathway leading to glucose exhaustion in the TME and immunosuppression. Together, these findings suggest that the IMMETCOLS signature provides a classifier of tumors from diverse origins, yielding three clusters with distinct immune-metabolic profiles, representing a new predictive tool for patient selection for specific immune-metabolic therapeutic approaches.Peer reviewe

Gamma-Secretase-Dependent and -Independent Effects of Presenilin1 on β-Catenin·Tcf-4 Transcriptional Activity

Presenilin1 (PS1) is a component of the γ-secretase complex mutated in cases of Familial Alzheimer's disease (FAD). PS1 is synthesized as a 50 kDa peptide subsequently processed to two 29 and 20 kDa subunits that remain associated. Processing of PS1 is inhibited by several mutations detected in FAD patients. PS1 acts as negative modulator of β-catenin·Tcf-4 transcriptional activity. In this article we show that in murine embryonic fibroblasts (MEFs) the mechanisms of action of the processed and non-processed forms of PS1 on β-catenin·Tcf-4 transcription are different. Whereas non-processed PS1 inhibits β-catenin·Tcf-4 activity through a mechanism independent of γ-secretase and associated with the interaction of this protein with plakoglobin and Tcf-4, the effect of processed PS1 is prevented by γ-secretase inhibitors, and requires its interaction with E- or N-cadherin and the generation of cytosolic terminal fragments of these two cadherins, which in turn destabilize the β-catenin transcriptional cofactor CBP. Accordingly, the two forms of PS1 interact differently with E-cadherin or β-catenin and plakoglobin: whereas processed PS1 binds E-cadherin with high affinity and β-catenin or plakoglobin weakly, the non-processed form behaves inversely. Moreover, contrarily to processed PS1, that decreases the levels of c-fos RNA, non-processed PS1 inhibits the expression c-myc, a known target of β-catenin·Tcf-4, and does not block the activity of other transcriptional factors requiring CBP. These results indicate that prevention of PS1 processing in FAD affects the mechanism of repression of the transcriptional activity dependent on β-catenin