Temporal Requirements of cMyc Protein for Reprogramming Mouse Fibroblasts

Exogenous expression of Oct4, Sox2, Klf4, and cMyc forces mammalian somatic cells to adopt molecular and phenotypic characteristics of embryonic stem cells, commencing with the required suppression of lineage-associated genes (e.g., Thy1 in mouse). Although omitting cMyc from the reprogramming cocktail minimizes risks of uncontrolled proliferation, its exclusion results in fold reductions in reprogramming efficiency. Thus, the feasibility of substituting cMyc transgene with (non-integrative) recombinant “pTAT-mcMyc” protein delivery was assessed, without compromising reprogramming efficiency or the pluripotent phenotype. Purification and delivery of semisoluble/particulate pTAT-mcMyc maintained Oct4-GFP+ colony formation (i.e., reprogramming efficiency) whilst supporting pluripotency by various criteria. Differential repression of Thy1 by pTAT-mcMyc ± Oct4, Sox2, and Klf4 (OSK) suggested differential (and non-additive) mechanisms of repression. Extending these findings, attempts to enhance reprogramming efficiency through a staggered approach (prerepression of Thy1) failed to improve reprogramming efficiency. We consider protein delivery a useful tool to decipher temporal/molecular events characterizing somatic cell reprogramming

CRISPR screens identify gene targets at breast cancer risk loci

Background: Genome-wide association studies (GWAS) have identified > 200 loci associated with breast cancer risk. The majority of candidate causal variants are in noncoding regions and likely modulate cancer risk by regulating gene expression. However, pinpointing the exact target of the association, and identifying the phenotype it mediates, is a major challenge in the interpretation and translation of GWAS. Results: Here, we show that pooled CRISPR screens are highly effective at identifying GWAS target genes and defining the cancer phenotypes they mediate. Following CRISPR mediated gene activation or suppression, we measure proliferation in 2D, 3D, and in immune-deficient mice, as well as the effect on DNA repair. We perform 60 CRISPR screens and identify 20 genes predicted with high confidence to be GWAS targets that promote cancer by driving proliferation or modulating the DNA damage response in breast cells. We validate the regulation of a subset of these genes by breast cancer risk variants. Conclusions: We demonstrate that phenotypic CRISPR screens can accurately pinpoint the gene target of a risk locus. In addition to defining gene targets of risk loci associated with increased breast cancer risk, we provide a platform for identifying gene targets and phenotypes mediated by risk variants.Natasha K. Tuano, Jonathan Beesley, Murray Manning, Wei Shi, Laura Perlaza, Jimenez, Luis F. Malaver, Ortega, Jacob M. Paynter, Debra Black, Andrew Civitarese, Karen McCue, Aaron Hatzipantelis, Kristine Hillman, Susanne Kaufmann, Haran Sivakumaran, Jose M. Polo, Roger R. Reddel, Vimla Band, Juliet D. French, Stacey L. Edwards, David R. Powell, Georgia Chenevix, Trench, and Joseph Rosenblu

Ruxolitinib in refractory acute and chronic graft-versus-host disease : a multicenter survey study

Graft-versus-host disease is the main cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. First-line treatment is based on the use of high doses of corticosteroids. Unfortunately, second-line treatment for both acute and chronic graft-versus-host disease, remains a challenge. Ruxolitinib has been shown as an effective and safe treatment option for these patients. Seventy-nine patients received ruxolitinib and were evaluated in this retrospective and multicenter study. Twenty-three patients received ruxolitinib for refractory acute graft-versus-host disease after a median of 3 (range 1-5) previous lines of therapy. Overall response rate was 69.5% (16/23) which was obtained after a median of 2 weeks of treatment, and 21.7% (5/23) reached complete remission. Fifty-six patients were evaluated for refractory chronic graft-versus-host disease. The median number of previous lines of therapy was 3 (range 1-10). Overall response rate was 57.1% (32/56) with 3.5% (2/56) obtaining complete remission after a median of 4 weeks. Tapering of corticosteroids was possible in both acute (17/23, 73%) and chronic graft-versus-host disease (32/56, 57.1%) groups. Overall survival was 47% (CI: 23-67%) at 6 months for patients with aGVHD (62 vs 28% in responders vs non-responders) and 81% (CI: 63-89%) at 1 year for patients with cGVHD (83 vs 76% in responders vs non-responders). Ruxolitinib in the real life setting is an effective and safe treatment option for GVHD, with an ORR of 69.5% and 57.1% for refractory acute and chronic graft-versus-host disease, respectively, in heavily pretreated patients

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Bovine pluripotent stem cells: strategies for generation and differentiation towards germ cells

The use of pluripotent stem cells for bovine transgenesis as well as the induction of primordial germ cell (PGC) differentiation from pluripotent bovine cells, represents conceptually feasible alternatives to reach high economic impact goals such as generating transgenic animals with improved or economically valuable traits or the modification of the genetic pool in an isolated population through germ cells transplantation. The goal of this study was to contribute towards the understanding of nuclear reprogramming and the germ cell specification in vitro in the specific case of Bos Taurus species (cattle) species. In this manuscript, key concepts on pluripotency, reprogramming and germ cell differentiation are introduced and reviewed (Chapter I). Focused mainly on the current murine and human models and what is known about cattle ; followed by a description on the materials and methods used in a regularly bases during the project (Chapter II). Then, the generation and characterization of three male biPSC cell lines by an integrative approach (lentiviral) with the potential to differentiate to several issues in vitro and in vivo is described followed by attempts to generated bovine induced pluripotent stem cells (biPSC) using a non-integrative (episomal) approach iPSC. Finally, different culture conditions and culture media were evaluated in terms of their effect on key pluripotent genes expression by the biPSC produced (Chapter III). The results demonstrate it is possible to increase the expression level of REX1, OCT4, NANOG and SOX2; key markers of pluripotency by pharmacological inhibition of the Glycogen synthase kinase-3 (GSK-3), the extracellular signal-regulated kinase (ERK) / mitogen-activated protein kinase (MAPK) pathways and DNA methyltransferases (DNMTs). The same effect is observed for the expression of OCT4 and REX1 merely by culture under LIF conditions. Next, a system for biPSC germ cell differentiation and isolation, based on VASA gene expression, was developed (Chapter IV). In this part of the work, an in silico analysis and in vitro validation of the putative regulatory elements upstream of the bovine vasa homolog gene (Bvh) was performed. This was through the modelling of regulatory elements by bioinformatics tools. Using this approach, a regulatory region of approximate 0.4 kb upstream of Bvh was identified. This region encloses a core of TFBS conserved, at the functional level, between at least three mammalian species. These findings were validated in human seminoma cells in vitro and subsequently the validated candidate regulatory sequences were used to generate male biPSCTg(bvh-EGFP) cell lines (germ cell reporter cell lines). The germ cell potential, across the cell lines generated, was evaluated (Chapter V) under distinct culture conditions (Hanging drop and suspension) and germ cell differentiation inducers such as Retinoic Acid (RA) and BMP4. The second part of the chapter involved analysis of the gene expression profile of the sorted population undergoing PGC specification. Using the culture conditions evaluated it was concluded that the biPSC generated not only had germ cell potential but also their rate of differentiation towards PGCs increases ten times in the presence of either RA or BMP4. Furthermore, those cells undergoing germ cell differentiation resemble an early post migratory state of PGC. The final chapter (Chapter VI) is a summary of the main findings from the previous three chapters. It centres on the potential of biPSC as a source of germ cells in vitro and the identity of the cells undergoing differentiation. Its identity is elucidated in the light of what is known from human and murine models. Finally, the limitations of this work and the future avenues of research are discussed

Bovine pluripotent stem cells: strategies for generation and differentiation towards germ cells

The use of pluripotent stem cells for bovine transgenesis as well as the induction of primordial germ cell (PGC) differentiation from pluripotent bovine cells, represents conceptually feasible alternatives to reach high economic impact goals such as generating transgenic animals with improved or economically valuable traits or the modification of the genetic pool in an isolated population through germ cells transplantation. The goal of this study was to contribute towards the understanding of nuclear reprogramming and the germ cell specification in vitro in the specific case of Bos Taurus species (cattle) species. In this manuscript, key concepts on pluripotency, reprogramming and germ cell differentiation are introduced and reviewed (Chapter I). Focused mainly on the current murine and human models and what is known about cattle ; followed by a description on the materials and methods used in a regularly bases during the project (Chapter II). Then, the generation and characterization of three male biPSC cell lines by an integrative approach (lentiviral) with the potential to differentiate to several issues in vitro and in vivo is described followed by attempts to generated bovine induced pluripotent stem cells (biPSC) using a non-integrative (episomal) approach iPSC. Finally, different culture conditions and culture media were evaluated in terms of their effect on key pluripotent genes expression by the biPSC produced (Chapter III). The results demonstrate it is possible to increase the expression level of REX1, OCT4, NANOG and SOX2; key markers of pluripotency by pharmacological inhibition of the Glycogen synthase kinase-3 (GSK-3), the extracellular signal-regulated kinase (ERK) / mitogen-activated protein kinase (MAPK) pathways and DNA methyltransferases (DNMTs). The same effect is observed for the expression of OCT4 and REX1 merely by culture under LIF conditions. Next, a system for biPSC germ cell differentiation and isolation, based on VASA gene expression, was developed (Chapter IV). In this part of the work, an in silico analysis and in vitro validation of the putative regulatory elements upstream of the bovine vasa homolog gene (Bvh) was performed. This was through the modelling of regulatory elements by bioinformatics tools. Using this approach, a regulatory region of approximate 0.4 kb upstream of Bvh was identified. This region encloses a core of TFBS conserved, at the functional level, between at least three mammalian species. These findings were validated in human seminoma cells in vitro and subsequently the validated candidate regulatory sequences were used to generate male biPSCTg(bvh-EGFP) cell lines (germ cell reporter cell lines). The germ cell potential, across the cell lines generated, was evaluated (Chapter V) under distinct culture conditions (Hanging drop and suspension) and germ cell differentiation inducers such as Retinoic Acid (RA) and BMP4. The second part of the chapter involved analysis of the gene expression profile of the sorted population undergoing PGC specification. Using the culture conditions evaluated it was concluded that the biPSC generated not only had germ cell potential but also their rate of differentiation towards PGCs increases ten times in the presence of either RA or BMP4. Furthermore, those cells undergoing germ cell differentiation resemble an early post migratory state of PGC. The final chapter (Chapter VI) is a summary of the main findings from the previous three chapters. It centres on the potential of biPSC as a source of germ cells in vitro and the identity of the cells undergoing differentiation. Its identity is elucidated in the light of what is known from human and murine models. Finally, the limitations of this work and the future avenues of research are discussed

Matemática Discreta-CE91-201900

El curso de Matemática Discreta es un curso de carrera que corresponde a la línea de Matemática para las carreras de Ingeniería de Sistemas y de Redes y Comunicaciones es de carácter teórico-práctico y se dicta en la modalidad semipresencial (blended). Está dirigido a estudiantes adultos trabajadores busca desarrollar la competencia general de razonamiento cuantitativo el cual le proporciona la capacidad de brindar explicaciones completas de información que se presenta en formas matemáticas convertir información en una representación matemática y la utiliza como argumento para sustentar una respuesta idea o proyecto así mismo le permite realizar operaciones matemáticas con éxito mostrando precisión en los resultados analiza información que contiene representaciones matemáticas para encontrar una solución y elaborar conclusiones correctas y podrá explicar los resultados de su razonamiento haciendo uso adecuado del lenguaje matemático. Este curso es parte de la formación integral de los estudiantes de Ingeniería de Sistemas y de Redes y Comunicaciones. El propósito de este curso es lograr que en el aprendizaje de los temas de Matemática Discreta el estudiante se dé cuenta de la relevancia de las ideas abstractas y por lo tanto se sienta motivado en la aplicación de estas ideas en computación

Inhibition of JAK-STAT ERK/MAPK and glycogen synthase kinase-3 induces a change in gene expression profile of bovineInduced pluripotent stem cells

Pluripotent stem cells (PSCs) fall in two states, one highly undifferentiated, the naïve state, and the primed state, characterized by the inability to contribute to germinal lineage. Several reports have demonstrated that these states can be modified by changes to the cell culture conditions. With the advent of nuclear reprogramming, bovine induced pluripotent stem cells (biPSCs) have been generated. These cells represent examples of a transient-intermediate state of pluripotency with remarkable characteristics and biotechnological potential. Herein, we generated and characterized biPSC. Next, we evaluated different culture conditions for the ability to affect the expression of the set of core pluripotent transcription factors in biPSC. It was found that the use of 6-bromoindirubin-3-oxime and Sc1 inhibitors alone or in combination with 5-AzaC induced significantly higher levels of expression of endogenous REX1, OCT4, NANOG, and SOX2. Furthermore, LIF increased the levels of expression of OCT4 and REX1, compared with those cultured with LIF + bFGF. By contrast, bFGF decreased the levels of expression for both REX1 and OCT4. These results demonstrate that the biPSC gene expression profile is malleable by modification of the cell culture conditions well after nuclear reprogramming, and the culture conditions may determine their differentiation potential