Evaluación de las diferentes metodologías para una mejora de la evaluación continua.

Este proyecto de innovación docente pretende evaluar el grado de valoración y satisfacción de los alumnos de la asignatura de Ingeniería Genética del Grado de Biotecnología en el uso de una evaluación continua basada en preguntas mensuales realizadas utilizando medios tradicionales (papel y bolígrafo) en comparación con una evaluación continua basada en preguntas diarias ludificadas utilizando las aplicaciones móviles

Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

This work was funded by the US National Institutes of Health (NIH) grant P01GM099117, and by the National Science Foundation Graduate Research Fellowship under the grant DGE1144152 (J.R.H. and K.M.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.United States Department of Health & Human Services National Institutes of Health (NIH) - USA P01GM099117National Science Foundation (NSF) DGE114415

Defining the first bona fide cell model for SMARCA4-deficient, undifferentiated tumor

The World Health Organization’s tumor classification guidelines are frequently updated and renewed as knowledge of cancer biology advances. For instance, in 2021, a novel lung tumor subtype named SMARCA4-deficient, undifferentiated tumor (SMARCA4-dUT, code 8044/3) was included. To date, there is no defined cell model for SMARCA4-dUT that could be used to help thoracic clinicians and researchers in the study of this newly defined tumor type. As this tumor type was recently described, it is feasible that some cell models formerly classified as lung adenocarcinoma (LUAD) could now be better classified as SMARCA4-dUT. Thus, in this work, we aimed to identify a bona fide cell model for the experimental study of SMARCA4-dUT. We compared the differential expression profiles of 36 LUAD-annotated cell lines and 38 cell lines defined as rhabdoid in repositories. These comparative results were integrated with the mutation and expression profiles of the SWI/SNF complex members, and they were surveyed for the presence of the SMARCA4-dUT markers SOX2, SALL4, and CD34, measured by RTqPCR and western blotting. Finally, the cell line with the paradigmatic SMARCA4-dUT markers was engrafted into immunocompromised mice to assess the histological morphology of the formed tumors and compare them with those formed by a bona fide LUAD cancer cell line. NCI-H522, formerly classified as LUAD, displayed expression profiles nearer to rhabdoid tumors than LUAD tumors. Furthermore, NCI-H522 has most of the paradigmatic features of SMARCA4-dUT: hemizygous inactivating mutation of SMARCA4, severe SMARCA2 downregulation, and high-level expression of stem cell markers SOX2 and SALL4. In addition, the engrafted tumors of NCI-H522 did not display a typical differentiated glandular structure as other bona fide LUAD cell lines (A549) do but had rather a largely undifferentiated morphology, characteristic of SMARCA4-dUT. Thus, we propose the NCI-H522 as the first bona fide cell line model of SMARCA4-dUT.Spanish Ministry of Science and Innovation (PID2021-126111OB-I00)Junta de Andalucía (PIGE-0213-2020, PI-0203-2022)University of Granada (B-CTS-480-UGR20)The Fundaci on Científica Asociaci on Española Contra el Cáncer (LAB-AECC-2018)FPU17/01258 fellowshipPrograma Operativo de Empleo Juvenil y de la Iniciativa de Empleo Juvenil (#04/2022-05)Grant from the Scientific Foundation of the Spanish Association Against Cancer in Granada (#PRDGR21428SAN

SWI/SNF proteins as targets in cancer therapy

Recent identification of synthetic lethal interactions involving several proteins of the SWI/SNF complex discussed in this Research Highlight has opened the possibility of new cancer therapeutic approaches.P.P.M. laboratory is funded by Ministry of Economy of Spain (SAF-2012- 37252), Junta de Andalucía (CICE-FEDER-BIO-1655, PeS-FEDER-PI-0903-2012), EU-MarieCurie (CIG-321926), CEI-BIOTIC (20 F12.6), Deutsche Leukämie-Stiftung and BBVA Foundation. P.P.M is a Ramon y Cajal Researcher (RYC-2011-07766). S S-O has a postdoctoral fellowship of Junta de Andalucía (CTS 03210) and the support of the CEI-BIOTIC (2014-MPBS33)

Activation‑induced cytidine deaminase causes recurrent splicing mutations in diffuse large B‑cell lymphoma

The online version contains supplementary material available at https://doi.org/10. 1186/s12943-​024-​01960-w.Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma. A major mutagenic process in DLBCL is aberrant somatic hypermutation (aSHM) by activation-induced cytidine deaminase (AID), which occurs preferentially at RCH/TW sequence motifs proximal to transcription start sites. Splice sequences are highly conserved, rich in RCH/TW motifs, and recurrently mutated in DLBCL. Therefore, we hypothesized that aSHM may cause recurrent splicing mutations in DLBCL. In a meta-cohort of > 1,800 DLBCLs, we found that 77.5% of splicing mutations in 29 recurrently mutated genes followed aSHM patterns. In addition, in whole-genome sequencing (WGS) data from 153 DLBCLs, proximal mutations in splice sequences, especially in donors, were significantly enriched in RCH/TW motifs (p 2,000; p < 0.0001) and confirmed its absence in 12 cancer types without aSHM (N > 6,300). Comparing sequencing data from mouse models with and without AID activity showed that the splice donor sequences were the top genomic feature enriched in AID-induced mutations (p < 0.0001). Finally, we observed that most AID-related splice site mutations are clonal within a sample, indicating that aSHM may cause early loss-of-function events in lymphomagenesis. Overall, these findings support that AID causes an overrepresentation of clonal splicing mutations in DLBCL.Grant PID2021-126111OB-I00 funded by the MCIN/AEI/10.13039/501100011033ERDF A way to make EuropeJunta de Andalucía (grants PI-0135–2020, and P20_00688)Spanish Association for Cancer Research (LABORATORY-AECC-2018)FPU19/00576 predoctoral fellowship funded by the Spanish Ministry of Science, Innovation, and Universitie

Choosing the right cell line for rectal cancer research

Up to date no effective method exists that predicts response to preoperative chemoradiation (CRT) in locally advanced rectal cancer (LARC). Nevertheless, identification of patients who have a higher likelihood of responding to preoperative CRT could be crucial in decreasing treatment morbidity and avoiding expensive and time-consuming treatments. Using the Gng4, c-Myc, Pola1, and Rrm1 signature, we were able to establish a model to predict response to CRT in rectal cancer with a sensitivity of 60% and 100% specificity. The aim of this study was to characterize c-Myc status in DNA, RNA and protein levels in 3 tumoral cell lines (SW480, SW620 and SW837) to establish the best cell line model and, subsequently, carry out genome silencing of c-Myc by means of RNA interference (iRNA). To study the expression levels of c-Myc, we used Polymerase Chain Reaction (PCR) amplifications and sequencing; quantitative real time PCR (qRT-PCR); and western blot analysis in each cell line. SW480 and SW620 showed a variation A > G in exon 2, which caused a substitution of aspargine to serine, and SW837 revealed a G > A transition in the same, which caused a mutation at codon 92. The three cell lines expressed c-Myc mRNA. SW837 showed a decrease of c-Myc expression levels compared with SW480, and SW620. At protein level, SW620 showed the highest expression of c-Myc. According to the results obtained, we can perform c-Myc gene silencing experiments to analyze the role of this biomarker in response to treatment

BCL7A is silenced by hypermethylation to promote acute myeloid leukemia

The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s40364‑ 023‑ 00472‑x. Additional file 1: Supplementary Figure 1. Diagram displaying CpG‑ methylation status around the BCL7A TSS. Genomic DNA from the NB4 cell line was subjected to bisulfite conversion and used for subsequent TA‑ cloning. Supplementary Figure 2. Schematic representation of the differ‑ ent lentiviral plasmids used in the experimental procedures. The specific region of the long isoform of BCL7A is colored in blue. Supplementary Figure 3. Western blot including the Decitabine (DAC) treatment over the NB4 cell line shown in Fig. 2c. Supplementary Figure 4. Protein‑protein interactions between BCL7A and SMARCA4 as determined by Mashtalir et al (2020). Supplementary Figure 5. DepMap AML cell lines collection data showing BCL7A Methylation Fraction (1kb upstream TSS) vs BCL7Aex‑ pression level. NB4 and M07e are marked. Supplementary Figure 6. Competition cell growth effect of BCL7A expression restoration on in vitro proliferation. Supplementary Table 1. Additional file 2: Supplementary Table 2. Differential expression analysis resultsP.P.M.’s laboratory is funded by Consejería de Universidad, Investigación e Innovación de la Junta de Andalucía and FEDER (P20‑00688), Aula de Investigación sobre la Leucemia infantil: Heroes contra la Leucemia, the Ministry of Science and Innovation of Spain (grant PID2021‑126111OB‑I00), Junta de Andalucía (grants PIGE‑0440–2019, PI‑0135–2020), the University of Granada (grants B‑CTS‑126‑UGR18, B‑CTS‑480‑UGR20, and E‑CTS‑304‑UGR20), and the Spanish Association for Cancer Research (LABORATORY‑AECC‑2018). J.R.P‑M, A.A, and M.S.B‑C were supported by fellowships FPU18/03709, FPU17/00067, and FPU19/00576 respectively funded by the Spanish Ministry of Science, Innovation and UniversitiesBackground Recent massive sequencing studies have revealed that SWI/SNF complexes are among the most fre‑ quently altered functional entities in solid tumors. However, the role of SWI/SNF in acute myeloid leukemia is poorly understood. To date, SWI/SNF complexes are thought to be oncogenic in AML or, at least, necessary to support leuke‑ mogenesis. However, mutation patterns in SWI/SNF genes in AML are consistent with a tumor suppressor role. Here, we study the SWI/SNF subunit BCL7A, which has been found to be recurrently mutated in lymphomas, but whose role in acute myeloid malignancies is currently unknown. Methods Data mining and bioinformatic approaches were used to study the mutational status of BCL7A and the correlation between BCL7A expression and promoter hypermethylation. Methylation‑specific PCR, bisulfite sequenc‑ ing, and 5‑aza‑2’‑deoxycytidine treatment assays were used to determine if BCL7A expression was silenced due to promoter hypermethylation. Cell competition assays after BCL7A expression restoration were used to assess the role of BCL7A in AML cell line models. Differential expression analysis was performed to determine pathways and genes altered after BCL7A expression restoration. To establish the role of BCL7A in tumor development in vivo, tumor growth was compared between BCL7A‑expressing and non‑expressing mouse xenografts using in vivo fluorescence imaging. Results BCL7A expression was inversely correlated with promoter methylation in three external cohorts: TCGA‑LAML (N = 160), TARGET‑AML (N = 188), and Glass et al. (2017) (N = 111). The AML‑derived cell line NB4 silenced the BCL7A expression via promoter hypermethylation. Ectopic BCL7A expression in AML cells decreased their competitive ability compared to control cells. Additionally, restoration of BCL7A expression reduced tumor growth in an NB4 mouse xenograft model. Also, differential expression analysis found that BCL7A restoration altered cell cycle pathways and modified significantly the expression of genes like HMGCS1, H1-0, and IRF7 which can help to explain its tumor sup‑ pressor role in AML. Conclusions BCL7A expression is silenced in AML by promoter methylation. In addition, restoration of BCL7A expres‑ sion exerts tumor suppressor activity in AML cell lines and xenograft models.Consejería de Universidad, Investigación e Innovación de la Junta de Andalucía and FEDER (P20‑00688)Ministry of Science and Innovation of Spain (grant PID2021‑126111OB‑I00)Junta de Andalucía (grants PIGE‑0440–2019, PI‑0135–2020)University of Granada (B‑CTS‑126‑UGR18, B‑CTS‑480‑UGR20, E‑CTS‑304‑UGR20)Spanish Association for Cancer Research (LABORATORY‑AECC‑2018)Spanish Ministry of Science, Innovation and Universities FPU18/03709, FPU17/00067, FPU19/0057

Maslinic Acid, a Triterpene from Olive, Affects the Antioxidant and Mitochondrial Status of B16F10 Melanoma Cells Grown under Stressful Conditions

Maslinic acid (MA) is a natural compound whose structure corresponds to a pentacyclic triterpene. It is abundant in the cuticular lipid layer of olives. MA has many biological and therapeutic properties related to health, including antitumor, anti-inflammatory, antimicrobial, antiparasitic, antihypertensive, and antioxidant activities. However, no studies have been performed to understand the molecular mechanism induced by this compound in melanoma cancer. The objective of this study was to examine the effect of MA in melanoma (B16F10) cells grown in the presence or absence of fetal bovine serum (FBS). We performed cell proliferation measurements, and the reactive oxygen species (ROS) measurements using dihydrorhodamine 123 (DHR 123) and activities of catalase, glucose 6-phosphate dehydrogenase, glutathione S-transferase, and superoxide dismutase. These changes were corroborated by expression assays. FBS absence reduced cell viability decreasing IC50 values of MA.The DHR 123 data showed an increase in the ROS level in the absence of FBS. Furthermore, MA had an antioxidant effect at lower assayed levels measured as DHR and antioxidant defense.However, at higher dosagesMAinduced cellular damage by apoptosis as seen in the results obtained.This study has been supported, in part, by funds of the consolidated Research Group BIO-157, from the General Secretariat of Universities, Research and Technology of the Ministry of Economy, Innovation, Science and Employment Government of the Junta de Andaluc´ıa (Spain), and by the Research Contract no. C-3650-00 under the program FEDER-INNTERCONECTA from the Spanish Government and European Union FEDER funds. Amalia P´erez-Jim´enez is a recipient of a postdoctoral research fellowship Torres- Quevedo no. PTQ 12-05739

The value of desmosomal plaque-related markers to distinguish squamous cell carcinoma and adenocarcinoma of the lung

An antibody panel is needed to definitively differentiate between adenocarcinoma (AC) and squamous cell carcinoma (SCC) in order to meet more stringent requirements for the histologic classification of lung cancers. Staining of desmosomal plaque-related proteins may be useful in the diagnosis of lung SCC. The specificity for SCC of membrane staining for PKP1, KRT15, and DSG3 was 97.4%, 94.6%, and 100%, respectively, and it was 100% when the markers were used together and in combination with the conventional markers (AUCs of 0.7619 for Panel 1 SCC, 0.7375 for Panel 2 SCC, 0.8552 for Panel 1 AC, and 0.8088 for Panel 2 AC). In a stepwise multivariate logistic regression model, the combination of CK5/6, p63, and PKP1 in membrane was the optimal panel to differentiate between SCC and AC, with a percentage correct classification of 96.2% overall (94.6% of ACs and 97.6% of SCCs). PKP1 and DSG3 are related to the prognosis. PKP1, KRT15, and DSG3 are highly specific for SCC, but they were more useful to differentiate between SCC and AC when used together and in combination with conventional markers. PKP1 and DSG3 expressions may have prognostic value.MEFV was supported by PAIDI programme, Group BIO309, Junta de Andalucía

Multi‑omic alterations of the SWI/SNF complex define a clinical subgroup in lung adenocarcinoma

PPM's lab is funded by the Ministry of Economy of Spain (SAF2015-67919-R), Junta de Andalucia (P20-00688, PI-0135-2020, PIGE-0213-2020, PIGE-04402019, PI-0245-2017), University of Granada (B-CTS-480-UGR20), International Association for the Study of Lung Cancer (IASLC), and Spanish Association for Cancer Research (LAB-AECC-2018). PP is supported by a PhD "La Caixa Foundation"LCF/BQ/DE15/10360019 Fellowship. AA is supported by an FPU17/00067 fellowship. IFC was supported by a PhD FPI-fellowship (BES-2013-064596). DJG was supported by a "Fundacion Benefica Anticancer Santa Candida y San Francisco Javier"predoctoral fellowship. MSBC and CC's work is supported by the project DPI2017-84439-R Ministry of Economy of Spain and FEDER and by the fellowship "Beca de Iniciacion a la Investigacion del Plan Propio de Investigacion 2019" by University of Granada. MSBC is supported by an FPU19/00576 predoctoral fellowship. CNIO Proteomics Unit is a member of Proteored PRB3 and is supported by grant PT17/0019, of the PE I + D + i 2013-2016, funded by ISCIII and ERDF.SWI/SNF complexes are major targets of mutations in cancer. Here, we combined multiple “-omics” methods to assess SWI/SNF composition and aberrations in LUAD. Mutations in lung SWI/SNF subunits were highly recurrent in our LUAD cohort (41.4%), and over 70% of the mutations were predicted to have functional impact. Furthermore, SWI/ SNF expression in LUAD suffered an overall repression that could not be explained exclusively by genetic alterations. Finally, SWI/SNF mutations were associated with poorer overall survival in TCGA-LUAD. We propose SWI/SNF-mutant LUAD as a separate clinical subgroup with practical implications.Spanish Government SAF2015-67919-R DPI2017-84439-RJunta de Andalucia P20-00688 PI-0135-2020 PIGE-0213-2020 PIGE-0440-2019 PI-0245-2017University of Granada B-CTS-480-UGR20International Association for the Study of Lung Cancer (IASLC)Spanish Association for Cancer Research LAB-AECC-2018La Caixa Foundation LCF/BQ/DE15/10360019PhD FPI-fellowship BES-2013-064596"Fundacion Benefica Anticancer Santa Candida y San Francisco Javier" predoctoral fellowshipEuropean Commissionfellowship "Beca de Iniciacion a la Investigacion del Plan Propio de Investigacion 2019" by University of Granada Instituto de Salud Carlos III PT17/0019European Commission PT17/0019 FPU17/00067 FPU19/0057