Resultados de investigación

Kdm1af/f, Kdm5cf/f and CamKIIα-CreERT2 mouse strains have been previously described , they are available at public repositories (Jackson Lab stock #23969; MMRRC stock 066789-UCD; and EMMA EM:02125, respectively). Mice were maintained on a C57BL/6J genetic background. The recombinant protein CreERT2 is dependent on tamoxifen (TMX) administration, and its expression is restricted to principal neurons of the forebrain, where CamKIIα is expressed. CaMKIIα–driven, TMX induced Cre recombination results in the elimination of exons 5 and 6 in Kdm1af/f and exons 11 and 12 in Kdm5cf/f . Mice were 2 to 3-month-old at the time of TMX administration., Loss or reduced expression of lysine demethylases (KDMs) is linked to neurodevelopmental disorders and intellectual disability. Given the phenotypic similarities between KDM1A and KDM5C deficient mice, and the convergence of both enzymes in maintaining a repressive state via H3K4 demethylation, we examined their functional interaction using double-inducible, forebrain-specific knockouts (dKDM-ifKOs). These mice showed transcriptional and epigenetic dysregulation beyond the additive effects of individual knockouts, including stronger ectopic expression of non-neuronal genes in hippocampal neurons. Thousands of de novo H3K4me3-enriched regions emerged, indicating synergistic disruption of chromatin regulation. In line with these molecular changes, dKDM-ifKOs displayed more severe behavioral impairments than the single ifKOs, along with altered hippocampal expression of ion channels and increased excitability of CA1 pyramidal neurons. These findings underscore the joint role of ID-linked KDMs in regulating cell-type-specific gene silencing and H3K4 methylation levels to safeguard neuronal identity and responsiveness, as well as cognitive function, Peer reviewed

Kdm1af/f, Kdm5cf/f and CamKIIα-CreERT2 mouse strains have been previously described , they are available at public repositories (Jackson Lab stock #23969; MMRRC stock 066789-UCD; and EMMA EM:02125, respectively). Mice were maintained on a C57BL/6J genetic background. The recombinant protein CreERT2 is dependent on tamoxifen (TMX) administration, and its expression is restricted to principal neurons of the forebrain, where CamKIIα is expressed. CaMKIIα–driven, TMX induced Cre recombination results in the elimination of exons 5 and 6 in Kdm1af/f and exons 11 and 12 in Kdm5cf/f . Mice were 2 to 3-month-old at the time of TMX administration., Loss or reduced expression of lysine demethylases (KDMs) is linked to neurodevelopmental disorders and intellectual disability. Given the phenotypic similarities between KDM1A and KDM5C deficient mice, and the convergence of both enzymes in maintaining a repressive state via H3K4 demethylation, we examined their functional interaction using double-inducible, forebrain-specific knockouts (dKDM-ifKOs). These mice showed transcriptional and epigenetic dysregulation beyond the additive effects of individual knockouts, including stronger ectopic expression of non-neuronal genes in hippocampal neurons. Thousands of de novo H3K4me3-enriched regions emerged, indicating synergistic disruption of chromatin regulation. In line with these molecular changes, dKDM-ifKOs displayed more severe behavioral impairments than the single ifKOs, along with altered hippocampal expression of ion channels and increased excitability of CA1 pyramidal neurons. These findings underscore the joint role of ID-linked KDMs in regulating cell-type-specific gene silencing and H3K4 methylation levels to safeguard neuronal identity and responsiveness, as well as cognitive function, Peer reviewed

Kdm5cf/f, and CamKIIα-CreERT2 mouse strains are available at public repositories (MMRRC stock 066789-UCD; and EMMA EM:02125, respectively). Mice were maintained on a C57BL/6J genetic background. The recombinant protein CreERT2 is dependent on tamoxifen (TMX) administration, and its expression is restricted to principal neurons of the forebrain, where CamKIIα is expressed. CaMKIIα–driven, TMX induced Cre recombination results in the elimination of exons 11 and 12 in Kdm5cf/f . Mice were 2 to 3-month-old at the time of TMX administration., Loss or reduced expression of lysine demethylases (KDMs) is linked to neurodevelopmental disorders and intellectual disability. Given the phenotypic similarities between KDM1A and KDM5C deficient mice, and the convergence of both enzymes in maintaining a repressive state via H3K4 demethylation, we examined their functional interaction using double-inducible, forebrain-specific knockouts (dKDM-ifKOs). These mice showed transcriptional and epigenetic dysregulation beyond the additive effects of individual knockouts, including stronger ectopic expression of non-neuronal genes in hippocampal neurons. Thousands of de novo H3K4me3-enriched regions emerged, indicating synergistic disruption of chromatin regulation. In line with these molecular changes, dKDM-ifKOs displayed more severe behavioral impairments than the single ifKOs, along with altered hippocampal expression of ion channels and increased excitability of CA1 pyramidal neurons. These findings underscore the joint role of ID-linked KDMs in regulating cell-type-specific gene silencing and H3K4 methylation levels to safeguard neuronal identity and responsiveness, as well as cognitive function, Peer reviewed

The study included a total of 39 blood samples, comprising 23 from de novo, drug-naïve Parkinson’s disease (PD) patients and 16 from healthy, age- and sex-matched controls. Following quality control assessments, one sample was excluded due to failing labeling control thresholds, resulting in a final dataset of 38 samples. Each patient represented a biological replicate. Peripheral Blood Mononuclear Cells (PBMCs) were isolated, and RNA was extracted and assessed for integrity before transcriptome analysis using the Clariom S Array (Affymetrix). Data preprocessing involved normalization and filtering, leading to a final dataset of 18,091 probes. -- Expression profiling by array, This study investigated gene expression differences in Parkinson’s disease (PD) using blood samples from 23 newly diagnosed, drug-naïve PD patients and 16 healthy controls recruited from two hospitals in Spain. Participants met diagnostic criteria for early-stage PD (Hoehn and Yahr I-II) and had no family history of the disease. Ethical approval and informed consent were obtained. Peripheral Blood Mononuclear Cells (PBMCs) were isolated, frozen, and processed for RNA extraction. RNA integrity was assessed before whole transcriptome analysis using the Clariom S Array (Affymetrix). Data processing was conducted in R using BioConductor packages. Quality control measures led to the exclusion of one sample, and data were normalized using Robust Multi-array Average (RMA). Lowly expressed genes and probes not mapping to known genes were filtered out, resulting in 38 samples with 18,091 probes, Peer reviewed

Peer reviewed

Experiment type: Expression profiling by high throughput sequencing. -- Total RNA was obtained from three biological replicates corresponding to i) parental cell lines (bloodstream S16 or procyclic 449), or ii) cell lines in which DRBD3 was depleted by RNAi (bloodstream and procyclic). In addition, three biological replicates from a bloodstream cell line in which ZC3H41 was depleted by RNAi was included as a control. -- Organism: Trypanosoma brucei brucei, Trypanosomes are unicellular eukaryotes that rely heavily on post-transcriptional mechanisms to control gene expression. DRBD3 is an RNA-binding protein known to play important roles in mRNA processing, stability, transport and translation. It was found to associate with grumpy, a long non-coding RNA (lncRNA) recently characterized in Trypanosoma brucei. Here, we explore the role of DRBD3 in lncRNA metabolism and show that its depletion leads to the upregulation of a specific subset of approximately one hundred lncRNAs in both bloodstream and procyclic forms, likely through the activation of cryptic splice sites. The effect of DRBD3 depletion on lncRNA expression appears to be mostly indirect, and results from reduced levels of the poly(A) polymerase PAP1 following DRBD3 silencing. In addition to its impact on lncRNAs, DRBD3 loss also affects the processing of protein-coding genes, leading to alternative trans-splicing and protein truncation. Furthermore, we demonstrate that DRBD3 regulates the splicing of the newly identified intron in the transcript encoding the RNA-binding protein RBP20, and is important for maintaining the balance between trans- and cis-splicing. Our results position DRBD3 as a high-level regulatory factor that shapes the expression landscape of both coding and non-coding genes in trypanosomes, Peer reviewed

5 Files (2 .txt; 3 .gpzip). License cc-by-4.0 Creative Commons Attribution 4.0 International., Contents of PanOryza pan-genes release v1.0 : 1. The Input fasta and gff folder contains the genomic DNA sequences and the annotation information of the genomes used for running GET_PANGENES pipeline. - Nipponbare_gene_to_transcript_map.txt : tabular details of the pangene identifier and the corresponding gene ids from the 3 Nipponbare annotations (RAPDB, MSU and Gramene). 2. Os4530.POR.1 contains the pangene cluster sequences - genomic(gdna.fna) , cDNA(cDNA.fna), coding sequence(cds.fna), amino acids (cds.faa) and the output files of GET_PANGENES pipeline. - POR.1.clusters_summary : Summarised tabular details of the pangene clusters , gene ids of the rice genome under that cluster, protein length etc. 3. Pan_gene_matrix_transcripts.txt : Pan-gene matrix with re-ordered pangene identifier in 1st column and pan-gene cluster members in subsequent columns. 4. manuscript_data : Workspace files (.RData) to reproduce the manuscript analyses. The codes and other tables are available at GitHub. 5. README.txt, The dataset provides the pan-gene sets release v1.0 for the asian rice developed by the PanOryza consortium., Developed by the PanOryza consortium. Members: 1. University of Liverpool, 2. Estación Experimental de Aula Dei, 3. European Bioinformatics Institute, 4. Oregon State University, 5. International Rice Research Institute, 6. Cold Spring Harbor Laboratory, 7. King Abdullah University of Science and Technology, 8. Huazhong Agricultural University, 9. United States Department of Agriculture, 10. Institute for Systems Biology, 11. University of Arizona, Peer reviewed

Sample Processing Protocol Nuclear extracts of mESCs were perepared as previosly described in Kloet et al. 2016. 2mg of lysate was mixed with 6ug of anti-FLAG antibody (Merck. F3165) and 50uL of Protein G mangnetic beads (Dynabeads). The magnetic beads were cleaned three times with 500 µl of 200 mM ammonium bicarbonate and 60 µl of 6M Urea / 200mM ammonium bicarbonate were added. Samples were then reduced with dithiothreitol (30 nmol, 37 ºC, 60 min), alkylated in the dark with iodoacetamide (60 nmol, 25 ºC, 30 min) and diluted to 1M urea with 200 mM ammonium bicarbonate for trypsin digestion (1 µg, 37ºC, 8h, Promega). After digestion, peptide mix was acidified with formic acid and desalted with a MicroSpin C18 column (The Nest Group, Inc) prior to LC-MS/MS analysis. The samples were then analysed using a LTQ-Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled to an EASY-nLC 1200 (Thermo Fisher Scientific (Proxeon, Odense, Denmark). Peptides were loaded directly onto the analytical column and were separated by reversed-phase chromatography using a 50-cm column with an inner diameter of 75 μm, packed with 2 μm C18 particles. Chromatographic gradients started at 95% buffer A and 5% buffer B with a flow rate of 300 nl/min and gradually increased to 25% buffer B and 75% A in 52 min and then to 40% buffer B and 60% A in 8 min. After each analysis, the column was washed for 10 min with 100% buffer B. Buffer A: 0.1% formic acid in water. Buffer B: 0.1% formic acid in 80% acetonitrile. The mass spectrometer was operated in positive ionization mode with nanospray voltage set at 2.4 kV and source temperature at 305°C. The acquisition was performed in data-dependent adquisition (DDA) mode and full MS scans with 1 micro scans at resolution of 120,000 were used over a mass range of m/z 350-1400 with detection in the Orbitrap mass analyzer. Auto gain control (AGC) was set to ‘standard’ and injection time to ‘auto’. In each cycle of data-dependent acquisition analysis, following each survey scan, the most intense ions above a threshold ion count of 10000 were selected for fragmentation. The number of selected precursor ions for fragmentation was determined by the “Top Speed” acquisition algorithm and a dynamic exclusion of 60 seconds. Fragment ion spectra were produced via high-energy collision dissociation (HCD) at normalized collision energy of 28% and they were acquired in the ion trap mass analyzer. AGC and injection time were set to ‘Standard’ and ‘Dynamic’, respectively and isolation window of 1.4 m/z was used. Digested bovine serum albumin (New England Biolabs) was analysed between each sample to avoid sample carryover and to assure stability of the instrument. QCloud has been used to control instrument longitudinal performance during the project. -- Data Processing Protocol Acquired spectra were analysed using the Proteome Discoverer software suite (v1.4, Thermo Fisher Scientific) and the Mascot search engine (v2.6, Matrix Science). The data were searches against a Swiss-Prot mouse database (as in March 2021, 17082 entries) plus a list of common contaminants and all the corresponding decoy entries. For peptide identification a precursor ion mass tolerance of 7 ppm was used for MS1 level, trypsin was chosen as enzyme and up to three missed cleavages were allowed. The fragment ion mass tolerance was set to 0.5 Da for MS2 spectra. Oxidation of methionine and N-terminal protein acetylation were used as variable modifications whereas carbamidomethylation on cysteines was set as a fixed modification. False discovery rate (FDR) in peptide identification was set to a maximum of 5%. SAINT express algorithm was used to score protein-protein interactions and STRING software was used to identify functional clusters among FLAG-BMAL1-interacting proteins. --, Immunoprecipitation with anti-FLAG antibody in nuclear extracts of FLAG-BMAL1 expressing mESCs. LC MS/MS was used to analyze the interactome of BMAL1 in mESCs., Peer reviewed

Refer to individual Series. This SuperSeries is composed of the SubSeries listed below. Figure 1: scRNAseq of a bulk sample 1014 NRAS*. Sample GSM8195555. Figure 2: RNAseq. Samples GSM8195543 to GSM8195554. Figures 4 - 6: scRNAseq of CX3CR1+/CD45+ samples from NRAS* MBM. Samples from GSM8706608 to GSM8706613., Melanoma is one of the solid tumor types with the highest risk of brain metastasis. However, the biology of melanoma brain metastasis and the contribution of the brain immune microenvironment to the responses to therapies remain insufficiently characterized. By using preclinical models and single-cell transcriptomics, we identify microglia, as critical regulators of melanoma biology in the brain. We show that activation of the Rela/NF-kB pathway in microglia promotes melanoma brain metastasis and that targeting this pathway elicits microglia reprogramming towards a pro-inflammatory phenotype that enhances anti-tumor immunity and reduces brain metastatic burden. Additionally, canonical and pro-inflammatory microglial markers in melanoma brain metastasis correlate with better responses to immune checkpoint inhibitors in patients and we show that Rela/NF-kB targeting improves responses to these therapies in the brain. Thus, we propose targeting Rela/NF-kB in activated microglia as a strategy to promote anti-tumor immunity and to improve the response to immune checkpoint inhibitors in melanoma brain metastasis., Peer reviewed

We designed a MERSCOPE panel targeting 265 genes using the Vizgen Gene Panel Design Portal. Formalin-fixed paraffin-embedded (FFPE) samples from three distinct MMTV-PyMT tumors were processed according to Vizgen MERSCOPE protocols. The resulting data were utilized to analyze transcriptional heterogeneity at the spatial level within the tumors., Disseminated cancer cells rarely succeed in forming macrometastases, yet they continue to be the most life-threatening cause of patient mortality. The determinants driving metastatic competence, however, are still poorly understood. Using data from human breast cancer patients and genetic mouse models, we uncover a non-linear relationship between the expression of the EMT transcription factor Prrx1 in the primary tumor and metastatic outcome. Multi-omics analyses in mouse tumors reveal that Prrx1 functions as a dual regulator, promoting invasive behavior while simultaneously inducing dormancy. Cells with intermediate Prrx1 levels exhibit the highest metastatic fitness, whereas extreme levels (negative or too high) lead to less metastasis, typical of a hormetic behavior, and respectively due to insufficient invasiveness or to dormancy. These findings provide mechanistic insight into the regulation of dormancy and its link to metastatic potential. Additionally, we propose that combined signatures of invasion and proliferation allow robust prognosis prediction in breast cancer patients. Our study further highlights Prrx1-driven heterogeneity, established in the primary tumor, as a key determinant of metastatic competence., Peer reviewed