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Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients., Research in the EDA and OMT labs are supported by “Asociación Española Contra el Cáncer” (AECC). EDA lab is also supported by the Ministry of Economy and Competitiveness of Spain-FEDER (CIBERONC, PI2000003, RD06/0020/0059). DGD and LHP are supported by CIBERONC (CB16/12/00361)S. LHP is funded by the Consejería de Salud de la Junta de Andalucía (PI-0013-2018). SRV is supported by “Asociación Alba Pérez lucha contra el cáncer infantil”. The OMT lab is supported by the Catalan Agency for the Management of University and Research Grants (AGAUR 2017 SGR 332).

Enhancers activate transcription through long-distance interactions with their cognate promoters within a particular subtopologically associated domain (sub-TAD). The TCRα enhancer (Eα) is located at the sub-TAD boundary between the TCRα and DAD1 genes and regulates transcription toward both sides in an ∼1-Mb region. Analysis of Eα activity in transcribing the unrearranged TCRα gene at the 5′-sub-TAD has defined Eα as inactive in CD4−CD8− thymocytes, active in CD4+CD8+ thymocytes, and strongly downregulated in CD4+ and CD8+ thymocytes and αβ T lymphocytes. Despite its strongly reduced activity, Eα is still required for high TCRα transcription and expression of TCRαβ in mouse and human T lymphocytes, requiring collaboration with distant sequences for such functions. Because VαJα rearrangements in T lymphocytes do not induce novel long-range interactions between Eα and other genomic regions that remain in cis after recombination, strong Eα connectivity with the 3′-sub-TAD might prevent reduced transcription of the rearranged TCRα gene. Our analyses of transcriptional enhancer dependence during T cell development and non-T lineage tissues at the 3′-sub-TAD revealed that Eα can activate the transcription of specific genes, even when it is inactive to transcribe the TCRα gene at the 5′-sub-TAD. Hence distinct requirements for Eα function are necessary at specific genes at both sub-TADs, implying that enhancers do not merely function as chromatin loop anchors that nucleate the formation of factor condensates to increase gene transcription initiated at their cognate promoters. The observed different regulated Eα activity for activating specific genes at its flanking sub-TADs may be a general feature for enhancers located at sub-TAD boundaries.

The study of microorganisms is a field of great interest due to their environmental (e.g., soil contamination) and biomedical (e.g., parasitic diseases, autism) importance. The advent of revolutionary next-generation sequencing techniques, and their application to the hypervariable regions of the 16S, 18S or 23S ribosomal subunits, have allowed the research of a large variety of organisms more in-depth, including bacteria, archaea, eukaryotes and fungi. Additionally, together with the development of analysis software, the creation of specific databases (e.g., SILVA or RDP) has boosted the enormous growth of these studies. As the cost of sequencing per sample has continuously decreased, new protocols have also emerged, such as shotgun sequencing, which allows the profiling of all taxonomic domains in a sample. The sequencing of hypervariable regions and shotgun sequencing are technologies that enable the taxonomic classification of microorganisms from the DNA present in microbial communities. However, they are not capable of measuring what is actively expressed. Conversely, we advocate that metatranscriptomics is a “new” technology that makes the identification of the mRNAs of a microbial community possible, quantifying gene expression levels and active biological pathways. Furthermore, it can be also used to characterise symbiotic interactions between the host and its microbiome. In this manuscript, we examine the three technologies above, and discuss the implementation of different software and databases, which greatly impact the obtaining of reliable results. Finally, we have developed two easy-to-use pipelines leveraging Nextflow technology. These aim to provide everything required for an average user to perform a metagenomic analysis of marker genes with QIMME2 and a metatranscriptomic study using Kraken2/Bracken., regional Andalusian Government, POSTDOC_21 _00394

To identify potential miRNAs involved in the development/progression of type 2 diabetes (T2D) we performed data screening and integration of microRNA (miRNA) and messenger RNA (mRNA) expression profiles available in public data repositories. We retrieved two independent sequencing datasets: GSE52314 (small RNA-Seq) and GSE50244 (RNA-Seq). Then, we identified Differentially Expressed Genes (DEGs) and Differentially Expressed miRNAs (DEMs) from non-diabetic (ND) vs. people with T2D. The integrative analysis of identified DEGs and DEMs revealed 303 interactions involving 33 miRNAs and 256 genes. Functional enrichment analysis showed these interactions could play a functional role in carbohydrate, lipid, and nucleotide impaired metabolism. Some interactions we identified could also be relevant at an early stage of T2D. We also performed a similar integration analysis using a single study from a rat model, including both RNA-Seq and small RNA-Seq in the same pancreatic tissue: CRA000791. Interestingly, in this analysis one identified miRNA was also identified in the integrative analysis in human transcriptome: hsa-miR-27a-5p. Moreover, functional enrichment analysis showed that the target genes are mainly enriched in biological processes and pathways related to lipid and nucleotide metabolism. The miRNAs and interactions we identified could be relevant to gene expression regulation in T2D. Although further validation is necessary for extensive use in clinics, they provide new and useful knowledge to develop potential novel strategies for early diagnosis, prevention, and treatment of T2D., This study was partially supported by a grant (PUE 2017 N° 22920170100082CO) provided by the National Scientific and Technical Research Council and a mobility scholarship provided by the Postgraduate Ibero-American University Association – AUIP. EAL was recipient of a postdoctoral fellowship from regional Andalusian Government (2020_DOC_00541).

Patients with common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, are characterized by hypogammaglobulinemia, poorly protective vaccine titers and increased susceptibility to infections. New pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), might constitute a particular threat to these immunocompromised patients since many of them experience a slower recovery and do not achieve full response to SARS-CoV-2 vaccines. To define the molecular basis of the altered immune responses caused by SARS-CoV-2 infection in CVID patients, we generated longitudinal single-cell datasets of peripheral blood immune cells along viral infection and recovery. We sampled the same individuals before, during and after SARS-CoV-2 infection to model their specific immune response dynamics while removing donor variability. We observed that COVID-19 CVID patients show defective canonical NF-κB pathway activation and dysregulated expression of BCR-related genes in naïve B cells, as well as enhanced cytotoxic activity but incomplete cytokine response in NK and T cells. Moreover, monocytes from COVID-19 CVID patients show persistent activation of several inflammasome-related genes, including the pyrin and NLRC4 inflammasomes. Our results shed light on the molecular basis of the prolonged clinical manifestations observed in these immunodeficient patients upon SARS-CoV-2 infection, which might illuminate the development of tailored treatments for COVID-19 CVID patients., We thank the CERCA Program/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. This publication is part of the Human Cell Atlas: www.humancellatlas.org/publications. This study was funded by ”la Caixa” Foundation under the grant agreement LCF/PR/HR22/52420002, Spanish Ministry of Science and Innovation (grant number PID2020-117212RB-I00/AEI/10.13038/501100011033) (E.B.), by the Wellcome Trust Grant 206194 and 108413/A/15/D (R.V.-T.), Instituto de Salud Carlos III (ISCIII), Ref. AC18/00057, associated with i-PAD project (ERARE European Union program) (E.B.), and the Chan Zuckerberg Initiative (grant 2020-216799) (R.V.-T. and E.B.). This publication has also been supported by the Unstoppable campaign of the Josep Carreras Leukaemia Foundation. We are indebted to the donors for participating in this research.

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-function al enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus con tributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and pro line. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of seques tered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the poten tial of mutation-induced leakage in metabolic engineering is noted, Human Frontier Science Program RGP0041/2017, Spanish Government RTI2018-097142-B-100, Ministry of Science and Innovation, Spain (MICINN) 80NSSC18K1277, European Commission, Junta de Andalucia, Regional Andalusian Government E-BIO-464-UGR-20 2020_DOC_00541

ObjectivesGiant cell arteritis (GCA) is a complex systemic vasculitis mediated by the interplay between both genetic and epigenetic factors. Monocytes are crucial players of the inflammation occurring in GCA. Therefore, characterisation of the monocyte methylome and transcriptome in GCA would be helpful to better understand disease pathogenesis.MethodsWe performed an integrated epigenome-and transcriptome-wide association study in CD14+ monocytes from 82 patients with GCA, cross-sectionally classified into three different clinical statuses (active, in remission with or without glucocorticoid (GC) treatment), and 31 healthy controls.ResultsWe identified a global methylation and gene expression dysregulation in GCA monocytes. Specifically, monocytes from active patients showed a more proinflammatory phenotype compared with healthy controls and patients in remission. In addition to inflammatory pathways known to be involved in active GCA, such as response to IL-6 and IL-1, we identified response to IL-11 as a new pathway potentially implicated in GCA. Furthermore, monocytes from patients in remission with treatment showed downregulation of genes involved in inflammatory processes as well as overexpression of GC receptor-target genes. Finally, we identified changes in DNA methylation correlating with alterations in expression levels of genes with a potential role in GCA pathogenesis, such asITGA7andCD63, as well as genes mediating the molecular response to GC, includingFKBP5, ETS2,ZBTB16andADAMTS2.ConclusionOur results revealed profound alterations in the methylation and transcriptomic profiles of monocytes from GCA patients, uncovering novel genes and pathways involved in GCA pathogenesis and in the molecular response to GC treatment.

This article belongs to the Special Issue Bioactive Compounds: From Diet to Therapeutic Use., Although genomes from many edible mushrooms are sequenced, studies on fungal micro RNAs (miRNAs) are scarce. Most of the bioinformatic tools are designed for plants or animals, but the processing and expression of fungal miRNAs share similarities and differences with both kingdoms. Moreover, since mushroom species such as Agaricus bisporus (A. bisporus, white button mushroom) are frequently consumed as food, controversial discussions are still evaluating whether their miRNAs might or might not be assimilated, perhaps within extracellular vesicles (i.e., exosomes). Therefore, the A. bisporus RNA-seq was studied in order to identify potential de novo miRNA-like small RNAs (milRNAs) that might allow their later detection in diet. Results pointed to 1 already known and 37 de novo milRNAs. Three milRNAs were selected for RT-qPCR experiments. Precursors and mature milRNAs were found in the edible parts (caps and stipes), validating the predictions carried out in silico. When their potential gene targets were investigated, results pointed that most were involved in primary and secondary metabolic regulation. However, when the human transcriptome is used as the target, the results suggest that they might interfere with important biological processes related with cancer, infection and neurodegenerative diseases., This research was funded by grants from the Spanish “Agencia Estatal de Investigación” and European FEDER Funds (PID2019-109369RB-I00 to A.D., AGL2018-78922-R to FRM and C.S.-R.). E.A.-L. was recipient of a postdoctoral fellowship from Regional Andalusian Government (2020_DOC_00541).

© 2021 Martínez-Blanco, Domínguez-Pantoja, Botía-Sánchez, Pérez-Cabrera, Bello-Iglesias, Carrillo-Rodríguez, Martin-Morales, Lario-Simón, Pérez-Sánchez-Cañete, Montosa-Hidalgo, Guerrero-Fernández, Longobardo-Polanco, Redondo-Sánchez, Cornet-Gomez, Torres-Sáez, Fernández-Ibáñez, Terrón-Camero, Andrés-León, O’Valle, Merino, Zubiaur and Sancho., The absence of the mouse cell surface receptor CD38 in Cd38−/− mice suggests that this receptor acts as a positive regulator of inflammatory and autoimmune responses. Here, we report that, in the context of the chronic graft-versus-host disease (cGVHD) lupus inducible model, the transfer of B6.C-H2bm12/KhEg(bm12) spleen cells into co-isogenic Cd38−/− B6 mice causes milder lupus-like autoimmunity with lower levels of anti-ssDNA autoantibodies than the transfer of bm12 spleen cells into WT B6 mice. In addition, significantly lower percentages of Tfh cells, as well as GC B cells, plasma cells, and T-bet+CD11chi B cells, were observed in Cd38−/− mice than in WT mice, while the expansion of Treg cells and Tfr cells was normal, suggesting that the ability of Cd38−/− B cells to respond to allogeneic help from bm12 CD4+ T cells is greatly diminished. The frequencies of T-bet+CD11chi B cells, which are considered the precursors of the autoantibody-secreting cells, correlate with anti-ssDNA autoantibody serum levels, IL-27, and sCD40L. Proteomics profiling of the spleens from WT cGVHD mice reflects a STAT1-driven type I IFN signature, which is absent in Cd38−/− cGVHD mice. Kidney, spleen, and liver inflammation was mild and resolved faster in Cd38−/− cGVHD mice than in WT cGVHD mice. We conclude that CD38 in B cells functions as a modulator receptor that controls autoimmune responses., S and MZ received financial support through “Proyecto del Plan Estatal”: SAF2017–89801-R. The IPBLN-CSIC Proteomics Unit belonged to ProteoRed-ISCIII (PRB2; PRB3) and was supported by grants PT13/0001/0011 (IPBLN-CSIC) and PT17/0019/0010 (CIB-CSIC; IPBLN-CSIC). RM: Project: SAF2017-82905-R. FO'V: Cátedra MIS IMPLANT-UGR. The stay of AC-G in Sancho’s lab was supported by a fellowship-contract JAE-Intro (CSIC). The stay of MD-P in Sancho’s lab was supported by a 1-year post-doctoral fellowship (Reference No. 502492) from the Consejo Nacional de Ciencia y Tecnología (CONACYT) of México. EA-L was recipient of a postdoctoral fellowship from the regional Andalusian Government.

Multiple sclerosis (MS) is a complex and demyelinating disease of the central nervous system. One of the challenges of the post-genome-wide association studies (GWAS) era is to understand the molecular basis of statistical associations to reveal gene networks and potential therapeutic targets. The L3MBTL3 locus has been associated with MS risk by GWAS. To identify the causal variant of the locus, we performed fine mapping in a cohort of 3440 MS patients and 1688 healthy controls. The variant that best explained the association was rs6569648 (P = 4.13E-10, odds ratio = 0.71, 95% confidence interval (CI) = 0.64–0.79), which tagged rs7740107, located in intron 7 of L3MBTL3. The rs7740107 (A/T) variant has been reported to be the best expression and splice quantitative trait locus (eQTL and sQTL) of the region in up to 35 human genotype-tissue expression (GTEx) tissues. By sequencing RNA from blood of 17 MS patients and quantification by digital qPCR, we determined that this eQTL/sQTL originated from the expression of a novel short transcript starting in intron 7 near rs7740107. The short transcript was translated into three proteins starting at different translation initiation codons. These N-terminal truncated proteins lacked the region where L3MBTL3 interacts with the transcriptional regulator Recombination Signal Binding Protein for Immunoglobulin Kappa J Region which, in turn, regulates the Notch signalling pathway. Our data and other functional studies suggest that the genetic mechanism underlying the MS association of rs7740107 affects not only the expression of L3MBTL3 isoforms, but might also involve the Notch signalling pathway.

Circular RNAs (circRNAs) are a class of single-stranded RNAs derived from exonic, intronic, and intergenic regions from precursor messenger RNAs (pre-mRNA), where a noncanonical back-splicing event occurs, in which the 5' and 3' ends are attached by covalent bond. CircRNAs participate in the regulation of gene expression at the transcriptional and posttranscriptional level primarily as miRNA and RNA-binding protein (RBP) sponges, but also involved in the regulation of alternative RNA splicing and transcription. CircRNAs are widespread and abundant in plants where they have been involved in stress responses and development. Through the analysis of all publications in this field in the last five years, we can summarize that the identification of these molecules is carried out through next generation sequencing studies, where samples have been previously treated to eliminate DNA, rRNA, and linear RNAs as a means to enrich circRNAs. Once libraries are prepared, they are sequenced and subsequently studied from a bioinformatics point of view. Among the different tools for identifying circRNAs, we can highlight CIRI as the most used (in 60% of the published studies), as well as CIRCExplorer (20%) and find_circ (20%). Although it is recommended to use more than one program in combination, and preferably developed specifically to treat with plant samples, this is not always the case. It should also be noted that after identifying these circular RNAs, most of the authors validate their findings in the laboratory in order to obtain bona fide results.

Background and purpose Liver fibrosis induced by chronic hepatic injury remains as a major cause of morbidity and mortality worldwide. Identification of susceptibility/prognosis factors and new therapeutic tools for treating hepatic fibrotic disorders are urgent medical needs. Cortistatin is a neuropeptide with potent anti-inflammatory and anti-fibrotic activities in lung that binds to receptors that are expressed in liver fibroblasts and hepatic stellate cells. We evaluated the capacity of cortistatin to regulate liver fibrosis. Experimental approach We experimentally induced liver fibrosis in mice by chronic CCl4 exposition and bile duct ligation and evaluated the histopathological signs and fibrotic markers. Key results Hepatic expression of cortistatin inversely correlated with liver fibrosis grade in mice and humans with hepatic disorders. Cortistatin-deficient mice showed exacerbated signs of liver damage and fibrosis and increased mortality rates when challenged to hepatotoxic and cholestatic injury. Compared to wild-type mice, non-parenchymal liver cells isolated from cortistatin-deficient mice showed increased presence of cells with activated myofibroblast phenotypes and a differential genetic signature that is indicative of activated hepatic stellate cells and periportal fibroblasts and of myofibroblasts with active contractile apparatus. Cortistatin treatment reversed in vivo and in vitro these exaggerated fibrogenic phenotypes and protected from progression to severe liver fibrosis in response to hepatic injury. Conclusion and implications We identify cortistatin as an endogenous molecular break of liver fibrosis and its deficiency as a potential poor-prognosis marker for chronic hepatic disorders that course with fibrosis. Cortistatin-based therapies emerge as attractive strategies for ameliorating severe hepatic fibrosis of various etiologies.

Ewing sarcoma (EWS) is an aggressive developmental sarcoma driven by a fusion gene, EWSR1-FLI1. However, little is known about the regulation of EWSR1-FLI1 chimeric fusion gene expression. Here, we demonstrate that active nuclear HDAC6 in EWS modulates acetylation status of specificity protein 1 (SP1), consequently regulating SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. Overexpression of HDAC6 in primary EWS tumor clinical samples correlates with a poor prognosis. Notably, a combination treatment of a selective HDAC6 inhibitor and doxorubicin (standard of care in EWS) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for EWS patients.

Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS., Research in the E.D.A. lab is supported by Asociación Española Contra el Cáncer (AECC), the Ministry of Science of Spain-FEDER (CIBERONC, PI1700464, PI2000003, RD06/0020/0059)S. D.G.D. and L.H.P. are supported by CIBERONC (CB16/12/00361). D.G.D., M.J.R. and L.H.P. are PhD researchers funded by the Consejería de Salud, Junta de Andalucía (PI-0197-2016, ECAI F2-0012-2018 and PI-0013-2018, respectively).

Circular RNAs (circRNAs) are a class of single-stranded RNAs derived from exonic, intronic, and intergenic regions from precursor messenger RNAs (pre-mRNA), where a noncanonical back-splicing event occurs, in which the 50and 30ends are attached by covalent bond. CircRNAs participate in the regulation of gene expression at the transcriptional and posttranscriptional level primarily as miRNA and RNA-binding protein (RBP) sponges, but also involved in the regulation of alternative RNA splicing and transcription. CircRNAs are widespread and abundant in plants where they have been involved in stress responses and development. Through the analysis of all publications in this field in the last five years, we can summarize that the identification of these molecules is carried out through next generation sequencing studies, where samples have been previously treated to eliminate DNA, rRNA, and linear RNAs as a means to enrich circRNAs. Once libraries are prepared, they are sequenced and subsequently studied from a bioinformatics point of view. Among the different tools for identifying circRNAs, we can highlight CIRI as the most used (in 60% of the published studies), as well as CIRCExplorer (20%) and find_circ (20%). Although it is recommended to use more than one program in combination, and preferably developed specifically to treat with plant samples, this is not always the case. It should also be noted that after identifying these circular RNAs, most of the authors validate their findings in the laboratory in order to obtain bona fide results.

STUDY QUESTION Does endometrium harbour functionally active microorganisms and whether the microbial composition differs between proliferative and mid-secretory phases? SUMMARY ANSWER Endometrium harbours functionally alive microorganisms including bacteria, viruses, archaea and fungi whose composition and metabolic functions change along the menstrual cycle. WHAT IS KNOWN ALREADY Resident microbes in the endometrium have been detected, where microbial dysfunction has been associated with reproductive health and disease. Nevertheless, the core microorganismal composition in healthy endometrium is not determined and whether the identified bacterial DNA sequences refer to alive/functionally active microbes is not clear. Furthermore, whether there are cyclical changes in the microbial composition remains an open issue. STUDY DESIGN, SIZE, DURATION RNA sequencing (RNAseq) data from 14 endometrial paired samples from healthy women, 7 samples from the mid-secretory phase and 7 samples from the consecutive proliferative phase were analysed for the microbial RNA sequences. PARTICIPANTS/MATERIALS, SETTING, METHODS The raw RNAseq data were converted into FASTQ format using SRA Toolkit. The unmapped reads to human sequences were aligned to the reference database Kraken2 and visualised with Krona software. Menstrual phase taxonomic differences were performed by R package metagenomeSeq. The functional analysis of endometrial microbiota was obtained with HUMANn2 and the comparison between menstrual phases was conducted by one-way ANOVA. Human RNAseq analysis was performed using miARma-Seq and the functional enrichment analysis was carried out using gene set enrichment analysis (GSEA; HumanCyc). The integration of metabolic pathways between host and microbes was investigated. The developed method of active microbiota mapping was validated in independent sample set. MAIN RESULTS AND THE ROLE OF CHANCE With the novel metatranscriptomic approach, we mapped the entire alive microbiota composing of >5300 microorganisms within the endometrium of healthy women. Microbes such as bacteria, fungi, viruses and archaea were identified. The validation of three independent endometrial samples from different ethnicity confirmed the findings. Significant differences in the microbial abundances in the mid-secretory vs. proliferative phases were detected with possible metabolic activity in the host-microbiota crosstalk in receptive phase endometrium, specifically in the prostanoid biosynthesis pathway and L-tryptophan metabolism. LARGE SCALE DATA The raw RNAseq data used in the current study are available at GEO GSE86491 and at BioProject PRJNA379542. LIMITATIONS, REASONS FOR CAUTION These pioneering results should be confirmed in a bigger sample size. WIDER IMPLICATIONS OF THE FINDINGS Our study confirms the presence of active microbes, bacteria, fungi, viruses and archaea in the healthy human endometrium with implications in receptive phase endometrial functions, meaning that microbial dysfunction could impair the metabolic pathways important for endometrial receptivity. The results of this study contribute to the better understanding of endometrial microbiota composition in healthy women and its possible role in endometrial functions. In addition, our novel methodological pipeline for analysing alive microbes with transcriptional and metabolic activities could serve to inspire new analysis approaches in reproductive medicine., This work is supported by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526- R; FEDER/Junta de Andalucía-Consejería de Economía y Conocimiento: MENDO (B-CTS-500-UGR18) and by the University of Granada Plan Propio de Investigacio ́n 2016 - Excellence actions: Unit of Excellence on Exercise and Health (UCEES) (SOMM17/6107/UGR). A.S.-L. and N.M.M. are funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-0854409 and FPU19/01638). S.A. has received honoraria for lectures from Merck. The funder had no role in this study.

System sclerosis (SSc) is a genetically complex autoimmune disease mediated by the interplay between genetic and epigenetic factors in a multitude of immune cells, with CD4+ T lymphocytes as one of the principle drivers of pathogenesis. In this study, we obtained DNA methylation and expression profiles of CD4+ T cells from 48 SSc patients and 16 healthy controls. Consequently, we identified 9112 and 3929 differentially methylated CpGs positions (DMPs) and differentially expressed genes (DEGs) respectively. These DMPs and DEGs are enriched in functional categories related to inflammation and T cell biology. Furthermore, correlation analysis identified 17,500 possible DMP-DEG interaction pairs within a window of 5 Mb, and utilizing promoter capture Hi-C data, we confirmed that 212 CD4+ T cel specific pairs of DMP-DEG physically interact involving CTCF. Finally, utilizing SSc GWAS data, we identified four important SSc-associated susceptibility loci, TNIP1 (rs3792783), GSDMB (rs9303277), IL12RB1 (rs2305743) and CSK (rs1378942), that physically interact with DMP-DEG pairs cg17239269-ANXA6, cg19458020-CCR7, cg10808810-JUND and cg11062629-ULK3 respectively. Overall, our study reveals a solid link between genetic, epigenetic and transcriptional deregulation in CD4+ T cells of SSc patients, providing a novel integrated view of SSc pathogenic determinants.

One of the current greatest challenges of Chagas disease is the establishment of biomarkers to assess the efficacy of drugs in a short period of time. In this context, the reactivity of sera from 66 adults with chronic indeterminate Chagas disease (IND) for a set of four Trypanosoma cruzi antigens (KMP11, PFR2, HSP70, and 3973(d)) was analyzed before and after benznidazole treatment. The results showed that the reactivity against these antigens decreased at 9, 24, and 48 months after treatment. Moreover, the 42.4% and 68.75% of IND patients met the established standard criteria of therapeutic efficacy (STEC at 24 and 48 months posttreatment, respectively. Meeting the STEC implied that there was a continuous decrease in the reactivity of the patient sera against the four antigens after treatment and that there was a substantial decrease in the reactivity for at least two of the antigens. This important decrease in reactivity may be associated with a drastic reduction in the parasite load, but it is not necessarily associated with a parasitological cure. After treatment, a positive PCR result was only obtained in patients who did not meet the STEC. The percentage of granzyme B+ /perforin(+) CD8(+) T cells was significantly higher in patients who met the STEC than in those who did not meet the STEC (35.2% versus 2.2%; P < 0.05). Furthermore, the patients who met the STEC exhibited an increased quality of the multifunctional response of the antigen-specific CD8(+) T cells compared with that in the patients who did not meet the STEC., This work was financially supported by grants SAF2016-81003-R, SAF2016-80998-R, and RTC-2014-2130 from the Programa Estatal I + D + i (MINECO); the Network of Tropical Diseases Research RICET (RD16/0027/0005 and RD16/0027/0016); and FEDER. This publication is part of the PhD thesis of the student Elena Perez-Anton at the University of Granada in the Biomedicine Program.

Fusions transcripts have been proven to be strong drivers for neoplasia-associated mutations, although their incidence in T-cell lymphoblastic lymphoma needs to be determined yet. Using RNA-Seq we have selected 55 fusion transcripts identified by at least two of three detection methods in the same tumour. We confirmed the existence of 24 predicted novel fusions that had not been described in cancer or normal tissues yet, indicating the accuracy of the prediction. Of note, one of them involves the proto oncogene TAL1. Other confirmed fusions could explain the overexpression of driver genes such as COMMD3-BMI1, LMO1 or JAK3. Five fusions found exclusively in tumour samples could be considered pathogenic (NFYG-TAL1, RIC3-TCRBC2, SLC35A3-HIAT1, PICALM MLLT10 and MLLT10-PICALM). However, other fusions detected simultaneously in normal and tumour samples (JAK3-INSL3, KANSL1-ARL17A/B and TFG-ADGRG7) could be germ-line fusions genes involved in tumour-maintaining tasks. Notably, some fusions were confirmed in more tumour samples than predicted, indicating that the detection methods underestimated the real number of existing fusions. Our results highlight the potential of RNA-Seq to identify new cryptic fusions, which could be drivers or tumour-maintaining passenger genes. Such novel findings shed light on the searching for new T-LBL biomarkers in these haematological disorders., Spanish Ministry of Economy and Competitiveness (SAF2015-70561-R; MINECO/FEDER, EU); the Autonomous Community of Madrid, Spain (B2017/BMD-3778; LINFOMAS-CM) and the Spanish Association Against Cancer (AECC, 2018; PROYE18054PIRI). Institutional grants from the Fundación Ramón Areces and Banco de Santander