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Activation of the IKK kinase complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-kB has remained elusive. Analysis of IKK-dependent substrates after UV-treatment revealed that BRD4 phosphorylation by IKKa is required for chromatin-binding dynamics upon damage. Moreover, IKKa induces the NF-kB-dependent transcription of LIF leading to STAT3 activation, association of BRD4 to STAT3 and recruitment to specific target genes. IKKa abrogation results in defective BRD4 and STAT3 function leading to irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKKa activity and JAK/STAT pathway enhanced the therapeutic potential of 5-FU plus irinotecan in CRC patient-derived organoids and is curative in a chemotherapy-resistant CRC xenograft model. Coordinated expression of LIF and IKKa is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKKa, BRD4 and JAK/STAT signaling with clinical relevance.

Current therapeutic approaches for Sézary syndrome (SS) do not achieve a significant improvement in long-term survival of patients, and they are mainly focused on reducing blood tumor burden to improve quality of life. Eradication of SS is hindered by its genetic and molecular heterogeneity. Determining effective and personalized treatments for SS is urgently needed. The present work compiles the current methods for SS patient–derived xenograft (PDX) generation and management to provide new perspectives on treatment for patients with SS. Mononuclear cells were recovered by Ficoll gradient separation from fresh peripheral blood of patients with SS (N = 11). A selected panel of 26 compounds that are inhibitors of the main signaling pathways driving SS pathogenesis, including NF-kB, MAPK, histone deacetylase, mammalian target of rapamycin, or JAK/STAT, was used for in vitro drug sensitivity testing. SS cell viability was evaluated by using the CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated one positive hit using SS patient–derived Sézary cells xenotransplanted (PDX) into NOD-SCID-γ mice. In vitro data indicated that primary malignant SS cells all display different sensitivities against specific pathway inhibitors. In vivo validation using SS PDX mostly reproduced the responses to the histone deacetylase inhibitor panobinostat that were observed in vitro. Our investigations revealed the possibility of using high-throughput in vitro testing followed by PDX in vivo validation for selective targeting of SS tumor cells in a patient-specific manner.

SummaryRecent findings are challenging the classical hematopoietic model in which long-term hematopoietic stem cells (LT-HSC) are the base of the hematopoietic system. Clonal dynamics analysis of the hematopoietic system indicate that LT-HSC are not the main contributors of normal hemapoiesis in physiological conditions and the hematopoietic system is mainly maintained by multipotent progenitors (MPPs, hereafter HPC) and LT-HSCs are mostly in a non-active state. The first HSCs emerge from the aorta-gonad and mesonephros (AGM) region along with hematopoietic progenitors (HPC) within hematopoietic clusters. Molecular pathways that determine the HSC fate instead of HPC are still unknown, although inflammatory signaling, including NF-κB has been implicated in the development of HSCs. Here, we identify a chromatin binding function for IκBα (also known as the inhibitor of NF-κB) that is Polycomb repression complex 2 (PRC2)-dependent and specifically determines dormant vs proliferating HSCs from the onset of their emergence in the AGM. We find a specific reduction of LT-HSCs in the IκBα knockout new-born pups. This defect is manifested at the FL stage already, and traceable to the first emerging HSCs in the E11.5 AGM, without affecting the general HPC population. IκBα deficient LT-HSCs express dormancy signature genes, are less proliferative and can robustly respond to activation stimuli such asin vitroculture and serial transplantation. At the molecular level, we find decreased PRC2-dependent H3K27me3 at the promoters of several retinoic acid signaling elements in the IκBα - deficient aortic endothelium and E14.5 FL LT-HSCs. Additionally, IκBα binding itself is found in the promoters of retinoic acid receptors rarα in the AGM, and rarγ in the LT-HSC of FL. Overall, we demonstrate that the retinoic acid pathway is over-activated in the hematopoietic clusters of IκBα-deficient AGMs leading to premature dormancy of LT-HSCs that persists in the FL LT-HSCs.

Activation of the IKK kinase complex has recurrently been linked to colorectal cancer (CRC) initiation and progression. However, identification of downstream effectors other than NF-kB has remained elusive. Analysis of IKK-dependent substrates after UV-treatment revealed that BRD4 phosphorylation by IKKa is required for chromatin-binding dynamics upon damage. Moreover, IKKa induces the NF-kB-dependent transcription of LIF leading to STAT3 activation, association of BRD4 to STAT3 and recruitment to specific target genes. IKKa abrogation results in defective BRD4 and STAT3 function leading to irreparable DNA damage and apoptotic cell death upon different stimuli. Simultaneous inhibition of BRAF-dependent IKKa activity and JAK/STAT pathway enhanced the therapeutic potential of 5-FU plus irinotecan in CRC patient-derived organoids and is curative in a chemotherapy-resistant CRC xenograft model. Coordinated expression of LIF and IKKa is a poor prognosis marker for CRC patients. Our data uncover a functional link between IKKa, BRD4 and JAK/STAT signaling with clinical relevance.

Understanding the molecular mechanisms that contribute to the appearance of chemotherapy resistant cell populations is necessary to improve cancer treatment. We have now investigated the role of β-catenin/CTNNB1 in the evolution of T-Acute Lymphoblastic Leukemia (T-ALL) patients and its involvement in therapy resistance. We have identified a specific gene signature that is directly regulated by β-catenin, TCF/LEF factors and ZBTB33/Kaiso in T-ALL cell lines, which is highly and significantly represented in 5 out of 6 refractory patients from a cohort of 40 children with T-ALL. By subsequent refinement of this gene signature, we found that a subset of β-catenin target genes involved with RNA-processing function are sufficient to segregate T-ALL refractory patients in three independent cohorts. We demonstrate the implication of β-catenin in RNA and protein synthesis in T-ALL and provide experimental evidence that β-catenin is crucial for the cellular response to chemotherapy, mainly in the cellular recovery phase after treatment.We propose that combination treatments involving chemotherapy plus β-catenin inhibitors will enhance chemotherapy response and prevent disease relapse in T-ALL patients.

IκBs exert principal functions as cytoplasmic inhibitors of NF-kB transcription factors. Additional roles for IκB homologues have been described, including chromatin association and transcriptional regulation. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds to histones H2A and H4 in the stem cell and progenitor cell compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unknown. Here, we show that serine 32-36 phosphorylation of IκBα favors its binding to nucleosomes and demonstrate that p-IκBα association with H4 depends on the acetylation of specific H4 lysine residues. The N-terminal tail of H4 is removed during intestinal cell differentiation by proteolytic cleavage by trypsin or chymotrypsin at residues 17-19, which reduces p-IκBα binding. Inhibition of trypsin and chymotrypsin activity in HT29 cells increases p-IκBα chromatin binding but, paradoxically, impaired goblet cell differentiation, comparable to IκBα deletion. Taken together, our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular basis for the understanding of the restricted nuclear distribution of p-IκBα in specific stem cell compartments. We want to thank the Bigas’ and Espinosa’s lab members for constructive discussions and suggestions and technical support. This work has been funded by Instituto de Salud Carlos III FEDER (PI19/0013), Agencia Estatal de Investigación, Spain (PID2019-104695RB-I00) to A.B., NIH/GM085490 to G.G., BIO2017-83650-P to JVF and Generalitat de Catalunya 2017SGR135. LM is a predoctoral fellow of 2015FI-B00806 and 2016FI-B1 00110 and MF has financial support by the Universitat de Vic-Universitat Central de Catalunya PhD fellowships program. The authors thankfully acknowledge the computer resources at Pirineus and the technical support provided by the Spanish Supercomputer Network (BCV-2020-1-0001).

Current therapy against colorectal cancer is based on DNA-damaging agents that eradicate highly proliferative malignant cells. Whether sublethal chemotherapy affects tumor cell behavior and impacts on patient outcome is primarily unstudied. We now show that sublethal chemotherapy imposes a quiescent-like state to p53 wildtype human colorectal cancer (CRC) cells that is linked to the acquisition of a fetal phenotype downstream of YAP1, similar to that observed after intestinal damage. CRC cells displaying this fetal phenotype exhibit tumor- initiating activity comparable to untreated cells but superior metastatic capacity. Notably, nuclear YAP1 accumulation, or detection of the fetal signature in tumors predict poor prognosis in CRC patients carrying p53 wildtype tumors. Collectively, our results uncover a potential adverse response of tumor cells to suboptimal chemotherapy, and identify nuclear YAP1 and fetal conversion of colorectal tumors as biomarkers for prognosis and therapy prescription.Statement of significanceChemotherapy induces a quiescent-like phenotype to colorectal cancer cells that is linked to the acquisition of a YAP1-dependent fetal signature. Notably, this signature is predictive of patient outcome in different cohorts of human colorectal cancer.

IκBs exert a principal function as cytoplasmic inhibitors of the NF-kB transcription factors. Additional functions for IκB homologues have been described including association to chromatin and transcriptional regulatioin. Phosphorylated and SUMOylated IκBα (pS-IκBα) binds histones H2A and H4 in the stem and progenitor compartment of skin and intestine, but the mechanisms controlling its recruitment to chromatin are largely unstudied.We here show that serine 32-36 phosphorylation of IκBα favors its binding with nucleosomes and demonstrated that p-IκBα association to H4 is favored by acetylation at specific H4 lysine residues. N-terminal tail of H4 is lost during intestinal cell differentiation by proteolytic cleavage at residues 17-19 imposed ny trypsin or chymotrypsin, which interferes p-IκBα binding. Paradoxically, inhibition of trypsin and chymotrypsin activity in HT29 cells increased p-IκBα chromatin binding and impaired goblet cell differentiation, comparable to IκBα deletion. Together our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular base for the restricted nuclear distribution of p-IκBα at specific stem cell compartments.

Supplemental Digital Content is available in the text, HIV-associated periodontal diseases (PD) could serve as a source of chronic inflammation. Here, we sought to characterize the oral microbial signatures of HIV+ and HIV– individuals at different levels of PD severity. This cross-sectional study included both HIV+ and HIV– patients with varying degrees of PD. Two tooth, 2 cheek, and 1 saliva samples were obtained for microbiome analysis. Mothur/SILVADB were used to classify sequences. R/Bioconductor (Vegan, PhyloSeq, and DESeq2) was employed to assess overall microbiome structure differences and differential abundance of bacterial genera between groups. Polychromatic flow cytometry was used to assess immune activation in CD4 and CD8 cell populations. Around 250 cheek, tooth, and saliva samples from 50 participants (40 HIV+ and 10 HIV–) were included. Severity of PD was classified clinically as None/Mild (N), Moderate (M), and Severe (S) with 18 (36%), 16 (32%), and 16 (32%) participants in each category, respectively. Globally, ordination analysis demonstrated clustering by anatomic site (R2 = 0.25, P

Background Chromosomal inversions have been pervasive during the evolution of the genus Drosophila, but there is significant variation between lineages in the rate of rearrangement fixation. D. mojavensis, an ecological specialist adapted to a cactophilic niche under extreme desert conditions, is a chromosomally derived species with ten fixed inversions, five of them not present in any other species. Results In order to explore the causes of the rapid chromosomal evolution in D. mojavensis, we identified and characterized all breakpoints of seven inversions fixed in chromosome 2, the most dynamic one. One of the inversions presents unequivocal evidence for its generation by ectopic recombination between transposon copies and another two harbor inverted duplications of non-repetitive DNA at the two breakpoints and were likely generated by staggered single-strand breaks and repair by non-homologous end joining. Four out of 14 breakpoints lay in the intergenic region between preexisting duplicated genes, suggesting an adaptive advantage of separating previously tightly linked duplicates. Four out of 14 breakpoints are associated with transposed genes, suggesting these breakpoints are fragile regions. Finally two inversions contain novel genes at their breakpoints and another three show alterations of genes at breakpoints with potential adaptive significance. Conclusions D. mojavensis chromosomal inversions were generated by multiple mechanisms, an observation that does not provide support for increased mutation rate as explanation for rapid chromosomal evolution. On the other hand, we have found a number of gene alterations at the breakpoints with putative adaptive consequences that directly point to natural selection as the cause of D. mojavensis rapid chromosomal evolution.

Background: Previous studies in mice indicated that Paneth cells and c-Kit-positive goblet cells represent the stem cell niche of the small intestine and colon, respectively, partly by supporting Wnt and Notch activation. Whether these cell populations play a similar role in human intestinal cancer remains unexplored. Methods: We performed histopathological evaluation and immunohistochemical analysis of early colorectal adenomas and carcinoma adenoma from patients at the Hospital del Mar in Barcelona. We then determined the possible correlation between the different parameters analyzed and with patient outcomes. Results: Paneth cells accumulate in a subset of human colorectal adenomas directly associated with Notch and Wnt/β-catenin activation. Adenoma areas containing Paneth cells display increased vessel density in the lamina propria and higher levels of the stem cell marker EphB2. In an in-house cohort of 200 colorectal adenoma samples, we also observed a significant correlation between the presence of Paneth cells and Wnt activation. Kaplan–Meier analysis indicated that early adenoma patients carrying Paneth cell-positive tumors display reduced disease-free survival compared with patients with Paneth cell-free lesions. Conclusions: Our results indicate that Paneth cells contribute to the initial steps of cancer progression by providing the stem cell niche to adenoma cells, which could be therapeutically exploited.

Increasing evidence supports a potential role for STAT3 as a tumor driver in cutaneous T-cell lymphomas (CTCL). The mechanisms leading to STAT3 activation are not fully understood; however, we recently found that miR-124, a known STAT3 regulator, is robustly silenced in MF tumor-stage and CTCL cells. Objective: We studied here whether deregulation of miR-124 contributes to STAT3 pathway activation in CTCL. Methods: We measured the effect of ectopic mir-124 expression in active phosphorylated STAT3 (p-STAT3) levels and evaluated the transcriptional impact of miR-124-dependent STAT3 pathway regulation by expression microarray analysis. Results: We found that ectopic expression of miR-124 results in massive downregulation of activated STAT3 in different CTCL lines, which resulted in a significant alteration of genetic signatures related with gene transcription and proliferation such as MYC and E2F. Conclusions: Our study highlights the importance of the miR-124/STAT3 axis in CTCL and demonstrates that the STAT3 pathway is regulated through epigenetic mechanisms in these cells. Since deregulated STAT3 signaling has a major impact on CTCL initiation and progression, a better understanding of the molecular basis of the miR-124/STAT3 axis may provide useful information for future personalized therapies.

Background: The identification of new biomarkers is essential to making it possible to predict the degree of protection following vaccination. Very few human studies have focused on baseline characteristics including microbiota and gene expression to underlie vaccine immune responsiveness. We investigated the host whole-blood transcriptome and microbiome before vaccination, to assess the likelihood of their involvement in an effective MVA-neutralizing antibody response (MVA-Nab) two months later. Results: We based our analyses on data obtained ­from a randomized clinical study in which participants (n=10) were vaccinated with the MVA-HIV clade B vaccine (MVA-B). Samples were collected at 2-time points prior vaccination (week-2, w0) to study blood transcriptome. Skin wrap (site of vaccination) and stool microbiota were analysed for diversity and abundance (16S rRNAseq). MVA-neutralizing antibody responses were measured at week 8. The levels of MVA-Nab responses were positively correlated with an abundance of Eubacterium in stool and Prevotella in skin. The simultaneous investigation of blood transcriptome and host microbiota before vaccination showed that genus diversity and bacterial abundance at that time correlated significantly with the expression of genes involved in B cell development stages. The combination of gene expression and microbiota makes it possible to forecast strong responders to MVA-B vaccination.Conclusion: To our knowledge, this is the first study integrating host blood gene expression and microbiota before vaccination to predict the intensity of humoral response months later. The genes identified are involved in B cell differentiation might open an avenue of research in this field to optimize vaccination strategies.

The intestinal epithelium is a paradigm of adult tissue in constant regeneration that is supported by intestinal stem cells (ISCs). The mechanisms regulating ISC homeostasis after injury are poorly understood. We previously demonstrated that IκBα, the main regulator of NF‐κB, exerts alternative nuclear functions as cytokine sensor in a subset of PRC2‐regulated genes. Here, we show that nuclear IκBα is present in the ISC compartment. Mice deficient for IκBα show altered intestinal cell differentiation with persistence of a fetal‐like ISC phenotype, associated with aberrant PRC2 activity at specific loci. Moreover, IκBα‐deficient intestinal cells produce morphologically aberrant organoids carrying a PRC2‐dependent fetal‐like transcriptional signature. DSS treatment, which induces acute damage in the colonic epithelium of mice, results in a temporary loss of nuclear P‐IκBα and its subsequent accumulation in early CD44‐positive regenerating areas. Importantly, IκBα‐deficient mice show higher resistance to damage, likely due to the persistent fetal‐like ISC phenotype. These results highlight intestinal IκBα as a chromatin sensor of inflammation in the ISC compartment.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions. We thank Thomas Graf (CRG), Minhong Yan (Genentech), Georges Lacaud (CRUK Manchester Institut) and Ralf Adams (Max Planck Institute for Molecular Medicine, Münster) for important mice models and reagents. Thanks to Catherine Robin, Timo Zimmermann and, Raul Gómez-Riera for imaging advice. Thanks to the PRBB facilities: animal facility, flow cytometry, CNAGCRG genomics, confocal microscopy for critical technical assistance. Thanks to Marta Garrido and Luis Galán for experimental support and all the members of the lab for helpful critical discussions. This research was funded by the Ministerio de Economía y Competitividad (SAF2016 -75613-R) and Generalitat de Catalunya, Agència de Gestió d’Ajuds Universitaris i de Recerca (AGAUR) (2017 SGR 135) and PERIS-SLT002/16/00299 to AB and ERC AdG 341096 to ED. CP was a recipient of Juan de la Cierva fellowship (FJCI2014-19870), RT is a recipient of Beatriu de Pinos (2016 BP 00021), JG is a recipient of PERIS (SLT002/16/00070).

SummaryMammalian IκB proteins (IκBs) exert their main function as negative regulators of NF-κB, a central signaling pathway controlling immunity and inflammation. An alternative chromatin role for IκBs has been shown to affect stemness and cell differentiation. However, the involvement of NF-κB in this function has not been excluded. NFKI-1 and IKB-1 are IκB homologs inCaenorhabditis elegans, which lacks NF-κB nuclear effectors. We found thatnfki-1andikb-1mutants display developmental defects that phenocopy mutations in Polycomb and UTX-1 histone demethylase, suggesting a role forC. elegansIκBs in chromatin regulation. Further supporting this possibility(i)we detected NFKI-1 in the nucleus of cells;(ii)NFKI-1 and IKB-1 bind to histones and Polycomb proteins,(iii)and associate with chromatinin vivo, and(iv)mutations innfki-1andikb-1alter chromatin marks. Based on these results, we propose that ancestral IκB inhibitors modulate Polycomb activity at specific gene subsets with an impact on development.

Evolutionary rates for protein-coding genes are determined not only by natural selection but also by multiple genomic factors including mutation rates, recombination, gene expression levels, and chromosomal location. To investigate the joint effects of different genomic determinants on protein evolution, we compared the coding sequences of 9017 single-copy orthologs between 2 cactophilic species from the Drosophila subgenus, Drosophila mojavensis and D. buzzatii, whose genomes have been previously sequenced. We assessed the impact of 7 genomic determinants, that is, chromosome type, recombination, chromosomal inversions, expression breadth, expression level, gene length, and the number of exons, on divergence rates of protein-coding genes to understand patterns of evolutionary variation. Integrative analysis of these factors revealed that 1) X-linked and autosomal genes evolve at significantly different rates in agreement with the faster-X hypothesis, 2) genes located on the dot chromosome and pericentromeric regions have higher divergence rates, 3) genes located at chromosomes with more fixed inversions have higher pairwise divergence than those located at nearly collinear chromosomes, and 4) gene expression patterns can be considered the strongest determinant of protein evolution. In addition, the number of exons and protein length had a significant effect on pairwise divergence at synonymous sites. All in all, our results show the relative importance of each genomic factor on the rates of protein evolution and functional constraint in these 2 cactophilic Drosophila species.

β-Catenin/CTNNB1 is critical for leukemia initiation or the stem cell capacity of several hematological malignancies. This review focuses on a general evaluation of β-catenin function in normal T-cell development and T-cell acute lymphoblastic leukemia (T-ALL). The integration of the existing literature offers a state-of-the-art dissection of the complexity of β-catenin function in leukemia initiation and maintenance in both Notch-dependent and independent contexts. In addition, β-catenin mutations are screened for in T-ALL primary samples, and it is found that they are rare and with little clinical relevance. Transcriptional analysis of Wnt family members (Ctnnb1, Axin2, Tcf7, and Lef1) and Myc in different publicly available T-ALL cohorts indicates that the expression of these genes may correlate with T-ALL subtypes and/or therapy outcomes.

Background In rhesus macaques, simian immunodeficiency virus infection is followed by expansion of enteric viruses but has a limited impact on the gut bacteriome. To understand the longitudinal effects of HIV-1 infection on the human gut microbiota, we prospectively followed 49 Mozambican subjects diagnosed with recent HIV-1 infection (RHI) and 54 HIV-1-negative controls for 9–18 months and compared them with 98 chronically HIV-1-infected subjects treated with antiretrovirals (n = 27) or not (n = 71). Results We show that RHI is followed by increased fecal adenovirus shedding, which persists during chronic HIV-1 infection and does not resolve with ART. Recent HIV-1 infection is also followed by transient non-HIV-specific changes in the gut bacterial richness and composition. Despite early resilience to change, an HIV-1-specific signature in the gut bacteriome—featuring depletion of Akkermansia, Anaerovibrio, Bifidobacterium, and Clostridium—previously associated with chronic inflammation, CD8+ T cell anergy, and metabolic disorders, can be eventually identified in chronically HIV-1-infected subjects. Conclusions Recent HIV-1 infection is associated with increased fecal shedding of eukaryotic viruses, transient loss of bacterial taxonomic richness, and long-term reductions in microbial gene richness. An HIV-1-associated microbiome signature only becomes evident in chronically HIV-1-infected subjects. Electronic supplementary material The online version of this article (10.1186/s40168-019-0687-5) contains supplementary material, which is available to authorized users.

Human immunodeficiency virus (HIV)-1 infection causes severe gut and systemic immune damage, but its effects on the gut microbiome remain unclear. Previous shotgun metagenomic studies in HIV-negative subjects linked low-microbial gene counts (LGC) to gut dysbiosis in diseases featuring intestinal inflammation. Using a similar approach in 156 subjects with different HIV-1 phenotypes, we found a strong, independent, dose-effect association between nadir CD4+ T-cell counts and LGC. As in other diseases involving intestinal inflammation, the gut microbiomes of subjects with LGC were enriched in gram-negative Bacteroides, acetogenic bacteria and Proteobacteria, which are able to metabolize reactive oxygen and nitrogen species; and were depleted in oxygen-sensitive methanogenic archaea and sulfate-reducing bacteria. Interestingly, subjects with LGC also showed increased butyrate levels in direct fecal measurements, consistent with enrichment in Roseburia intestinalis despite reductions in other butyrate producers. The microbiomes of subjects with LGC were also enriched in bacterial virulence factors, as well as in genes associated with beta-lactam, lincosamide, tetracycline, and macrolide resistance. Thus, low nadir CD4+ T-cell counts, rather than HIV-1 serostatus per se, predict the presence of gut dysbiosis in HIV-1 infected subjects. Such dysbiosis does not display obvious HIV-specific features; instead, it shares many similarities with other diseases featuring gut inflammation. Fundació Glòria Soler Fundació Catalunya-La Pedrera Gala SIDA 2015-2016 Nit per la Recerca a la Catalunya Central 2015 edition People in Red-Barcelona 2016 edition RED de SIDA RD16/0025/0041 ISCIII European Regional Develpment Fund (ERDF) Agencia de Gestio d´Ajuts Universitaris i de Recerca (AGAUR) Secretaria d´Universitats i Recerca del Departament d´Economia i Coneixement de la Generalitat de Catalunya Ministerio de Economia y Competitividad. España Universidad de Whashington

The intestinal epithelium is a paradigm of adult tissue in constant regeneration that is supported by intestinal stem cells (ISC). The mechanisms regulating ISC homeostasis after injury are poorly understood. We previously demonstrated that IκBα, not only controls NF-κB activation, but also exerts nuclear functions as cytokine sensor in a subset of PRC2-regulated genes. We have now detected nuclear phosphorylated IκBα (P-IκBα) in the ISC compartment where it binds highly histone methylated genomic regions. Mice deficient for IκBα show aberrant distribution of the H3K27me3 mark, and altered intestinal cell differentiation with persistence of a fetal-like ISC gene expression signature. In vitro, IκBα deficient intestinal cells produce morphologically aberrant organoids carrying a PRC2, Notch and IFN-dependent fetal-like transcriptional signature. Induction of the fetal-like phenotype by DSS treatment is associated with loss of nuclear P-IκBα in the damaged colonic epithelium and its subsequent accumulation in early CD44 positive regenerating areas. Importantly, IκBα deficient animals show higher resistance to damage, likely due to the persistent fetal-like phenotype. These results point out intestinal IκBα as a chromatin sensor of inflammation in the ISC compartment.

Alternative splicing is one of the multiple post-transcriptional mechanisms that contribute to functional diversification in eukaryotic cells. Cancer-associated alternative splicing events have been reported in different tumor types, establishing new putative targets for diagnostic tools and therapies. Here, we explore the transcriptional program at exon resolution of T-Cell Acute Lymphoblastic Leukemia (T-ALL), an aggressive hematological malignancy characterized by the outgrowth of immature T-cells. To date, we have characterized the alternative splicing configuration of more than 120 RNA-Seq samples from four different T-ALL cohorts, normal T-cell maturation stages, and two cell lines, using vastdb. Exon skipping and intron retention are the most frequent events across all samples, with similar incidence in both cancer driver and non-driver genes. Interestingly, we observe that intron retention is significantly higher in immature and mature T-cells compared to leukemic cells. Conversely, exon skipping is higher in leukemic samples. These results suggest an impairment in the splicing machinery that might be driven by RNA binding proteins (RBPs) deregulation in leukemia. To further investigate that, we aim to discover which are the RBPs associated with isoform changes by constructing co-expression networks. The computational assessment of T-ALL specific isoforms will eventually help to improve the diagnosis, as well as the discovery of putative neoantigens targeted by CAR therapy.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease comprising up to 25% of all acute leukemia cases. Despite intensive chemotherapy, up to 50% of T-ALL patients fail to achieve a complete remission, making relapse and resistance to therapy the greatest challenge in the treatment of this disease. Activating mutations in Notch are the most prevalent genetic alterations in T-ALL. However, genomic studies suggest that other signaling pathways are also pivotal. We previously showed that β-catenin, a key element in hematopoietic stem cell regulation, is required for T-cell leukemia initiation through Myc transcription modulation downstream of Notch1. To further explore the molecular mechanisms that underlie β-catenin dependent leukemia initiation and maintenance, here we take a comprehensive genomic strategy integrating: 1) the chromatin occupancy of β-catenin and its associated transcription factors TCF and LEF, in T-ALL cell lines by ChIP-Seq, including external data in different models, 2) the transcriptomic map of cells upon β-catenin gene silencing by RNA-Seq, and 3) a gene co-expression network using transcriptome data from public T-ALL primary samples. We identified almost 400 DNA-binding sites located in regulatory regions for β-catenin, co-occupied with TCF and LEF in T-ALL cell lines and, intriguingly, highly enriched for ZBTB33 DNA motif. Interestingly, our data showed a prominent role of β-catenin in regulating RNA processing functions. Also, we further characterize β-catenin modulated genes in different leukemic mouse models. Surprisingly, we detected a low coincidence of β-catenin chromatin binding as compared to human cell lines. Altogether these results allow for a better comprehension of β-catenin activity and its essentiality in T-ALL leukemogenesis, and eventually the opportunity to design β-catenin target therapies for T-ALL treatment.

The impact of host genetics on gut microbial dynamics is debated. No study to date has investigated the possible role of host genetics in shaping the gut microbiota in HIV-1 infected subjects. With the aim of generating preliminary data to inform future host genetic studies, we performed an exploratory host exome analysis of 147 subjects either infected or at risk of becoming infected with HIV-1 from the MetaHIV cohort in Barcelona. Using a DNA microarray chip, we sought to identify host genetic variants associated to three specific microbial features with a potentially inheritable component, and which were previously found to be associated with gut dysbiosis in HIV infection, i.e.: gut enterotype, presence of methanogenic archaea and microbial gene richness. After correction for multiple comparisons, we did not observe any statistically significant association between the host’s genetic landscape and the explored gut microbiome traits. These findings will help design future, adequately-powered studies to assess the influence of host genetics in the microbiome of HIV-1-infected subjects.

We investigated rates of chromosomal evolution in Drosophila mojavensis using whole-genome sequence information from D. mojavensis, Drosophila buzzatii, and Drosophila virilis. Drosophila mojavensis is a cactophilic species of the repleta group living under extreme ecological conditions in the deserts of the Southwestern United States and Northwestern Mexico. The genome of D. buzzatii, another member of the repleta group, was recently sequenced and the largest scaffolds anchored to all chromosomes using diverse procedures. Chromosome organization between D. mojavensis and D. buzzatii was compared using MUMmer and GRIMM software. Our results corroborate previous cytological analyses that indicated chromosome 2 differed between these 2 species by 10 inversions, chromosomes X and 5 differed by one inversion each, and chromosome 4 was homosequential. In contrast, we found that chromosome 3 differed by 5 inversions instead of the expected 2 that were previously inferred by cytological analyses. Thirteen of these inversions occurred in the D. mojavensis lineage: 12 are fixed and one of them is a polymorphic inversion previously described in populations from Sonora and Baja California, Mexico. We previously investigated the breakpoints of chromosome 2 inversions fixed in D. mojavensis. Here we characterized the breakpoint regions of the 5 inversions found in chromosome 3 in order to infer the molecular mechanism that generated each inversion and its putative functional consequences. Overall, our results reveal a number of gene alterations at the inversion breakpoints with putative adaptive consequences that point to natural selection as the cause for fast chromosomal evolution in D. mojavensis.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in the embryonic aorta. In most vertebrates, hemogenic cells protrude into the aortic lumen forming clusters where the hematopoietic features are acquired. Here we used advanced live imaging techniques to visualize the origin and 3D-organization of intraortic hematopoietic clusters (IAHC) in the mouse embryo and how they evolve over time. Using organotypic slice cultures, clonal analysis and mathematical modelling we show that IAHC formation is a two-step process. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the cluster. Next, cells from the surrounding hemogenic endothelium are also recruited into the IAHCs, increasing their size and heterogeneity. We found that the recruitment phase of IAHC formation is negatively controlled by a Notch and Delta-like-4 (Dll4) and its blockage promotes the entrance of new hemogenic Gfi1+ cells into the IAHC thus increasing the number of cells that acquire HSC activity. Mathematical modelling based on our data estimates the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions. Upon blockage of Dll4 the recruitment time was dramatically decreased, with changes in the number of cells composing a cluster. Transcriptome analysis at single cell indicates that cell identities are preserved after Dll4 blockage. Our data provides for the first time a detailed characterization of the aortic hematopoietic stem cell niche and describe its process of maturation, identifying the Notch ligand Dll4 as a major molecular player governing cluster formation and definitive HSCs production.

Altres ajuts: This work was supported by grant R01GM077582 to C.F from the National Institutes of Health, and by PIF-UAB fellowship to N.R. Many new Drosophila genomes have been sequenced in recent years using new-generation sequencing platforms and assembly methods. Transposable elements (TEs), being repetitive sequences, are often misassembled, especially in the genomes sequenced with short reads. Consequently, the mobile fraction of many of the new genomes has not been analyzed in detail or compared with that of other genomes sequenced with different methods, which could shed light into the understanding of genome and TE evolution. Here we compare the TE content of three genomes: D. buzzatii st-1, j-19, and D. mojavensis. We have sequenced a new D. buzzatii genome (j-19) that complements the D. buzzatii reference genome (st-1) already published, and compared their TE contents with that of D. mojavensis. We found an underestimation of TE sequences in Drosophila genus NGS-genomes when compared to Sanger-genomes. To be able to compare genomes sequenced with different technologies, we developed a coverage-based method and applied it to the D. buzzatii st-1 and j-19 genome. Between 10.85 and 11.16 % of the D. buzzatii st-1 genome is made up of TEs, between 7 and 7,5 % of D. buzzatii j-19 genome, while TEs represent 15.35 % of the D. mojavensis genome. Helitrons are the most abundant order in the three genomes. TEs in D. buzzatii are less abundant than in D. mojavensis, as expected according to the genome size and TE content positive correlation. However, TEs alone do not explain the genome size difference. TEs accumulate in the dot chromosomes and proximal regions of D. buzzatii and D. mojavensis chromosomes. We also report a significantly higher TE density in D. buzzatii and D. mojavensis X chromosomes, which is not expected under the current models. Our easy-to-use correction method allowed us to identify recently active families in D. buzzatii st-1 belonging to the LTR-retrotransposon superfamily Gypsy. The online version of this article (doi:10.1186/s12864-016-2648-8) contains supplementary material, which is available to authorized users.

Cactophilic Drosophila species provide a valuable model to study gene–environment interactions and ecological adaptation. Drosophila buzzatii and Drosophila mojavensis are two cactophilic species that belong to the repleta group, but have very different geographical distributions and primary host plants. To investigate the genomic basis of ecological adaptation, we sequenced the genome and developmental transcriptome of D. buzzatii and compared its gene content with that of D. mojavensis and two other noncactophilic Drosophila species in the same subgenus. The newly sequenced D. buzzatii genome (161.5 Mb) comprises 826 scaffolds (>3 kb) and contains 13,657 annotated protein-coding genes. Using RNA sequencing data of five life-stages we found expression of 15,026 genes, 80% protein-coding genes, and 20% noncoding RNA genes. In total, we detected 1,294 genes putatively under positive selection. Interestingly, among genes under positive selection in the D. mojavensis lineage, there is an excess of genes involved in metabolism of heterocyclic compounds that are abundant in Stenocereus cacti and toxic to nonresident Drosophila species. We found 117 orphan genes in the shared D. buzzatii–D. mojavensis lineage. In addition, gene duplication analysis identified lineage-specific expanded families with functional annotations associated with proteolysis, zinc ion binding, chitin binding, sensory perception, ethanol tolerance, immunity, physiology, and reproduction. In summary, we identified genetic signatures of adaptation in the shared D. buzzatii–D. mojavensis lineage, and in the two separate D. buzzatii and D. mojavensis lineages. Many of the novel lineage-specific genomic features are promising candidates for explaining the adaptation of these species to their distinct ecological niches., This work was supported by grants BFU2008-04988 and BFU2011-30476 from Ministerio de Ciencia e Innovación (Spain) to A.R., by an FPI fellowship to Y.G. and a PIF-UAB fellowship to N.R, and by the National Institute of General Medical Sciences of the National Institute of Health under award number R01GM071813 to E.B.

Bartonella bacilliformis is the causative agent of Carrion's disease, a highly endemic human bartonellosis in Peru. We performed a whole-genome assembly of two B. bacilliformis strains isolated from the blood of infected patients in the acute phase of Carrion's disease from the Cusco and Piura regions in Peru.

Introducción: La secuenciación masiva (NGS, Next generation sequencing) nos permite conocer la composición genética de un microorganismo, mediante la secuenciación completa de su genoma, o la composición de un conjunto de microorganismos presentes en una muestra, gracias a la metagenómica. Existen diferentes bases de datos y herramientas bioinformáticas para la búsqueda de genes de resistencia en datos obtenidos de NGS. El objetivo del estudio es comparar dos de las herramientas más utilizadas, ResFinder y Resistance Gene Identifier (RGI) así como sus bases de datos correspondientes, ResFinder y The Comprehensive Antimicrobial Resistance Database (CARD). Método: Se seleccionaron 3 conjuntos de datos de la siguiente manera: I) Secuencias de genes de resistencia a antimicrobianos de referencia, II) Datos de un único genoma completo, III) Datos de un metagenoma. Se realizó una comparativa de la composición de las bases de datos, del número de genes de referencia detectados y de la diversidad de estos en cada uno de los conjuntos de datos. Resultados: CARD tiene un mayor número de secuencias de referencia, ya que esta incluye genes cromosómicos y adquiridos, a diferencia de RestFinder que solo incluye adquiridos. En todos los conjuntos de datos RGI detecta un mayor número de genes de resistencia, si bien, en el set de genes de referencia se identificaron correctamente los mismos por ambas técnicas. Conclusión: La comparación de herramientas bioinformáticas para la detección de genes de resistencia nos permite conocer las ventajas e inconvenientes del uso de diferentes bases de datos y así poder realizar un análisis de los resultados más adecuado y no llegar a conclusiones erróneas. Introduction: Next generation sequencing (NGS) allows us to know the complete genetic sequence of a single isolate microorganism using the Whole Genome Sequencing, or microorganisms present in a complex sample, thanks to metagenomics. There are several databases and bioinformatics tools to search resistance genes in NGS data. The aim of this study was to compare two of the most used tools, ResFinder and Resistance Gene Identifier (RGI) and their respective databases, ResFinder and The Comprehensive Antimicrobial Resistance Database (CARD). Method: Three datasets were selected as follows: I) Antimicrobial resistance reference gene sequences; II) Whole genome sequencing data; III) Metagenomics sequencing data. A comparison between both databases and software was performed studying the composition of each database, number of reference genes detected and the diversity of this genes in their respective dataset. Results: CARD has a higher number of reference sequences because this database includes both chromosomic and acquired genes compared to RestFinder that only includes acquired genes. Both RGI and ResFinder identified correctly the same number of genes in the reference gene set. However, RGI detected more genes using the NGS data. Conclusion: The comparison of bioinformatics tools used for resistance gene detection allows us to know their advantages and disadvantages and, thanks to this, we can get to better approaches and to carry out a more exhaustive analysis avoid reaching to wrong conclusions.