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The generation of functional regulatory T cells (Tregs) is essential to keep tissue homeostasis and restore healthy immune responses in many biological and inflammatory contexts. Cannabinoids have been pointed out as potential therapeutic tools for several diseases. Dendritic cells (DCs) express the endocannabinoid system, including the cannabinoid receptors CB1 and CB2. However, how cannabinoids might regulate functional properties of DCs is not completely understood. We uncover that the triggering of cannabinoid receptors promote human tolerogenic DCs that are able to prime functional FOXP3+ Tregs in the context of different inflammatory diseases. Mechanistically, cannabinoids imprint tolerogenicity in human DCs by inhibiting NF-κB, MAPK and mTOR signalling pathways while inducing AMPK and functional autophagy flux via CB1- and PPARα-mediated activation, which drives metabolic rewiring towards increased mitochondrial activity and oxidative phosphorylation. Cannabinoids exhibit in vivo protective and anti-inflammatory effects in LPS-induced sepsis and also promote the generation of FOXP3+ Tregs. In addition, immediate anaphylactic reactions are decreased in peanut allergic mice and the generation of allergen-specific FOXP3+ Tregs are promoted, demonstrating that these immunomodulatory effects take place in both type 1- and type 2-mediated inflammatory diseases. Our findings might open new avenues for novel cannabinoid-based interventions in different inflammatory and immune-mediated diseases.

Purinergic signaling plays a pivotal role in physiological processes and pathological conditions. Over the past decades, conventional pharmacological, biochemical, and molecular biology techniques have been utilized to investigate purinergic signaling cascades. However, none of them is capable of spatially and temporally manipulating purinergic signaling cascades. Currently, optical approaches, including optopharmacology and optogenetic, enable controlling purinergic signaling with low invasiveness and high spatiotemporal precision. In this mini-review, we discuss optical approaches for controlling purinergic signaling and their applications in basic and translational science.

Background Allergoid–mannan conjugates are novel vaccines for allergen-specific immunotherapy being currently assayed in phase 2 clinical trials. Allergoid–mannan conjugates target dendritic cells (DCs) and generate functional forkhead box P3 (FOXP3)-positive Treg cells, but their capacity to reprogram monocyte differentiation remains unknown. Objective We studied whether allergoid–mannan conjugates could reprogram monocyte differentiation into tolerogenic DCs and the underlying molecular mechanisms. Methods Monocytes from nonatopic and allergic subjects were differentiated into DCs under conventional protocols in the absence or presence of allergoid–mannan conjugates. ELISA, real-time quantitative PCR, coculture, flow cytometry, and suppression assay were performed. Metabolic and epigenetic techniques were also used. Results Monocyte differentiation from nonatopic and allergic subjects into DCs in the presence of allergoid–mannan conjugates yields stable tolerogenic DCs. Lipopolysaccharide-stimulated mannan-tolDCs show a significantly lower cytokine production, lower TNF-α/IL-10 ratio, and higher expression of the tolerogenic molecules PDL1, IDO, SOCS1, SOCS3, and IL10; and they induce higher numbers of functional FOXP3+ Treg cells than conventional DC counterparts. Mannan-tolDCs shift glucose metabolism from Warburg effect and lactate production to mitochondrial oxidative phosphorylation. They also display epigenetic reprogramming involving specific histone marks within tolerogenic loci and lower expression levels of histone deacetylase genes. Mannan-tolDCs significantly increase the expression of the anti-inflammatory miRNA-146a/b and decrease proinflammatory miRNA-155. Conclusions Allergoid–mannan conjugates reprogram monocyte differentiation into stable tolerogenic DCs via epigenetic and metabolic reprogramming. Our findings shed light on the novel mechanisms by which allergoid–mannan conjugates might contribute to allergen tolerance induction during allergen-specific immunotherapy.

Graphical abstract, Engineered immunoglobulin-G molecules (IgGs) are of wide interest for the development of detection elements in protein-based biosensors with clinical applications. The strategy usually employed for the de novo design of such engineered IgGs consists on merging fragments of the three-dimensional structure of a native IgG, which is immobilized on the biosensor surface, and of an antibody with an exquisite target specificity and affinity. In this work conventional and accelerated classical molecular dynamics (cMD and aMD, respectively) simulations have been used to propose two IgG-like antibodies for COVID-19 detection. More specifically, the crystal structure of the IgG1 B12 antibody, which inactivates the human immunodeficiency virus-1, has been merged with the structure of the antibody CR3022 Fab tightly bounded to SARS-CoV-2 receptor-binding domain (RBD) and the structure of the S309 antibody Fab fragment complexed with SARS-CoV-2 RBD. The two constructed antibodies, named IgG1-CR3022 and IgG1-S309, respectively, have been immobilized on a stable gold surface through a linker. Analyses of the influence of both the merging strategy and the substrate on the stability of the two constructs indicate that the IgG1-S309 antibody better preserves the neutralizing structure than the IgG1-CR3022 one. Overall, results indicate that the IgG1-S309 is appropriated for the generation of antibody based sensors for COVID-19 diagnosis.

Summary RNA-based sensors for intracellular metabolites are a promising solution to the emerging issue of metabolic heterogeneity. However, their development, i.e., the conversion of an aptamer into an in vivo-functional intracellular metabolite sensor, still harbors challenges. Here, we accomplished this for the glycolytic flux-signaling metabolite, fructose-1,6-bisphosphate (FBP). Starting from in vitro selection of an aptamer, we constructed device libraries with a hammerhead ribozyme as actuator. Using high-throughput screening in yeast with fluorescence-activated cell sorting (FACS), next-generation sequencing, and genetic-environmental perturbations to modulate the intracellular FBP levels, we identified a sensor that generates ratiometric fluorescent readout. An abrogated response in sensor mutants and occurrence of two sensor conformations—revealed by RNA structural probing—indicated in vivo riboswitching activity. Microscopy showed that the sensor can differentiate cells with different glycolytic fluxes within yeast populations, opening research avenues into metabolic heterogeneity. We demonstrate the possibility to generate RNA-based sensors for intracellular metabolites for which no natural metabolite-binding RNA element exits.

The coating composition of nanomedicines is one of the main features in determining the medicines' fate, clearance, and immunoresponse in the body. To highlight the coatings' impact in pulmonary administration, two micellar superparamagnetic iron oxide nanoparticles (SPION) were compared. These nanoparticles are similar in size and charge but have different coatings: either phosphatidylcholine (PC-SPION) or bovine serum albumin (BSA-SPION). The aim of the study was to increase the understanding of the nano-bio interaction with the cellular and non-cellular components of the lung and underline valuable coatings either for local lung-targeted drug delivery in theranostic application or patient-friendly route systemic administration. PC-SPION and BSA-SPION were deposited in the alveoli by in vivo instillation and, despite the complexity of imaging the lung, SPION were macroscopically visualized by MRI. Impressively, PC-SPION were retained within the lungs for at least a week, while BSA-SPION were cleared more rapidly. The different lung residence times were confirmed by histological analysis and supported by a flow cytometry analysis of the SPION interactions with different myeloid cell populations. To further comprehend the way in which these nanoformulations interact with lung components at the molecular level, we used fluorescence spectroscopy, turbidity measurements, and dynamic light scattering to evaluate the interactions of the two SPION with surfactant protein A (SP-A), a key protein in setting up the NP behavior in the alveolar fluid. We found that SP-A induced aggregation of PC-SPION, but not BSA-SPION, which likely caused PC-SPION retention in the lung without inducing inflammation. In conclusion, the two SPION show different outcomes from interaction with SP-A leading to distinctive fate in the lung. PC-SPION hold great promise as imaging and theranostic agents when prolonged pulmonary drug delivery is required.

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function, mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

Type 2 diabetes mellitus (T2DM) is associated with impaired skeletal muscle function and degeneration of the skeletal muscles. However, the mechanisms underlying the degeneration are not well described in human skeletal muscle. Here we show that skeletal muscle of T2DM patients exhibit degenerative remodeling of the extracellular matrix that is associated with a selective increase of a subpopulation of fibro-adipogenic progenitors (FAPs) marked by expression of THY1 (CD90)—the FAPCD90+. We identify platelet-derived growth factor (PDGF) as a key FAP regulator, as it promotes proliferation and collagen production at the expense of adipogenesis. FAPsCD90+ display a PDGF-mimetic phenotype, with high proliferative activity, clonogenicity, and production of extracellular matrix. FAPCD90+ proliferation was reduced by in vitro treatment with metformin. Furthermore, metformin treatment reduced FAP content in T2DM patients. These data identify a PDGF-driven conversion of a subpopulation of FAPs as a key event in the fibrosis development in T2DM muscle. Sin financiación 31.373 JCR (2021) Q1, 8/195 Cell Biology 9.297 SJR (2021) Q1, 9/293 Cell Biology No data IDR 2021 UEM

Besides their direct effects on peripheral metabolic tissues, thyroid hormones (TH) act on the hypothalamus to modulate energy homeostasis. However, since most of the hypothalamic actions of TH have been addressed in studies with direct central administration, the estimation of the relative contribution of the central vs. peripheral effects in physiologic conditions of peripheral release (or administration) of TH remains unclear. In this study we used two different models of peripherally induced hyperthyroidism (i.e., T4 and T3 oral administration) to assess and compare the serum and hypothalamic TH status and relate them to the metabolic effects of the treatment. Peripheral TH treatment affected feeding behavior, overall growth, core body temperature, body composition, brown adipose tissue (BAT) morphology and uncoupling protein 1 (UCP1) levels and metabolic activity, white adipose tissue (WAT) browning and liver metabolism. This resulted in an increased overall uncoupling capacity and a shift of the lipid metabolism from WAT accumulation to BAT fueling. Both peripheral treatment protocols induced significant changes in TH concentrations within the hypothalamus, with T3 eliciting a downregulation of hypothalamic AMP-activated protein kinase (AMPK), supporting the existence of a central action of peripheral TH. Altogether, these data suggest that peripherally administered TH modulate energy balance by various mechanisms; they also provide a unifying vision of the centrally mediated and the direct local metabolic effect of TH in the context of hyperthyroidism.

RESUMEN La infección causada por haemosporidios en colibríes no ha sido estudiada en zonas agroforestales o urbanas de la vertiente occidental de la Cordillera Oriental de los Andes en el departamento de Santander, pese a existir evidencia de esta en otros grupos de aves. Con el fin de detectar e identificar los parásitos causales de infecciones por haemosporidios, se tomaron muestras de sangre de la vena yugular de colibríes en seis localidades. La presencia de infección se llevó a cabo por PCR y la identificación de los parásitos se hizo a partir de secuencias del gen mitocondrial Citocromo b (Cyt b). Se obtuvieron 86 muestras de sangre de 20 especies de colibríes. La prevalencia de infección en general fue del 43 % y en el 18 % de las muestras infectadas del colibrí Amazilia colirufa (Amazilia tzacatl) se identificaron secuencias de Haemoproteus archilochus correspondientes al linaje HUMHA4. Se reporta por primera vez para Colombia la presencia de H. archilochus en A. tzacatl, por medio de técnicas de biología molecular. Este parásito podría estar implicado en la haemoproteosis de colibríes en el país. ABSTRACT Haemosporidian infection in hummingbirds has not been studied in agroforestry or urban areas of the Eastern Andes' western slope, in the department of Santander, despite the existence of evidence of this in other groups of birds. To detect and identify the causative parasites of haemosporidium infections, we took blood samples from the jugular vein of hummingbirds in six locations. PCR carried out the diagnosis of the infection, and the identification of the parasites was made from sequences of the mitochondrial gene Cytochrome b (Cyt b). 86 blood samples were obtained from 20 species of hummingbirds. The prevalence of infection, in general, was 43 %, and in 18 % of the infected samples of Rufous-tailed Hummingbird (Amazilia tzacatl), sequences of HUMHA4 lineage from Haemoproteus archilochus were identified. The presence of H. archilochus in A. tzacatl is reported for the first time in Colombia using molecular biology techniques. This parasite could be implicated in the haemoproteosis of hummingbirds in the country.

© 2021 by the authors., C3G (RAPGEF1) is a guanine nucleotide exchange factor (GEF) for GTPases from the Ras superfamily, mainly Rap1, although it also acts through GEF-independent mechanisms. C3G regulates several cellular functions. It is expressed at relatively high levels in specific brain areas, playing important roles during embryonic development. Recent studies have uncovered different roles for C3G in cancer that are likely to depend on cell context, tumour type, and stage. However, its role in brain tumours remained unknown until very recently. We found that C3G expression is downregulated in GBM, which promotes the acquisition of a more mesenchymal phenotype, enhancing migration and invasion, but not proliferation. ERKs hyperactivation, likely induced by FGFR1, is responsible for this pro-invasive effect detected in C3G silenced cells. Other RTKs (Receptor Tyrosine Kinases) are also dysregulated and could also contribute to C3G effects. However, it remains undetermined whether Rap1 is a mediator of C3G actions in GBM. Various Rap1 isoforms can promote proliferation and invasion in GBM cells, while C3G inhibits migration/invasion. Therefore, other RapGEFs could play a major role regulating Rap1 activity in these tumours. Based on the information available, C3G could represent a new biomarker for GBM diagnosis, prognosis, and personalised treatment of patients in combination with other GBM molecular markers. The quantification of C3G levels in circulating tumour cells (CTCs) in the cerebrospinal liquid and/or circulating fluids might be a useful tool to improve GBM patient treatment and survival., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness [SAF2016-76588-C2-1-R and PID2019-104143RB-C22 to A.P.; SAF2016-76588-C2-2-R and PID2019-104143RB-C21 to C.G. and PID2019-104991RB-I00 to P.B.], and by two grants from the Council of Education of Junta de Castilla y León, Spain [SA017U16 and SA078P20 to C.G.]. All funding was cosponsored by the European FEDER Programme. S.M. was a recipient of a FPU fellowship from Spanish Ministry of Education. A.G.-U. is supported by Madrid Community Programme for Talent Attraction (2017-T1/BMD-5468).

Orientador: Laymert Garcia dos Santos Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Filosofia e Ciencias Humanas Resumo: A tese central deste trabalho é que a aceitação pública despertada pelo Projeto Genoma Humano só se explica pelo uso político e retórico de um determinismo genético crescentemente irreconciliável com os resultados empíricos da pesquisa genômica atual. A complexidade verificada no genoma humano e em suas interações com o meio desautoriza a manutenção de uma noção simples e unidirecional de causalidade, contrariamente ao pressuposto na idéia de gene como único portador de informação, esteio da doutrina do determinismo genético. Um complexo de metáforas informacionais e/ou lingüísticas continuo vivo nos textos publicados por biólogos moleculares na literatura científica, notadamente nos artigos veiculados nos periódicos de alto impacto Nature e Science de 15 e 16 fevereiro de 2001, respectivamente. Tais metáforas inspiram um tipo de discurso ambíguo que modula nuances variadas de retórica determinista, conforme se dirija aos próprios pares ou ao público leigo" O campo da genômica ainda está longe de rejeitar a conjunção problemática das noções de gene pré-formacionista e de gene como recurso desenvo/vimenta/ na base da metáfora do gene como informação. Essa fusão inspirada pela terminologia cibernética propicia uma versão asséptica de gene, distanciada da natureza, puramente sintática, móvel e virtual o bastante para circular desimpedida nos circuitos de produção de valor como recurso genético passível de garimpagem e de patenteamento. Críticos dã tecnociência devem desafiar o campo da genômica a reformular drasticamente as metáforas que dão suporte a seu programa hegemônico de pesquisa Abstract: The central thesis of this work is that the public support generated for the Human Genome Project and the hype surrounding it can be explained only by the political and rhetorical uses of genetic determinism, a notion which increasingly cannot be reconciled with the empirical results of on-going genomic research. The complexity that has been uncovered in the human genome and in its interactions with the environment implies that a simple and unidirectional notion of causality cannot be maintained, contrary to a presupposition of the idea of the gene as the sole carrier of iliformation, an idea that contributes to sustain the doctrine of genetic determinism. A complex of informational and/or linguistic metaphors lives on in the texts published by molecular biologists in the scientific press, most notably in the issues published February 15thand 16thof 2001 ofthe high impact journals Nature and Science, respectively. These metaphors generate an ambiguous type of discourse that modulates various nuances of deterministic rhetoric, depending on whether it addresses peers or the lay publico The field of genomics is still a long way ITom rejecting the questionable conflation of the notions of gene as preformation and gene as developmental resource which underpins the metaphor of gene as information. This conflation inspired by cybernetics terminology enables an aseptic version of the gene, separated ITom nature, portable and virtual enough to flow unimpeded through the channels ofvalue production as genetic resource suitable for mining and patenting. Critics of technoscience should challenge the field of genomics to drastically reshape the metaphors which have supported its hegemonic research agenda Doutorado Doutor em Ciências Sociais

Alzheimer's disease (AD) is characterized by non-linear, genetic-driven pathophysiological dynamics with high heterogeneity in biological alterations and disease spatial-temporal progression. Human in-vivo and post-mortem studies point out a failure of multi-level biological networks underlying AD pathophysiology, including proteostasis (amyloid-β and tau), synaptic homeostasis, inflammatory and immune responses, lipid and energy metabolism, oxidative stress. Therefore, a holistic, systems-level approach is needed to fully capture AD multi-faceted pathophysiology. Omics sciences - genomics, epigenomics, transcriptomics, proteomics, metabolomics, lipidomics - embedded in the systems biology (SB) theoretical and computational framework can generate explainable readouts describing the entire biological continuum of a disease. Such path in Neurology is encouraged by the promising results of omics sciences and SB approaches in Oncology, where stage-driven pathway-based therapies have been developed in line with the precision medicine paradigm. Multi-omics data integrated in SB network approaches will help detect and chart AD upstream pathomechanistic alterations and downstream molecular effects occurring in preclinical stages. Finally, integrating omics and neuroimaging data - i.e., neuroimaging-omics - will identify multi-dimensional biological signatures essential to track the clinical-biological trajectories, at the subpopulation or even individual level. Sin financiación 10.895 JCR (2021) Q1, 25/195 Cell Biology 3.523 SJR (2021) Q1, 1/35 Aging No data IDR 2021 UEM

Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors.

The gut associated lymphoid tissue (GALT) effector sites play a crucial role on the pathogenesis of many immune-mediated gastrointestinal diseases. The lymphocytes at these effector sites are principally T cells which present important morphological, phenotypical and functional differences. Flow cytometry (FC) is one of the most commonly used techniques to characterize intestinal lymphocytes in human and animal models. Published studies with a focus on dogs for intraepithelial lymphocytes (IEL) immunophenotyping exist in very limited numbers. Moreover, no lamina propria lymphocytes (LPL) isolation protocols in the canine species have been described for FC evaluation. In addition to immune intestinal dysregulation, imbalances in the peripheral blood immune system have been described in both human and animal gastrointestinal disorders. The aim of this study was to provide a protocol for canine IEL and LPL isolation for FC immunophenotyping of T cells subsets. Specifically, T helper, T cytotoxic, activated Th and Tc lymphocytes, regulatory, double negative, double positive, IFN-γ and IL-4 producing T cells, and to compare their respective populations between these effector sites and with the blood stream compartment in healthy dogs. The potential relationship of these cells distributions with age, sex and breed was also evaluated. This study included sixteen healthy dogs of different sexes and breeds with a mean age of 4.55 ± 2.93 years old. The selected protocols for the three immune compartments showed proper cell yield, purity, viability, and the absence of phenotypic and functional disturbances. Histologically, an adequate separation of the duodenal epithelium from the lamina propria was also observed. All the proposed T cells subsets were identified in the three immune compartments studied, showing some statistically significant differences in their distributions at these locations that highlight the importance of their individual evaluation. This study provides an adequate method for canine small intestine IEL and LPL isolation for FC immunophenotyping and is key for future studies on the gastrointestinal immune system associated with different canine diseases.

Multiple transcription factors have been shown to promote pancreatic β-cell differentiation, yet much less is known about negative regulators. Earlier epigenomic studies suggested that the transcriptional repressor REST could be a suppressor of endocrinogenesis in the embryonic pancreas. However, pancreatic Rest knockout mice failed to show abnormal numbers of endocrine cells, suggesting that REST is not a major regulator of endocrine differentiation. Using a different conditional allele that enables profound REST inactivation, we observed a marked increase in pancreatic endocrine cell formation. REST inhibition also promoted endocrinogenesis in zebrafish and mouse early postnatal ducts and induced β-cell-specific genes in human adult duct-derived organoids. We also defined genomic sites that are bound and repressed by REST in the embryonic pancreas. Our findings show that REST-dependent inhibition ensures a balanced production of endocrine cells from embryonic pancreatic progenitors. This research was supported by Ministerio de Ciencia, Innovación y Universidades (SAF2015-73226-JIN [Agencia Estatal de Investigación {AEI}/European Regional Development Fund, European Union {UE}] and RYC-2017-21950 [AEI/European Social Fund, UE] to M.R., and BFU2014-54284-R and RTI2018-095666-B-I00 to J.F.); the Medical Research Council (MR/L02036X/1), Wellcome Trust (WT101033), and European Research Council Advanced Grant (789055) (to J.F.); the Instituto de Salud Carlos III (CA18/00045 to J.L.M.); and a Spanish Ministry of Science, Innovation, and Universities (MCIU) fellowship (PTA2018-016371-I to M.M.). J.K.-C.'s and F.P.'s research was supported by L'Agence Nationale de la Recherche (ANR) grants, L'Institut Européen de Génomique du Diabète (EGID), ANR-10-LABX-0046, a French state fund managed by ANR under the frame program Investissements d'Avenir (I-SITE ULNE/ANR-16-IDEX-0004 ULNE to F.P.), and the European Consortium for Islet Transplantation funded by the Juvenile Diabetes Research Foundation International. Work in the Centre for Genomic Regulation was supported by the Centres de Recerca de Catalunya (CERCA) Programme, Generalitat de Catalunya, and Centro de Excelencia Severo Ochoa (SEV-2015-0510). Work at Institut d'Investigació Biomèdica de Bellvitge was supported by the CERCA Programme and Generalitat de Catalunya

This work was partially supported by grant number PR87/19-22587 from Santander Bank/Complutense University of Madrid

Many intercellular communication processes, known as quorum sensing (QS), are regulated by the autoinducers N-acyl-l-homoserine lactones (AHLs) in Gram-negative bacteria. The inactivation of these QS processes using different quorum quenching (QQ) strategies, such as enzymatic degradation of the autoinducers or the receptor blocking with non-active analogs, could be the basis for the development of new antimicrobials. This study details the heterologous expression, purification, and characterization of a novel N-acylhomoserine lactone acylase from Actinoplanes utahensis NRRL 12052 (AuAHLA), which can hydrolyze different natural penicillins and N-acyl-homoserine lactones (with or without 3-oxo substitution), as well as synthesize them. Kinetic parameters for the hydrolysis of a broad range of substrates have shown that AuAHLA prefers penicillin V, followed by C12-HSL. In addition, AuAHLA inhibits the production of violacein by Chromobacterium violaceum CV026, confirming its potential use as a QQ agent. Noteworthy, AuAHLA is also able to efficiently synthesize penicillin V, besides natural AHLs and phenoxyacetyl-homoserine lactone (POHL), a non-natural analog of AHLs that could be used to block QS receptors and inhibit signal of autoinducers, being the first reported AHL acylase capable of synthesizing AHLs.

The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1Δ cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1Δ mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.

Pregnancy is a unique immunological situation in which a fetus-bearing paternal histocompatibility antigens can survive in a maternal environment without apparent rejection. To face this challenge, cells of the uterine immune system show characteristic changes in absolute number and composition during pregnancy. Particularly relevant to this process are uterine natural killer (uNK) cells and their cell surface receptors, killer immunoglobulin-like receptors (KIRs). The main purpose of this review is to outline the current body of knowledge on the involvement of KIRs in the complications of pregnancy. Implantation depends on the invasion of embryonic trophoblast cells into maternal uterine tissue and remodeling of the uterine spiral arterioles, which is essential for placental perfusion and successful pregnancy. The proper interaction between maternal KIRs and their ligands human leukocyte antigen (HLA) class I molecules, expressed by the extravillous trophoblast cells, is crucial in this process. KIRs are a complex family that includes both activator and inhibitory receptors. The activation profile is genetically determined in each individual and leads to diverse levels of functionality for NK and T cells on engagement with specific HLA class I molecules. An association between different KIR alleles and HLA molecules has been reported in pregnancy complications, supporting the idea of a relevant role of these receptors in successful pregnancy. Sin financiación 2.829 JCR (2019) Q2, 21/82 Obstetrics & Ginecology, 14/29 Reproductive Biology, 88/177 Genetics & Heredity 1.140 SJR (2019) Q1, 405/2754 Medicine (miscellaneous), 35/185 Obstetrics and Gynecology, 16/76 Reproductive Medicine; Q2, 29/87 Developmental Biology, 120/346 Genetics, 40/99 Genetics (clinical) No data IDR 2019 UEM

Insight into the catalytic mechanism of Lactobacillus leichmannii nucleoside 2′-deoxyribosyltransferase (LlNDT) has been gained by calculating a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape of the reaction within the enzyme active site. Our results support an oxocarbenium species as the reaction intermediate and thus an S1 reaction mechanism in this family of bacterial enzymes. Our mechanistic proposal is validated by comparing experimental kinetic data on the impact of the single amino acid replacements Tyr7, Glu98 and Met125 with Ala, Asp and Ala/norLeu, respectively, and accounts for the specificity shown by this enzyme on a non-natural substrate. This work broadens our understanding of enzymatic C-N bond cleavage and C-N bond formation., Financial support from the FWF (Lise Meitner Program M 2260) to PAS-M, the Spanish Ministerio de Economía y Competitividad (SAF2015-64629-C2-2-R) to F. G. and Grants XSAN001906 from the Santander Foundation to J. F. L. is gratefully acknowledged. We thank the University of Vienna for computational resources at the Institute of Theoretical Chemistry.

A series of peptide receptors were designed using an in-silico approach for the recognition of the heme prosthetic group in carboxyhemoglobin (COHb). These peptide chains were designed from the amino acids present in the enzyme Cytochrome b561 (Cyt b561) active site. The evaluation of the interaction energy of the heme prosthetic group against the Cyt b561 active site was found to be −7.1 kcal/mol. However, after evaluating the interaction energies of the heme group against the different peptides representing the enzyme's active site, a higher affinity was discovered with one particular peptide chain (−8.2 kcal/mol). For the design, the peptide was built preserving the distance between the amino acids involved in the catalytic process of Cyt b561, using methylenes as spacers. Additionally, the peptide was polymerized with styrene, resulting in the following peptide chain poly(styrene)-A(CH2)5QW(CH2)5GF(CH2)5YW(CH2)6M(CH2)5HA(CH2)6G-(styrene)poly. This result promotes the rational design of peptide receptors in the detection and quantification of COHb from the recognition of its prosthetic group. Keywords: Active site, Amino acids, Molecular docking, Steric hindrance

Hypertension is a complex multifactorial disease characterized by a chronic increase of arterial pressure. Ninety percent of the cases are idiopathic and thus classified as essential hypertension. Uncontrolled arterial pressure has devasting consequences including cardiac insufficiency, stroke, dementia, chronic renal disease, ischemic heart disease and death. The hiPSC lines described here from six hypertensive patients and three controls were characterized according to established criteria and were shown to maintain pluripotency, differentiation into the three germ layers and genomic integrity. These cell lines can contribute to the understanding of the molecular mechanisms involved in hypertension in different cell types.

Over the last few years an intense activity in the areas of advanced microscopy and quantitative cell biology has put the focus on the morphogenetic events that shape embryos. The interest in these processes is taking place against the backdrop of genomic studies, particularly of global patterns of gene expression at the level of single cells, which cannot fully account for the way cells build tissues and organs. Here we discuss the need to integrate the activity of genes with that of cells and propose the need to develop a framework, based on cellular processes and cell interactions, that parallels that which has been created for gene activity in the form of Gene Regulatory Networks (GRNs). We begin to do this by suggesting elements for building Cell Regulatory Networks (CRNs). In the same manner that GRNs create schedules of gene expression that result in the emergence of cell fates over time, CRNs create tissues and organs i.e. space. We also suggest how GRNs and CRNs might interact in the building of embryos through feedback loops involving mechanics and tissue tectonics.

Background Paracoccidioidomycosis (PCM) is a systemic and endemic fungal infection in Latin American, mainly in Brazil. The majority of PCM cases occur in large areas in Brazil, comprising the South, Southeast and Midwest regions, with the latter demonstrating a higher incidence of the species Paracoccidioides lutzii. Methodology and main findings This study presents clinical, molecular and serological data of thirteen new PCM cases during 2016 to 2019 from the state of Mato Grosso do Sul, located in the Midwest region, Brazil. From these thirteen cases, sixteen clinical isolates were obtained and their genomic DNAs were subjected to genotyping by tub1 -PCR-RFLP. Results showed Paracoccidioides brasiliensis sensu stricto (S1) (11/16; 68.8%), Paracoccidioides restrepiensis (PS3) (4/16; 25.0%) and P. lutzii (1/16; 6.2%) as Paracoccidiodes species. Therefore, in order to understand whether the type of phylogenetic species that are circulating in the state influence the reactivity profile of serological tests, we performed double agar gel immunodiffusion (DID), using exoantigens from genotyped strains found in this series of PCM cases. Overall, our DID tests have been false negative in about 30% of confirmed PCM cases. All patients were male, most with current or previous rural activity, with ages ranging from 17 to 59 years, with 11 patients (84.6%) over 40 years of age. No clinical or epidemiological differences were found between Paracoccidioides species. However, it is important to note that the only case of P. lutzii died as an outcome. Conclusions This study suggests P. brasiliensis sensu stricto (S1) as the predominant species, showing its wide geographic distribution in Brazil. Furthermore, our findings revealed, for the first time, the occurrence of P. restrepiensis (PS3) in the state of Mato Grosso do Sul, Brazil. Despite our setbacks, it would be interesting to provide the complete sequencing of these clinical isolates to complement the molecular information presented., Author summary Paracoccidioides spp. complex and P. lutzii are etiological agents of paracoccidioidomycosis (PCM), one of the most important systemic mycosis of Latin America. This study aimed to describe the molecular epidemiology associated with clinical and serological data of Paracoccidiodes spp. in the Mato Grosso do Sul, located in the Midwest region, Brazil. Thus, for the first time in this state, the clinical Paracoccidioides species were molecularly identified. Previous findings have frequently been misinterpreted as proving that P. lutzii predominates in this region. In fact, we observed that this state differs from others in the Midwest, presenting a higher proportion of PCM cases due to P. brasiliensis sensu stricto (S1). In addition, we described the first report of P. restrepiensis (PS3) occurrence in the Brazilian Midwest region. Then, we presented an update on the occurrence of Paracoccidioides species in the Midwest region from Brazil. On the other hand, our results do not demonstrate clinical and epidemiological differences between Paracoccidioides spp. Nevertheless, this study reinforces that other researches should be carried out in order to evaluate an association of clinical manifestations and epidemiology with Paracoccidioides species.

From urinary tract infections to bacterial pneumonia, many diseases can now be treated through a course of antibiotics. Yet bacteria have evolved to respond to this threat, gaining new antibiotic resistance genes that allow them to evade the drugs. Addressing this growing issue requires to either discover new antibiotics, or to stop resistance before it emerges – a strategy that can only work if scientists know exactly how this mechanism takes place. For bacteria, it is a waste of resources to produce the proteins that confer resistance if antibiotics are absent. In fact, doing so can decrease their chance to survive and reproduce. A genetic element known as an integron can help to manage that burden. This piece of genetic information is formed of a succession of ‘cassettes’ containing antibiotic resistance genes. More proteins are made from the genes present at the start of the integron, compared to the ones towards the end. When bacteria encounter antibiotics, an enzyme called integrase is activated, allowing the organisms to shuffle the order of their cassettes in the integron. It is thought – but not yet proven – that this mechanism helps bacteria to activate their resistance ‘on demand’. To find out, Souque et al. engineered the bacteria Pseudomonas aeruginosa to carry a custom integron with three cassettes, each helping the organism to resist to a different antibiotic. In addition, only half of the bacteria had a working integrase and could therefore shuffle their gene cassettes. The organisms were then exposed to an increasing amount of the antibiotics for which the cassette in the last position provided resistance. The bacteria with a working integrase survived longer than those without, as they were able to shuffle their cassettes and move the useful antibiotic resistance gene into top position. In addition, the cassettes carrying the genes to resist to other types of antibiotics were excised from the genetic information and lost. Understanding integrons could guide future antibiotic treatment strategies, for instance by combining antibiotics with chemicals that block integrase activity. It might also be possible to force bacteria to delete resistance cassettes by cycling through different antibiotics.

Sticholysins are pore-forming toxins produced by sea anemones that are members of the actinoporin family. They exert their activity by forming pores on membranes, provided they have sphingomyelin. To assemble into pores, specific recognition, binding, and oligomerization are required. While recognition and binding have been extensively studied, delving into the oligomerization process and the stoichiometry of the pores has been more difficult. Here, we present evidence that these toxins are capable of oligomerizing in solution and suggesting that the interaction of sticholysin II (StnII) with its isoform sticholysin I (StnI) is stronger than that of StnI with itself. We also show that the stoichiometry of the final, thermodynamically stable StnI pores is, at least, heptameric. Furthermore, our results indicate that this association maintains its oligomerization number when StnII is included, indicating that the stoichiometry of StnII is also of that order, and not tetrameric, as previously thought. These results are compatible with the stoichiometry observed for the crystallized pore of FraC, another very similar actinoporin produced by a different sea anemone species. Our results also indicate that the stoichiometry of actinoporin pores in equilibrium is conserved regardless of the particular composition of a given pore ensemble, which we have shown for mixed sticholysin pores.

Introduction: Helicobacter pylori (H. pylori) is a Gram negative bacterium considered to be the etiologic agent of various gastric diseases. The prevalence of bacterial infection varies according to age, geographic location, ethnicity and socioeconomic status. The chronic infection caused by microorganism can favor the development of severe pathologies such as gastric adenocarcinoma. In this sense, early diagnosis is essential for a better prognosis and therapeutic success. Several diagnostic methods performed using invasive and non-invasive techniques, with different sensitivity and specificity, have been used in the detection of H. pylori. Objective: To compare the performance of the molecular and histopathological technique used in the diagnosis of H. pylori infection. Methods: 76 gastric tissue samples were collected from dyspeptic patients who underwent molecular and histopathological diagnosis. Molecular detection was performed using the ribosomal gene (16S rRNA) using the polymerase chain reaction (PCR) technique. Results: The PCR-based molecular diagnostic method detected the bacterium in 63.1% of the samples, while the histopathological test identified the microorganism in only 38.1% of gastric biopsies. The data demonstrated that the PCR technique was about 1.6 times more sensitive than the histopathological technique. Conclusion: The PCR technique was the most efficient diagnostic method for detecting H. pylori and can be implemented in the laboratory routine as a complementary test for the early detection of H. pylori. RESUMEN Introducción: Helicobacter pylori (H. pylori) es una bacteria Gram negativa considerada agente etiológico de diversas enfermedades gástricas. La prevalencia de la infección bacteriana varía según la edad, la ubicación geográfica, la etnia y el nivel socioeconómico. La infección crónica provocada por esos microorganismos puede favorecer el desarrollo de patologías graves como el adenocarcinoma gástrico. Por esa razón, el diagnóstico precoz es fundamental para un mejor pronóstico y éxito terapéutico. En la detección de H. pylori se han utilizado varios métodos de diagnóstico realizados mediante técnicas invasivas y no invasivas, con diferentes sensibilidad y especificidad. Objetivo: Comparar el desempeño de las técnicas moleculares e histopatológicas utilizadas en el diagnóstico de la infección por H. pylori. Métodos: Se recolectaron 76 muestras de tejido gástrico de pacientes dispépticos y se las sometieron a diagnóstico molecular e histopatológico. La detección molecular se realizó mediante el gen ribosómico (ARNr 16S) mediante la técnica de reacción en cadena de la polimerasa (PCR). Resultados: El método molecular de diagnóstico basado en PCR detectó la bacteria en el 63,1% de las muestras, mientras que la prueba histopatológica identificó el microorganismo en solo el 38,1% de las biopsias gástricas. Los datos demostraron que la técnica de PCR era aproximadamente 1,6 veces más sensible que la técnica histopatológica. Conclusión: La técnica de PCR fue el método diagnóstico más eficaz para la detección de H. pylori y puede implementarse en la rutina del laboratorio como prueba complementaria para la detección precoz de H. pylori. RESUMO Introdução: Helicobacter pylori (H. pylori) é uma bactéria Gram negativa considerada o agente etiológico de várias doenças gástricas. A prevalência da infecção bacteriana varia de acordo com idade, localização geográfica, etnia e status socioeconômico. A infecção crônica ocasionada por esse microrganismo pode favorecer o desenvolvimento de patologias severas, como o adenocarcinoma gástrico. Nesse sentido, o diagnóstico precoce é essencial para um melhor prognóstico e o sucesso terapêutico. Vários métodos de diagnóstico realizados com técnicas invasivas e não invasivas, com diferentes sensibilidade e especificidade, têm sido utilizados na detecção de H. pylori. Objetivo: Comparar o desempenho das técnicas molecular e histopatológica utilizadas no diagnóstico da infecção por H. pylori. Métodos: Setenta e seis amostras de tecido gástrico foram coletadas de pacientes dispépticos e submetidas ao diagnóstico molecular e histopatológico. A detecção molecular foi realizada utilizando o gene ribossomal (rRNA 16S) por meio da técnica de reação em cadeia da polimerase (PCR). Resultados: O método molecular de diagnóstico com base na PCR detectou a bactéria em 63,1% das amostras, enquanto o teste histopatológico identificou o microrganismo em apenas 38,1% das biópsias gástricas. Os dados demonstram que a técnica de PCR apresentou cerca de 1,6 vezes mais sensibilidade que a técnica histopatológica. Conclusão: A técnica de PCR foi o método de diagnóstico mais eficiente para detecção de H. pylori e pode ser implementada na rotina laboratorial como teste complementar para detecção precoce de H. pylori.

The regulation of AMPA-type receptor (AMPAR) abundance in the postsynaptic membrane is an important mechanism involved in learning and memory formation. Recent data suggest that one of the constituents of the AMPAR complex is carnitine palmitoyltransferase 1C (CPT1C), a brain-specific isoform located in the endoplasmic reticulum of neurons. Previous results had demonstrated that CPT1C deficiency disrupted spine maturation in hippocampal neurons and impaired spatial learning, but the role of CPT1C in AMPAR physiology had remained mostly unknown. In the present study, we show that CPT1C binds GluA1 and GluA2 and that the three proteins have the same expression profile during neuronal maturation. Moreover, in hippocampal neurons of CPT1C KO mice, AMPAR-mediated miniature excitatory postsynaptic currents and synaptic levels of AMPAR subunits GluA1 and GluA2 are significantly reduced. We show that AMPAR expression is dependent on CPT1C levels because total protein levels of GluA1 and GluA2 are decreased in CPT1C KO neurons and are increased in CPT1C-overexpressing neurons, whereas other synaptic proteins remain unaltered. Notably, mRNA levels of AMPARs remained unchanged in those cultures, indicating that CPT1C is post-transcriptionally involved. We demonstrate that CPT1C is directly involved in the de novo synthesis of GluA1 and not in protein degradation. Moreover, in CPT1C KO cultured neurons, GluA1 synthesis after chemical long term depression was clearly diminished, and brain-derived neurotrophic factor treatment was unable to phosphorylate the mammalian target of rapamycin (mTOR) and stimulate GluA1 protein synthesis. These data newly identify CPT1C as a regulator of AMPAR translation efficiency and therefore also synaptic function in the hippocampus.

Herein we report the first proof for the application of type II 2&prime, deoxyribosyltransferase from Lactobacillus delbrueckii (LdNDT) in suicide gene therapy for cancer treatment. To this end, we first confirm the hydrolytic ability of LdNDT over the nucleoside-based prodrugs 2&prime, deoxy-5-fluorouridine (dFUrd), 2&prime, deoxy-2-fluoroadenosine (dFAdo), and 2&prime, deoxy-6-methylpurine riboside (d6MetPRib). Such activity was significantly increased (up to 30-fold) in the presence of an acceptor nucleobase. To shed light on the strong nucleobase dependence for enzymatic activity, different molecular dynamics simulations were carried out. Finally, as a proof of concept, we tested the LdNDT/dFAdo system in human cervical cancer (HeLa) cells. Interestingly, LdNDT/dFAdo showed a pronounced reduction in cellular viability with inhibitory concentrations in the low micromolar range. These results open up future opportunities for the clinical implementation of nucleoside 2&prime, deoxyribosyltransferases (NDTs) in cancer treatment.

Graphical abstract, Highlights • PxXyn11B is the first crystal structure of a GH11 from Paenibacillus. • X-ray structure of PxXyn11B reveals its molecular architecture and a second binding site (SBS). • MD simulations shed light on structural determinants of uncommon PxXyn11B cooperative behavior. • Our results reveal molecular mechanism of SBS communication with the active site in GH11 scaffold settings., Glycoside hydrolases (GHs) are essential for plant biomass deconstruction. GH11 family consist of endo-β-1,4-xylanases which hydrolyze xylan, the second most abundant cell wall biopolymer after cellulose, into small bioavailable oligomers. Structural requirements for enzymatic mechanism of xylan hydrolysis is well described for GH11 members. However, over the last years, it has been discovered that some enzymes from GH11 family have a secondary binding sites (SBS), which modulate the enzymes activities, but mechanistic details of the molecular communication between the active site and SBS of the enzymes remain a conundrum. In the present work we structurally characterized GH11 xylanase from Paenibacillus xylanivorans A57 (PxXyn11B), a microorganism of agricultural importance, using protein crystallography and molecular dynamics simulations. The PxXyn11B structure was solved to 2.5 Å resolution and different substrates (xylo-oligosaccharides from X3 to X6), were modelled in its active and SBS sites. Molecular Dynamics (MD) simulations revealed an important role of SBS in the activity and conformational mobility of PxXyn11B, demonstrating that binding of the reaction products to the SBS of the enzyme stabilizes the N-terminal region and, consequently, the active site. Furthermore, MD simulations showed that the longer the ligand, the better is the stabilization within active site, and the positive subsites contribute less to the stabilization of the substrates than the negative ones. These findings provide rationale for the observed enzyme kinetics, shedding light on the conformational modulation of the GH11 enzymes via their SBS mediated by the positive molecular feedback loop which involve the products of the enzymatic reaction.

Antecedentes: La leishmaniasis es una enfermedad causada por un parásito del género Leishmania que afecta a millones de personas en todo el mundo. Los actuales sistemas diagnósticos de la leishmaniasis presentan importantes limitaciones, por lo que existe una gran demanda para desarrollar nuevos sistemas económicos, rápidos y que no requieran personal especializado. Los aptámeros son ácidos nucleicos de cadena sencilla capaces de interaccionar de forma específica y con alta afinidad con una molécula diana y se utilizan actualmente con fines de detección, diagnósticos y terapéuticos. Métodos: Partiendo de una población de aptámeros de ssDNA obtenida previamente contra la proteína histona H3 de Leishmania infantum (LiH3), identificamos dos aptámeros individuales, AptLiH3#4 y AptLiH3#10. A continuación, realizamos ensayos ELONA, western blot y slot blot para establecer la especificidad y afinidad de los aptámeros por la histona LiH3. También, realizamos ensayos ELONA utilizando péptidos correspondientes a secuencias superpuestas de LiH3 para mapear la interacción aptámero:LiH3. Además, desarrollamos diferentes sistemas de detección basados en ELONA utilizando los aptámeros como moléculas de reconocimiento y detección para determinar cuál es el que muestra una mayor sensibilidad. Resultados: Ambos aptámeros tienen alta afinidad y especificidad por LiH3 endógena en lisados de promastigotes. De los diferentes sistemas ensayados, hemos confirmado que el ELONA acoplado a PCR a tiempo real (ELONA-qPCR) es el más sensible, alcanzándose un límite de detección de 600 parásitos. Conclusiones: Los aptámeros identificados podrían ser utilizados como moléculas de bioreconocimiento de la histona LiH3, pero es necesario optimizar el sistema de detección para su uso en el diagnóstico de la leishmaniasis.

The K/BxN mouse model of rheumatoid arthritis (RA) closely resembles the human disease. In this model, arthritis results from activation of autoreactive KRN T cells recognizing the glycolytic enzyme glucose-6-phosphate isomerase (GPI) autoantigen, which provides help to GPI-specific B cells, resulting in the production of pathogenic anti-GPI antibodies that ultimately leads to arthritis symptoms from 4 weeks of age. Vasoactive intestinal peptide (VIP) is a neuropeptide broadly distributed in the central and peripheral nervous system that is also expressed in lymphocytes and other immune cell types. VIP is a modulator of innate and adaptive immunity, showing anti-inflammatory and immunoregulatory properties. Basically, this neuropeptide promotes a shift in the Th1/Th2 balance and enhances dedifferentiation of T regulatory cells (Treg). It has demonstrated its therapeutic effects on the collagen-induced arthritis (CIA) mouse model of RA. In the present hypothesis and theory article, we propose that the immunoregulatory properties of VIP may be due likely to the inhibition of T cell plasticity toward non-classic Th1 cells and an enhanced follicular regulatory T cells (Tfr) activity. The consequences of these regulatory properties are the reduction of systemic pathogenic antibody titers.

This article belongs to the Section Microbial Biotechnology., The highly xerotolerant bacterium classified as Exiguobacterium sp. Helios isolated from a solar panel in Spain showed a close relationship to Exiguobacterium sibiricum 255-15 isolated from Siberian permafrost. Xerotolerance has not been previously described as a characteristic of the extremely diverse Exiguobacterium genus, but both strains Helios and 255-15 showed higher xerotolerance than that described in the reference xerotolerant model strain Deinococcus radiodurans. Significant changes observed in the cell morphology after their desiccation suggests that the structure of cellular surface plays an important role in xerotolerance. Apart from its remarkable resistance to desiccation, Exiguobacterium sp. Helios strain shows several polyextremophilic characteristics that make it a promising chassis for biotechnological applications. Exiguobacterium sp. Helios cells produce nanoparticles of selenium in the presence of selenite linked to its resistance mechanism. Using the Lactobacillus plasmid pRCR12 that harbors a cherry marker, we have developed a transformation protocol for Exiguobacterium sp. Helios strain, being the first time that a bacterium of Exiguobacterium genus has been genetically modified. The comparison of Exiguobacterium sp. Helios and E. sibiricum 255-15 genomes revealed several interesting similarities and differences. Both strains contain a complete set of competence-related DNA transformation genes, suggesting that they might have natural competence, and an incomplete set of genes involved in sporulation; moreover, these strains not produce spores, suggesting that these genes might be involved in xerotolerance., This research was supported by grants BIO2015-66960-C3-1-R, BIO2016-79736-R and PID2019-110612RB-I00 from the Ministry of Economy and Competitiveness of Spain, RTI2018-095584-B-C41-42-43-44 from the Ministry of Science and Innovation of Spain, and by Grants CSIC 2017 2 0I 015. María Castillo has a fellowship from the State Program for the Promotion of Talent and its Employability in R&D&I supported by grant PRE2019-087933.

Human gut microbiota harbors numerous microbial species with molecular enzymatic potential that impact on the eubiosis/dysbiosis and health/disease balances. Microbiota species isolation and description of their specific molecular features remain largely unexplored. In the present study, we focused on the cultivation and selection of species able to tolerate or biodegrade the endocrine disruptor bisphenol A (BPA), a xenobiotic extensively found in food plastic containers. Chemical xenobiotic addition methods for the directed isolation, culturing, Whole Genome Sequencing (WGS), phylogenomic identification, and specific gene-encoding searches have been applied to isolate microorganisms, assess their BPA metabolization potential, and describe encoded catabolic pathways. BPA-tolerant strains were isolated from 30% of infant fecal microbial culture libraries analyzed. Most isolated strains were phylogenetically related to the operational taxonomic group Bacillus amyloliquefaciens spp. Importantly, WGS analysis of microbial representative strain, Bacillus sp. AM1 identified the four complete molecular pathways involved on BPA degradation indicating its versatility and high potential to degrade BPA. Pathways for Exopolysaccharide (EPS) and Polyhydroxyalkanates (PHA) biopolymer synthesis were also identified and phenotypically confirmed by transmission electronic microscopy (TEM). These microbial biopolymers could generally contribute to capture and/or deposit xenobiotics., GP/EFSA/ENCO/380 2018/03/G04: OBEMIRISK: Knowledge platform for assessing the risk of Bisphenols on gut microbiota and its role in obesogenic phenotype: looking for biomarkers, FEDERInfrastructure: IE_2019-198, APC was funded by EIN-2019-103082

[SPA] Las Solanáceas son una familia de plantas que van desde hierbas anuales hasta arbustos y árboles perennes. Incluyen muchas especies ornamentales apreciadas en todo el mundo, como el género Petunia, Browallia y Lycianthes, así como géneros de considerable impacto económico en el mercado mundial. Entre estos se encuentran la patata (S. tuberosum) tomate, (S .lycopersicum) y berenjena (S. melongena), tabaco (Nicotiana tabaccum) o pimiento (C. annuum) que incluye las variedades chili y pimiento dulce, dos de las más comercializadas en el mundo. El objetivo de esta tesis fue llevar a cabo un análisis genético comparativo del gen GIGANTEA (GI), para profundizar en el conocimiento de su expresión y regulación génica, así como el papel que desempeña en el control del desarrollo y ritmo circadiano en las Solanáceas. Para lograr los objetivos que nos propusimos analizamos en el sistema modelo Petunia x hybrida los dos parálogos préviamente identificados PhGI1 y PhGI2, a través de dos técnicas distintas: primero creando líneas de perdida de función, por cada uno de los dos parálogos, mediante técnica de ARN de interferencia y posteriormente mutantes knock-out mediante la técnica CRISPR/Cas9, para confirmar los resultados obtenidos. Se realizaron análisis de fenotipado de los caracteres vegetativos y reproductivos, genotipado y análisis de los componentes volátiles utilizando el método SPME desde el espacio de cabeza, seguido de GC/MS. Dentro de los estudios realizados hemos podido observar cómo los parálogos comparten funciones comunes. Ambos tienen efectos menores en el ritmo y la expresión de los genes del reloj. Esto es debido a que la función de GI ocurre a través de las interacciones proteína-proteína. PhGI1 y PhGI2 actúan como reguladores negativos del crecimiento vegetativo, controlando el tamaño y el contenido relativo de clorofila de las hojas, así como la longitud de entrenudos. Con respecto a los caracteres generativos, hemos identificado una nueva función aún no descrita hasta ahora. Líneas silenciadas para cada uno de los paralogos, presentaron flores abortadas caracterizadas por la senescencia temprana de los tejidos. Además, el número total y el tamaño de las flores fue menor en comparación con el fenotipo silvestre. Al mismo tiempo se observó como la cantidad y el perfil de los compuestos volátiles emitidos resultó afectado en las líneas silenciadas. La cantidad total de aromas emitidos durante 24 horas resultó inferior en comparación con el fenotipo silvestre y el perfil de aromas emitidos durante el día resultó alterado. Los resultados de está Tesis confirman que PhGI1 y PhGI2 están involucrados en el desarrollo floral. Observamos también diferentes fenotipos entre las líneas silenciadas para cada uno de los parálogos, particularmente en el control del tiempo de floración. Aunque el silenciamiento de PhGI1 no mostró ninguna diferencia en comparación con el fenotipo silvestre, plantas silenciadas en PhGI2 mostraron un retraso de entre dos/tres semanas más tarde en la floración, confirmándose como gen cuya pérdida de función provoca floración tardía, ya descrito en Arabidopsis thaliana. Estos datos confirmaron la hipótesis previa de que algunos genes duplicados podrían haber sufrido subfuncionalización o neofuncionalización. [ENG] Solanaceae are a family of plants ranging from annual grasses to evergreen shrubs and trees. They include many ornamental species appreciated around the world, such as the genus Petunia, Browallia and Lycianthes, as well as genera of considerable economic impact on the world trade such as potato (S. tuberosum), tomato (S .lycopersicum), eggplant (S. melongena), tobacco (Nicotiana tabaccum) and pepper (Capsicum annuum) that includes the chili pepper and bell pepper varieties, two of the most commercialized in the world. The objective of this thesis was to carry out a comparative genetic analysis of the GIGANTEA (GI) gene, to deepen the knowledge of its expression and gene regulation, as well as the role it plays in the control plant development and circadian rhythm in Solanaceae. In order to achieve the objectives that we set, we analyzed in the model system Petunia x hybrida the two paralogs previously identified PhGI1 and PhGI2, through two different techniques: first creating loss of function lines for each of the two paralogs, using the interference- RNA technique and subsequently knock-out mutants using the CRISPR/Cas9 technique to confirm the results obtained. Phenotypic analysis of vegetative and reproductive traits, genotyping and volatile components analysis were performed using the SPME method from the headspace, followed by GC/MS. Among the studies carried out we have been able to observe how the paralogs share common functions. Both have minor effects on the timing and expression of clock genes, this could be because GI molecular function occurs through protein-protein interactions. PhGI1 and PhGI2 act as negative regulators of vegetative growth, controlling the size and relative content of chlorophyll in the leaves, as well as the internode distance. Regarding generative characters, we have identified a new function non described so far. Both, PhGI1 and PhGI2, silenced plants presented aborted flowers characterized by early tissue senescence. Furthermore, the size and total number of the flowers were reduced compared to the wild type phenotypes. At the same time, it was observed how the amount and the profile of the volatile compounds emitted were affected in the silenced lines. The total amount of aromas emitted during 24 hours was lower compared to wild type phenotypes and the profile of aromas emitted during the day was altered. Our results confirm that PhGI1 and PhGI2 are involved in flower development. We also observed different behaviors between the silencing of each paralog, particularly in the control of the flowering time. Although PhGI1-silenced plants did not show difference compared to wild type phenotypes, PhGI2-silenced plants flowered two to three weeks later, confirming itself as a late-flowering mutant, as already described in Arabidopsis thaliana. These data confirmed the previously hypothesized idea that some duplicate may undergo subfunctionalization or neofunctionalization. Escuela Internacional de Doctorado de la Universidad Politécnica de Cartagena Universidad Politécnica de Cartagena Programa de Doctorado de Técnicas avanzadas en investigación y desarrollo agrario y alimentario

Temporal variability of the genetic structure and connectivity patterns of the blue and red shrimp Aristeus antennatus in the seven most important fishing grounds of the Western Mediterranean Sea, were assessed using twelve microsatellite loci during 2 consecutive years (2016 and 2017), in a total of 1403 adult individuals. A high level of geographical connectivity among groups was observed in the two studied years. In fact, no significant geographical differentiation was found in 2016 (FST = 0.0018, p > 0.05), whereas it was indicated in 2017 (FST = 0.0025, p FCT = 0.0006, p A. antennatus.

This work gives novel information about the antimicrobial effect and volatiles of melanoidins isolated from Maria biscuit, common and soft bread. Melanoidins were isolated from scraped and sieved crusts (1 mm), after gluten digestion, 10 kDa ultrafiltration, and diafiltration. Finally, they were freeze-dried. Headspace solid-phase dynamic extraction coupled with a gas chromatograph with a mass spectrometer was used to determine the volatile profiles. The antimicrobial effect was evaluated against isolated strains of the most relevant food spoilage and pathogen microorganisms, together with some molds and yeasts. Melanoidins from common bread exhibited the most extensive antimicrobial activities and showed the most composite volatile profile. No undesirable compounds, such as furfural and 5-hydroxy-methyl-furfural, were found in any of the melanoidins studied. The obtained data pointed out that bakery melanoidins can exert effective food technological properties as natural antimicrobials that can improve shelf-life and security of foodstuffs, together with a possible contribution to food aroma., Government of Autonomous Community of Castile-Leon and FEDER funds [JCyL/FEDER, BU243P18].

The Museu Imperial has an important historical collection from the Portuguese Royal Family in Brazil. The throne represents the imperial power and is considered one of the most important objects in the museum; however, the piece has been facing for decades a gradual deterioration, mainly in its fabrics. With the growing motivation to preserve this property, conservation and restoration processes were conducted in all parts of the throne. As a part of this treatment, a microbiological survey was conducted in selected parts of the object and the surrounding environment where it was placed. This biodeterioration study, control and detection of microbial species indicated a very pertinent answer in relation to the place it was exposed, and the restoration process was performed in order to solve the most pronounced damages aiming to restore the original characteristics of the artefact. The study indicated a high diversity of fungi and bacteria. It was observed a marked decrease, before and after restoration, being observed the recurrence of the genera Absidia, Cladosporium and Epicoccum. This reduction was not so pronounced for bacteria, being also observed the recurrence of some bacterial groups after restoration (Microbacterium, Staphylococcus and Kocuria). In the atmosphere new fungal species were found after restoration, indicating a natural recontamination of the exhibition area., O Museu Imperial tem uma importante coleção histórica da Família Real Portuguesa no Brasil. O trono representa o poder imperial, e é considerado uma das peças mais importantes do Museu. Entretanto, vinha enfrentando há décadas uma deterioração gradual, principalmente devido aos seus componentes em tecido. Devido ao interesse em preservar o objeto, processos de conservação e restauro foram executados em todas as partes do trono. Como parte deste tratamento, uma inspeção microbiológica foi executada em partes selecionadas do objeto, bem como no seu ambiente de guarda. Este estudo de biodeterioração, controle e detecção de espécies microbianas indicou uma clara resposta em relação ao local de guarda, e então o processo de restauro pode ser executado visando solucionar os danos mais pronunciados objetivando restaurar as características originais do artefacto. O estudo indicou uma alta diversidade de fungos e bactérias. Foi observada uma redução de espécies fúngicas detectadas no trono antes e após o restauro, observando-se a recorrência dos gêneros Absidia, Cladosporium e Epicoccum. Essa redução não foi muito pronunciada para as bactérias, observando-se, também, recorrência bacteriana após o restauro (Microbacterium, Staphylococcus e Kocuria). No ar, observou-se o aparecimento de novas espécies fúngicas após o restauro, indicando recontaminação natural na área expositiva.

22q11.2 deletion syndrome (22q11DS)—a neurodevelopmental condition caused by a hemizygous deletion on chromosome 22—is associated with an elevated risk of psychosis and other developmental brain disorders. Prior single-site diffusion magnetic resonance imaging (dMRI) studies have reported altered white matter (WM) microstructure in 22q11DS, but small samples and variable methods have led to contradictory results. Here we present the largest study ever conducted of dMRI-derived measures of WM microstructure in 22q11DS (334 22q11.2 deletion carriers and 260 healthy age- and sex-matched controls; age range 6–52 years). Using harmonization protocols developed by the ENIGMA-DTI working group, we identified widespread reductions in mean, axial and radial diffusivities in 22q11DS, most pronounced in regions with major cortico-cortical and cortico-thalamic fibers: the corona radiata, corpus callosum, superior longitudinal fasciculus, posterior thalamic radiations, and sagittal stratum (Cohen’s d’s ranging from −0.9 to −1.3). Only the posterior limb of the internal capsule (IC), comprised primarily of corticofugal fibers, showed higher axial diffusivity in 22q11DS. 22q11DS patients showed higher mean fractional anisotropy (FA) in callosal and projection fibers (IC and corona radiata) relative to controls, but lower FA than controls in regions with predominantly association fibers. Psychotic illness in 22q11DS was associated with more substantial diffusivity reductions in multiple regions. Overall, these findings indicate large effects of the 22q11.2 deletion on WM microstructure, especially in major cortico-cortical connections. Taken together with findings from animal models, this pattern of abnormalities may reflect disrupted neurogenesis of projection neurons in outer cortical layers. Sin financiación 15.992 JCR (2020) Q1, 10/295 Biochemistry & Molecular Biology 5.071 SJR (2020) Q1, 5/85 Cellular and Molecular Neuroscience No data IDR 2020 UEM

Venoms constitute complex mixtures of many different molecules arising from evolution in processes driven by continuous prey–predator interactions. One of the most common compounds in these venomous cocktails are pore-forming proteins, a family of toxins whose activity relies on the disruption of the plasmatic membranes by forming pores. The venom of sea anemones, belonging to the oldest lineage of venomous animals, contains a large amount of a characteristic group of pore-forming proteins known as actinoporins. They bind specifically to sphingomyelin-containing membranes and suffer a conformational metamorphosis that drives them to make pores. This event usually leads cells to death by osmotic shock. Sticholysins are the actinoporins produced by Stichodactyla helianthus. Three different isotoxins are known: Sticholysins I, II, and III. They share very similar amino acid sequence and three-dimensional structure but display different behavior in terms of lytic activity and ability to interact with cholesterol, an important lipid component of vertebrate membranes. In addition, sticholysins can act in synergy when exerting their toxin action. The subtle, but important, molecular nuances that explain their different behavior are described and discussed throughout the text. Improving our knowledge about sticholysins behavior is important for eventually developing them into biotechnological tools.

The question of how phenotypic and genomic complexity are inter‐related and how they are shaped through evolution is a central question in biology that historically has been approached from the perspective of animals and plants. In recent years, however, fungi have emerged as a promising alternative system to address such questions. Key to their ecological success, fungi present a broad and diverse range of phenotypic traits. Fungal cells can adopt many different shapes, often within a single species, providing them with great adaptive potential. Fungal cellular organizations span from unicellular forms to complex, macroscopic multicellularity, with multiple transitions to higher or lower levels of cellular complexity occurring throughout the evolutionary history of fungi. Similarly, fungal genomes are very diverse in their architecture. Deep changes in genome organization can occur very quickly, and these phenomena are known to mediate rapid adaptations to environmental changes. Finally, the biochemical complexity of fungi is huge, particularly with regard to their secondary metabolites, chemical products that mediate many aspects of fungal biology, including ecological interactions. Herein, we explore how the interplay of these cellular, genomic and metabolic traits mediates the emergence of complex phenotypes, and how this complexity is shaped throughout the evolutionary history of Fun T.G. acknowledges support from the Spanish Ministry of Science and Innovation for grants ‘Centro de Excelencia Severo Ochoa’ and PGC2018‐099921‐B‐I00, cofunded by the European Regional Development Fund (ERDF); from the CERCA Programme/Generalitat de Catalunya; from the Catalan Research Agency (AGAUR) SGR423, and grants from the European Union's Horizon 2020 research and innovation programme under the grant agreement ERC‐2016‐724173, and the Marie Sklodowska‐Curie grant agreement No H2020‐MSCA‐IF‐2017‐793699. T.G.'s research group also receives support from a INB Grant (PT17/0009/0023 ‐ ISCIII‐SGEFI/ERDF).

Introducción. El género Meloidogyne contiene muchas especies y el diagnóstico correcto de estas debe basarse en el uso de múltiples herramientas, mismas que en laboratorios de diagnóstico permiten una evaluación correcta y confiable, especialmente cuando podría tratarse de nematodos cuarentenarios. La identificación adecuada de un fitonematodo es importante no solo para establecer una apropiada estrategia de control, sino que también para evitar su diseminación a otras áreas donde podría provocar daños que se traducirían en pérdidas económicas. Objetivo. Realizar estudios taxonómicos y moleculares para confirmar la especie de Meloidogyne, asociada a una plantación de café en la localidad de San Pedro de Barva, Heredia. Materiales y métodos. Se recolectaron muestras de suelo y raíz en una plantación de café ubicada en Barva, Heredia, Costa Rica, las cuales se procesaron en el laboratorio de Nematología de la Escuela de Ciencias Agrarias de la Universidad Nacional (UNA), por el método centrifugación-flotación en solución azucarada y técnicas moleculares. Resultados. En promedio se contabilizaron 30 J2 en 100 cc-1 en suelo y 1000 J2 en 10 g-1 de raíces del nematodo agallador. Tanto las técnicas taxonómicas como moleculares permitieron identificar que las hembras y juveniles extraídos de raíces de café correspondieron a Meloidogyne exigua. El análisis de las secuencias generadas mediante PCR y el uso de la enzima Dral confirmaron la identidad de la especie con un 99 % de similitud al compararla con secuencias de Costa Rica y Nicaragua, reportadas en el Genbank. Conclusión. Mediante el uso de diferentes técnicas morfométricas y moleculares se identificó a M. exigua asociada a una plantación de café. Introduction. The genus Meloidogyne contains many species and their correct diagnosis of these must be based on the use of multiple tools, which in diagnostic laboratories allow a correct and reliable evaluation, specially for quarantine nematodes. The proper identification of a phytonematode is important not only to establish an appropriate control strategy, but also to avoid its spread to other areas where it could cause damage that would result in economic losses. Objective. To perform taxonomic and molecular studies to confirm the Meloidogyne species, associated to a coffee plantation located in Barva, Heredia. Materials and methods. Soil and root samples were collected in a coffee plantation located in Barva, Heredia, Costa Rica, which were processed in the Nematology laboratory of the School of Agricultural Sciences at the Universidad Nacional (UNA), using the centrifugation-flotation method in sugary solution and molecular techniques. Results. On average, 30 J2 100 cc-1 were counted in soil and 1000 J2 10 g-1 of roots of the root-knot nematode. Taxonomic and molecular techniques allowed to identify that the females and juveniles extracted from coffee roots corresponded to Meloidogyne exigua. Analysis of the sequences generated by PCR and the use of the DraI enzyme confirmed the identity of the species with 99 % similarity when compared to sequences from Costa Rica and Nicaragua, reported in the Genbank. Conclusion. Using different morphometric and molecular techniques, M. exigua associated with a coffee plantation was identified. Universidad Nacional, Costa Rica Escuela de Ciencias Agrarias

Background Cratera is a genus of land planarians endemic to the Brazilian Atlantic forest. The species of this genus are distinguished from each other by a series of external and internal characters, nonetheless they represent a challenging taxonomic issue due to the extreme alikeness of the species analysed in the present work. To resolve these difficulties, we have performed morphological analyses and used three nuclear markers (ribosomal 18S and 28S, Elongation Factor, a new anonymous marker named Tnuc813) and two mitochondrial fragments (Cytochrome c oxidase subunit I gene, and a fragment encompasing NADH deshydrogenase subunit 4 gene, trnF and the beginning of the Cytochrome c oxidase I gene) in an integrative taxonomic study. Methods To unveil cryptic species, we applied a molecular species delimitation approach based on molecular discovery methods, followed by a validation method. The putative species so delimited were then validated on the basis of diagnostic morphological features. Results We discovered and described four new species, namely Cratera assu, C. tui, C. boja, and C. imbiri. A fifth new species, C. paraitinga was not highly supported by molecular evidence, but was described because its morphological attributes are unique. Our study documents for the genus Cratera the presence of a number of highly similar species, a situation that is present also in other genera of land planarians. The high number of poorly differentiated and presumably recent speciation events might be explained by the recent geological history of the area.

Uniparental disomy (UPD) is an underestimated cause of autosomal recessive disorders. In this study, we aim to raise awareness about the possibility of UPD in mitochondrial disorders - where it is a hardly described event -, by functionally characterizing a novel variant in a structural subunit of complex I (CI) of the mitochondrial oxidative phosphorylation system. Using next-generation sequencing, we identified a new intronic homozygous c.350 + 5G > A variant in the NDUFS4 gene in a one-year-old girl (being alive at the age of 7) belonging to a non-consanguineous family presenting with encephalopathy, psychomotor delay, lactic acidosis and a single CI deficiency, a less severe phenotype than those previously reported in most NDUFS4 patients. One parent lacked the variant, and microsatellite genotyping showed complete paternal uniparental isodisomy of the non-imprinted chromosome 5. We demonstrated in patient's skeletal muscle and fibroblasts splicing abnormalities, low expression of NDUFS4, undetectable NDUFS4 protein, defects in cellular respiration (decreased oxygen consumption and ATP production), and impaired assembly or stability of mitochondrial supercomplexes containing CI. Our findings support that c.350 + 5G > A variant is pathogenic, and reinforce that UPD, although rare, should be considered as a possible cause of mitochondrial diseases in order to provide accurate genetic counselling. Sin financiación 4.797 JCR (2020) Q2, 47/145 Endocrinology & Metabolism 1.329 SJR (2020) Q1, 101/438 Biochemistry No data IDR 2019 UEM

Según datos de la Organización Mundial de la Salud (OMS), la enfermedad de Chagas es una de las principales enfermedades desatendidas del subcontinente latinoamericano. El agente causante es el parásito protozoario Tripanosoma cruzi, transmitido a humanos por insectos triatominos de distintas especies. Debido a la ausencia de vacunas y tratamientos efectivos para la etapa crónica de la enfermedad, el control del vector sigue siendo el medio elegido para reducir el riesgo de transmisión. Después de casi 30 años de tratamiento con insecticidas piretroides, han emergido poblaciones de Triatoma infestans con altísimos niveles de resistencia, asociadas a fallas en las campañas de control, lo que plantea la necesidad de programas para el manejo de resistencia a nivel regional. Un requerimiento fundamental para ello es la detección temprana de la dispersión de poblaciones e individuos resistentes, así como del surgimiento de nuevos focos. Los piretroides ejercen su acción insecticida modificando el funcionamiento fisiológico de canales proteicos de Na+ dependientes de voltaje (NaV), presentes en la membrana de células excitables. Se conoce como kdr (knockdown resistance) a la reducción en la sensibilidad a piretroides causada por mutaciones puntuales en el gen NaV. El mecanismo de resistencia tipo kdr ha sido descrito en numerosas especies de insectos de interés económico y sanitario. La gran mayoría de las mutaciones correlacionadas con resistencia a piretroides se encuentra en el dominio II de esta proteína. El diseño de ensayos moleculares para detección de mutaciones asociadas a resistencia habilita la detección temprana de dispersión y surgimiento de nuevos focos, al permitir detectar la presencia de dichas mutaciones en insectos individuales, cuando la frecuencia poblacional de las mismas aún es baja. La detección temprana no puede conseguirse con ensayos de toxicidad, que detectan resistencia a nivel poblacional y no individual. En este sentido, en la primera parte de esta Tesis se realizó una evaluación de la sensibilidad y una optimización de los métodos moleculares de detección de mutaciones asociadas a resistencia, seguida de un estudio de la presencia de mutaciones para la región IIS4-IIS6 del gen del canal TiNav presentes en distintas poblaciones de T. infestans resistentes a piretroides provenientes de la ecoregión del Gran Chaco. Por otro lado, a través de una secuencia de TiNav detectada en una base de datos transcriptómica depositada en vector base (https://www.vectorbase.org/), complementada con el clonado y pirosecuenciación de regiones concretas del gen, se obtuvo la secuencia nucleotídica completa del mismo, y se realizó una caracterización bioinformática de la secuencia aminoacídica. Los resultados de esta parte del trabajo podrían tener aplicabilidad en el manejo de resistencia dentro de las campañas de control primario de Chagas. Durante el transcurso de la primera parte de la Tesis hemos encontrado que, aunque las mutaciones en el gen TiNav parecen ser la principal causa de resistencia a piretroides en T. infestans provenientes del Gran Chaco, no toda la resistencia es explicable por estos polimorfismos; distintas poblaciones con la misma frecuencia en una mutación kdr presentaron tasas de resistencia muy variables. Esto puede indicar que existen otros mecanismos de resistencia potencialmente involucrados en el fenómeno, tales como procesos de detoxificación y cambios en la penetrancia cuticular. En la segunda parte de esta Tesis, se realizó un estudio comparativo de la expresión diferentes enzimas detoxificativas entre una población sensible y una resistente de T. infestans provenientes del Gran Chaco. También se estudió la expresión génica en una población sensible expuesta a deltametrina, a fin de estudiar su posible papel en la respuesta detoxificativa a insecticidas piretroides, y posiblemente en la resistencia. Los experimentos mostraron una sobreexpresión de un citocromo P450 del clado 4 en la población resistente de T. infestans del Gran Chaco. No detectamos cambios en la expresión de las enzimas estudiadas cinco horas después de una topicación con deltametrina. Se estudió el rol de una Glutatión Transferasa del clado Delta (deltaGST) en la detoxificación de deltametrina. Se realizaron estudios bioinformáticos y de fisiología molecular usando como modelo Rhodnius prolixus. Se registró un aumento en la letalidad causada por dosis bajas de deltametrina cuando la expresión del gen de gst delta fue disminuida significativamente con técnicas de ARN de interferencia. La llamativa conservación en la estructura, función y farmacología del canal de sodio dependiente de voltaje a través del reino animal, genera que aquellos insecticidas que tengan como blanco esta molécula posean también una toxicidad potencial para otras especies. En este sentido, y teniendo en cuenta que las moléculas del sistema neuroendocrino (neuropéptidos y sus receptores) han sido propuestos como blanco de insecticidas, en el Capítulo 3 se estudió la expresión diferencial de genes precursores de diferentes neuropéptidos entre una población de T. infestans sensible a piretroides y otra resistente, a fin de obtener indicios de su posible papel en procesos asociados a resistencia. Por otra parte, se comparó el efecto de deltametrina en la inducción de la expresión de genes precursores de neuropéptidos en una población susceptible de T. infestans. En el Capítulo 4, a partir de secuencias ortólogas en Anopheles gambiae, Apis mellifera, Bombyx mori, Drosophila melanogaster y Tribolium castaneum, se identificaron genes de receptores de neuropéptidos en transcriptomas de T. infestans, Triatoma dimidiata y Triatoma pallidipennis generados en nuestro laboratorio, así como en el genoma de R. prolixus, por medio de búsquedas en bases de datos y análisis filogenéticos. Se espera que esta última parte del trabajo de Tesis aporte conocimientos para ampliar los horizontes en la investigación de posibles nuevos blancos de insecticidas, que sean capaces de reemplazar o complementar a los neurotóxicos dentro de estrategias de manejo integrado de plagas., Facultad de Ciencias Exactas

© 2020 by the authors., The complexity of hepatocellular carcinoma (HCC) challenges the identification of disease-relevant signals. C3G, a guanine nucleotide exchange factor for Rap and other Ras proteins, plays a dual role in cancer acting as either a tumor suppressor or promoter depending on tumor type and stage. The potential relevance of C3G upregulation in HCC patients suggested by database analysis remains unknown. We have explored C3G function in HCC and the underlying mechanisms using public patient data and in vitro and in vivo human and mouse HCC models. We found that C3G is highly expressed in progenitor cells and neonatal hepatocytes, whilst being down-regulated in adult hepatocytes and re-expressed in human HCC patients, mouse HCC models and HCC cell lines. Moreover, high C3G mRNA levels correlate with tumor progression and a lower patient survival rate. C3G expression appears to be tightly modulated within the HCC program, influencing distinct cell biological properties. Hence, high C3G expression levels are necessary for cell tumorigenic properties, as illustrated by reduced colony formation in anchorage-dependent and -independent growth assays induced by permanent C3G silencing using shRNAs. Additionally, we demonstrate that C3G down-regulation interferes with primary HCC tumor formation in xenograft assays, increasing apoptosis and decreasing proliferation. In vitro assays also revealed that C3G down-regulation enhances the pro-migratory, invasive and metastatic properties of HCC cells through an epithelial-mesenchymal switch that favors the acquisition of a more mesenchymal phenotype. Consistently, a low C3G expression in HCC cells correlates with lung metastasis formation in mice. However, the subsequent restoration of C3G levels is associated with metastatic growth. Mechanistically, C3G down-regulation severely impairs HGF/MET signaling activation in HCC cells. Collectively, our results indicate that C3G is a key player in HCC. C3G promotes tumor growth and progression, and the modulation of its levels is essential to ensure distinct biological features of HCC cells throughout the oncogenic program. Furthermore, C3G requirement for HGF/MET signaling full activation provides mechanistic data on how it works, pointing out the relevance of assessing whether high C3G levels could identify HCC responders to MET inhibitors, This work was funded by grants from the Spanish Ministry of Economy and Competitiveness [SAF2013-48210-C2-2-R and SAF2016-76588-C2-1-R to AP; and SAF2013-48210-C2-1-R and SAF2016-76588-C2-2-R to CG], and by two grants from the Council of Education of Junta de Castilla y León, Spain [SA157A12-1 and SA017U16 to CG]. All funding was cosponsored by the European FEDER Program. CS was supported by a fellowship from Complutense University from Madrid and a short-term FEBS’ fellowship. S.M. is a recipient of a FPU fellowship from Spanish Ministry of Education. A G-U is supported by Madrid Community Program for Talent Attraction. P.B. received support from BBVA (Becas Leonardo 2018, BBM-TRA-0041) and F.M. from FdF (Fondation de France; 2014_00051580 and 2016_00067080), ARC (Association pour la Recherche sur le Cancer; PJA20181208172), and GEFLUC – Les Entreprises contre le Cancer. M.A. was supported by a FdF fellowship.

espanolObjetivo. Determinar las caracteristicas biologicas y moleculares de las cepas de Panstrongylussp. nativas del norte del Peru. Metodos. Estudio observacional, descriptivo. Se utilizo material biologico procedente de 5 diferentes regiones del norte del Peru, y por muestreo intencional se selecciono una muestra de 50 especimenes. Para la caracterizacion de los especimenes a nivel molecular, se uso el marcador ITS-2 del ADN ribosomal, mediante la tecnica del PCR. Resultados. El 100% de las muestras fueron clasificadas como Panstrongylus herreri. A nivel molecular se observaron 3 patrones diferentes de la banda ITS-2: 960pb, 800pb y 750pb. Conclusion. 100% de las muestras fueron Panstrongylus herreri, capturados en ambientes intradomiciliarios de regiones del norte del Peru, con un patron de ITS-2 EnglishObjective. To determine the biological and molecular characteristics of the Panstrongylus sp. native to northern Peru. Methods. Observational, descriptive study. Biological material from 5 different regions of northern Peru was used, and a sample of 50 specimens was selected by intentional sampling. For the characterization of the specimens at the molecular level, the ITS-2 marker of ribosomal DNA was used, using the PCR technique. Results. 100% of the samples were classified as Panstrongylus herreri. At the molecular level, 3 different patterns of the ITS-2 band were observed: 960bp, 800bp and 750bp. Conclusion. 100% of the samples were Panstrongylus herreri, captured in intra-household environments in northern regions of Peru, with an ITS-2 pattern.