Structural and RNAi characterization of the German cockroach lipophorin receptor, and the evolutionary relationships of lipoprotein receptors

This article is available from: http://www.biomedcentral.com/1471-2199/8/53[Background] Lipophorin receptors (LpRs) have been described in a number of insects, but functional studies have been reported only in locusts and mosquitoes. The aim of the present work was to characterize the LpR of the cockroach Blattella germanica, not only molecularly but also functionally using RNAi techniques, and to place LpRs in a phylogenetical context among lipoprotein receptors.[Results] We cloned a putative LpR from B. germanica (BgLpR) using RT-PCR methods. Two isoforms of BgLpR that differ from each other by an insertion/deletion of 24 amino acids were obtained from the fat body and the ovary. A phylogenetical analysis of lipoprotein receptors showed that BgLpR grouped with other sequences annotated as LpR in a cluster placed as a sister group of vertebrate low density lipoprotein receptors (LDLR) + lipoprotein receptor-related proteins 8 (LPR8) + vitellogenin receptors (VgR) + very low density lipoprotein receptors (VLDLR). The two BgLpR isoforms are expressed in different adult female tissues (fat body, ovary, brain, midgut, muscle) and in embryos. In ovaries and fat body, the two isoforms are similarly expressed during the first gonadotrophic cycle. mRNA levels in the fat body increase in parallel to vitellogenesis, whereas they decrease in the ovaries. BgLpR protein levels increase in parallel to vitellogenesis in both organs. Treatment with juvenile hormone increases BgLpR protein. RNAi experiments show that females with lower BgLpR expression have less lipophorin in the growing oocytes with respect to controls.[Conclusion] The two isoforms of BgLpR are structurally similar to other LpRs. Phylogenetical analyses show that LpRs and the group formed by vertebrate LDLR + LPR8 + VgR + VLDLR, diverged from a common ancestor and diversified in parallel. The different expression patterns in the fat body and the ovary, comparing mRNA and protein, indicate that the corresponding mechanisms regulating BgLpR expression are different. Experiments with JH III suggest that such a hormone regulates the expression of BgLpR posttranscriptionally. RNAi experiments indicate that BgLpR is a functional lipophorin receptor.Financial support from the Ministry of Education and Science, Spain (projects BOS2002-03359, BFU2005-00264, AGL2002-01169, AGL2005- 00773) and the Generalitat de Catalunya (2001 SGR 003245) is gratefully acknowledged. L.C. is recipient of pre-doctoral research grants (I3P) from CSIC. We thank Prof. Coby Schal (North Carolina State University) for generous gifts of anti-lipophorin antibody.Peer reviewe

Ciberfeminismo y TIC a través de un ABP interdisciplinar

EL presente TFM conforma un proyecto de innovación que consiste en un cambio sustancial en la metodología a la hora de impartir los contenidos curriculares de los bloques 3 y 6 (Organización, diseño y producción de información digital; y Publicación y difusión de contenidos) de la asignatura TIC de 4º de la ESO. Además, este proyecto introduce la perspectiva de género en la temática del proyecto de manera clara y concisa, elemento transversal del currículo con el que se pretende conseguir un mayor conocimiento y concienciación con la problemática que supone la desigualdad de género y los estereotipos de género en la sociedad. La asamblea feminista que ya poseía el propio centro educativo y sus redes sociales han sido el hilo conductor que han permitido diseñar un ABP interdisciplinar en el que el alumnado sea protagonista del diseño y publicación del contenido multimedia de dichas redes sociales. Este proyecto pretende la mejora del aprendizaje propio de la asignatura TIC dándole un sentido y un valor al contenido multimedia creado por el alumnado; fomentar el conocimiento y la concienciación sobre temas relacionados con la igualdad de género a través del propio proceso de investigación del ABP; y dar a conocer y fomentar la participación del alumnado en la asamblea feminista del instituto.<br /

Hábitos de alimentación y actividad física de la población infantil en una zona rural de Zaragoza

La alimentación es uno de los principales determinantes del estado de salud del ser humano y el factor extrínseco más importante para su desarrollo. Una alimentación adecuada es fundamental a lo largo de toda la vida, pero durante la infancia es particularmente crucial, pues las carencias y desequilibrios nutricionales en esta etapa tienen consecuencias negativas no sólo en la salud del propio niño, sino que pueden condicionar su salud durante la vida adulta, aumentando el riesgo de desarrollar trastornos crónicos (cáncer, hipertensión arterial, cardiopatía isquémica y otras enfermedades cardiovasculares, enfermedades cerebrovasculares, diabetes mellitus tipo 2, obesidad, osteoporosis) , las cuales constituyen las principales causas de mortalidad y/o morbilidad y discapacidad en España. Se llevó a cabo un estudio descriptivo transversal mediante una encuesta nutricional en una muestra de niños de 9 a 11 años (n=90) del colegio Mamés Esperabé de Ejea de los Caballeros, con el principal objetivo de evaluar la situación de salud, alimentación y ejercicio físico. La encuesta utilizada incluía tres partes; datos generales y sociodemográficos, ejercicio físico, alimentación y hábitos alimentarios. Al finalizar el estudio observamos que la prevalencia de sobrepeso-obesidad de los alumnos de 9-10 años es de 46,1%, sin embargo, en el rango de 10-12 años es de un 9,8%. La prevalencia de sobrepeso-obesidad en niños es similar a la de niñas. Observamos un abuso de conductas sedentarias y un porcentaje alto de alum nos norealiza 5 comidas al dí

Base-Mediated Nitrophenyl Reductive Cyclization for the Synthesis of Hexahydro-2,6-methano-1-benzazocines

A Diels-Alder reaction leading to 4-nitrophenylcyclohexanones followed by a newly developed base-mediated reductive cyclization of the resulting ketone tethered to the nitrobenzene moiety gives access to the hexahydro-2,6-methano-1-benzazocine ring system present in several biologically interesting natural products such as aspernomine. The scope of the reaction was explored with eight substituted nitrobenzenes, obtaining yields of up to 87%. The highest cytotoxicity was observed in benzazocine 4h, bearing an enone moiety, which was active against eight cancer cell lines

Circulating cell-free DNA methylation mirrors alterations in cerebral patterns in epilepsy

Background: DNA methylation profiling of circulating cell-free DNA (cfDNA) has rapidly become a promising strategy for biomarker identification and development. The cell-type-specific nature of DNA methylation patterns and the direct relationship between cfDNA and apoptosis can potentially be used non-invasively to predict local alterations. In addition, direct detection of altered DNA methylation patterns performs well as a biomarker. In a previous study, we demonstrated marked DNA methylation alterations in brain tissue from patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS). Results: We performed DNA methylation profiling in cfDNA isolated from the serum of MTLE patients and healthy controls using BeadChip arrays followed by systematic bioinformatic analysis including deconvolution analysis and integration with DNase accessibility data sets. Differential cfDNA methylation analysis showed an overrepresentation of gene ontology terms and transcription factors related to central nervous system function and regulation. Deconvolution analysis of the DNA methylation data sets ruled out the possibility that the observed differences were due to changes in the proportional contribution of cortical neurons in cfDNA. Moreover, we found no overrepresentation of neuron- or glia-specific patterns in the described cfDNA methylation patterns. However, the MTLE-HS cfDNA methylation patterns featured a significant overrepresentation of the epileptic DNA methylation alterations previously observed in the hippocampus. Conclusions: Our results support the use of cfDNA methylation profiling as a rational approach to seeking non-invasive and reproducible epilepsy biomarkers.This study has been supported by R+D+i project PID2020-117212RB-I00 funded by MCIN/AEI/10.13039/501100011033. This work has also been par‑ tially supported by a BICE Tecnifar Grant. RM-F is funded by an FCT (Fundação para a Ciência e Tecnologia) fellowship (SFRH/BD/137900/2018). UMIB is funded by FCT Portugal (UIDB/00215/2020 and UIDP/00215/2020) and ITR (LA/P/006/2020).info:eu-repo/semantics/publishedVersio

Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins

Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5'UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS

SIRT1/2 orchestrate acquisition of DNA methylation and loss of histone H3 activating marks to prevent premature activation of inflammatory genes in macrophages

Sirtuins 1 and 2 (SIRT1/2) are two NAD-dependent deacetylases with major roles in inflammation. In addition to deacetylating histones and other proteins, SIRT1/2-mediated regulation is coupled with other epigenetic enzymes. Here, we investigate the links between SIRT1/2 activity and DNA methylation in macrophage differentiation due to their relevance in myeloid cells. SIRT1/2 display drastic upregulation during macrophage differentiation and their inhibition impacts the expression of many inflammation-related genes. In this context, SIRT1/2 inhibition abrogates DNA methylation gains, but does not affect demethylation. Inhibition of hypermethylation occurs at many inflammatory loci, which results in more drastic upregulation of their expression upon macrophage polarization following bacterial lipopolysaccharide (LPS) challenge. SIRT1/2-mediated gains of methylation concur with decreases in activating histone marks, and their inhibition revert these histone marks to resemble an open chromatin. Remarkably, specific inhibition of DNA methyltransferases is sufficient to upregulate inflammatory genes that are maintained in a silent state by SIRT1/2. Both SIRT1 and SIRT2 directly interact with DNMT3B, and their binding to proinflammatory genes is lost upon exposure to LPS or through pharmacological inhibition of their activity. In all, we describe a novel role for SIRT1/2 to restrict premature activation of proinflammatory genes

Epstein–Barr virus-mediated transformation of B cells induces global chromatin changes independent to the acquisition of proliferation

Epstein-Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation

Vitamin C enhances NF-κB-driven epigenomic reprogramming and boosts the immunogenic properties of dendritic cells

Dendritic cells (DCs), the most potent antigen-presenting cells, are necessary for effective activation of naïve T cells. DCs' immunological properties are modulated in response to various stimuli. Active DNA demethylation is crucial for DC differentiation and function. Vitamin C, a known cofactor of ten-eleven translocation (TET) enzymes, drives active demethylation. Vitamin C has recently emerged as a promising adjuvant for several types of cancer; however, its effects on human immune cells are poorly understood. In this study, we investigate the epigenomic and transcriptomic reprogramming orchestrated by vitamin C in monocyte-derived DC differentiation and maturation. Vitamin C triggers extensive demethylation at NF-κB/p65 binding sites, together with concordant upregulation of antigen-presentation and immune response-related genes during DC maturation. p65 interacts with TET2 and mediates the aforementioned vitamin C-mediated changes, as demonstrated by pharmacological inhibition. Moreover, vitamin C increases TNFβ production in DCs through NF-κB, in concordance with the upregulation of its coding gene and the demethylation of adjacent CpGs. Finally, vitamin C enhances DC's ability to stimulate the proliferation of autologous antigen-specific T cells. We propose that vitamin C could potentially improve monocyte-derived DC-based cell therapies