mRNA expression analysis of the SUMO pathway genes in the adult mouse retina

Sumoylation is a reversible post-translational modification that regulates different cellular processes by conjugation/deconjugation of SUMO moieties to target proteins. Most work on the functional relevance of SUMO has focused on cell cycle, DNA repair and cancer in cultured cells, but data on the inter-dependence of separate components of the SUMO pathway in highly specialized tissues, such as the retina, is still scanty. Nonetheless, several retinal transcription factors (TFs) relevant for cone and rod fate, as well as some circadian rhythm regulators, are regulated by sumoylation. Here we present a comprehensive survey of SUMO pathway gene expression in the murine retina by quantitative RT-PCR and in situ hybridization (ISH). The mRNA expression levels were quantified in retinas obtained under four different light/dark conditions, revealing distinct levels of gene expression. In addition, a SUMO pathway retinal gene atlas based on the mRNA expression pattern was drawn. Although most genes are ubiquitously expressed, some patterns could be defined in a first step to determine its biological significance and interdependence. The wide expression of the SUMO pathway genes, the transcriptional response under several light/dark conditions, and the diversity of expression patterns in different cell layers clearly support sumoylation as a relevant post-translational modification in the retina. This expression atlas intends to be a reference framework for retinal researchers and to depict a more comprehensive view of the SUMO-regulated processes in the retina

Expression atlas of the Deubiquitinating enzymes in the adult mouse retina, their evolutionary diversification and phenotypic roles

Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

SUMO a la retina

[cat] La sumoïlació és una modificació posttraduccional (PTM) que consisteix en la conjugació covalent i reversible d’una proteïna SUMO (Small ubiquitin-like modifier) al residu lisina d’una proteïna diana. Igual que la ubiquitina, el cicle metabòlic de SUMO consta d’una conjugació, on hi participen tres tipus diferents d’enzims (E1 activadors, E2 conjugadors i E3 lligases), i d’una deconjugació, que es dóna a través de l’acció de proteases. A nivell funcional, la sumoïlació regula processos cel·lulars tan diversos com la replicació, la reparació del DNA, la localització, activitat i degradació de proteïnes, o la transcripció, entre d’altres. Els estudis existents sobre la sumoïlació se centraven en la modificació de proteïnes diana concretes, pel que mancaven estudis de la interdependència dels elements de la maquinària de SUMO en la regulació i el manteniments de teixits especialitzats. Un d’aquests teixits és la retina, que recobreix l’ull per la seva cara interna i s’encarrega de la captació i el processat de l’estímul lumínic. Forma part del sistema nerviós central, pel que es tracta de teixit neuronal, format per tres capes nuclears de neurones i les seves connexions. Les cèl·lules encarregades d’iniciar la cadena de la fototransducció són els fotoreceptors: cons i bastons; que comparteixen una mateixa estructura, i que difereixen en els fotopigments que hi contenen, fet que els dóna diferents funcionalitats lumíniques. Per aquest motiu, un dels objectius d’aquesta tesi va ser l’estudi de l’expressió dels gens de la via de SUMO a la retina de ratolí adult, per mitjà de la seva quantificació per RT-qPCR i de la seva localització en talls de retina per hibridació in situ. Es van detectar l’expressió de tots els gens de la maquinària de SUMO, amb nivells variables en concordança amb la seva importància dins del cicle. . A més, atès que la llum és un dels marcadors externs que controlen els ritmes circadiaris de l’organisme, es va estudiar l’expressió dels gens de la via de SUMO en diferents condicions del cicle de llum/foscor, detectant alguns patrons diferencials d’entre el conjunt de gens estudiats. Pel que fa a la localització de l’expressió, els gens de la via de SUMO van donar diferents patrons de localització a les diverses capes de la retina, coincidint tots a la capa de fotoreceptors. A continuació, es va estudiar la sumoïlació del factor de transcripció (TF) de fotoreceptors NR2E3, que juga un paper central en el desenvolupament i manteniment dels bastons, activant els gens específics de bastons i silenciant els de cons. Aquesta repressió està regulada, en part, per la seva sumoïlació a través de la E3 lligasa PIAS3, tot i que aquesta no és la única que modificava el TF. En aquest context, es va plantejar l’estudi de noves interaccions del TF amb altres E3 lligases de SUMO. Per mitjà de la tècnica de BRET es va poder trobar una nova interacció amb HDAC4, posteriorment confirmada per CoIP. A més, l’estudi comparatiu de la forma humana d’NR2E3 WT amb el seu mutant no sumoïlable (123) va permetre detectar diferències d’interacció amb PIAS3, més baixa amb el TF mutant. D’altra banda, la tècnica del BioID va permetre el descobriment d’una segona interacció amb una E3 lligasa de SUMO: CBX4. Per últim, amb la comparació de les formes WT i no sumoïlable d’NR2E3 es va detectar una nova conjugació de SUMO amb el TF, que no es veia alterada per cap de les E3 lligases descrites com a interactores. En conjunt, aquest treball demostra la importància de la sumoïlació en la regulació i el manteniment de teixits i tipus cel·lulars, com ho són la retina i els fotoreceptors.[eng] Sumoylation is a reversible post-translational modification that regulates different cellular processes by conjugation/deconjugation of SUMO moieties to target proteins. Most work on the functional relevance of SUMO has focused on cell cycle, DNA repair and cancer in cultured cells, but data on the inter-dependence of separate components of the SUMO pathway in highly specialized tissues, such as the retina, is still scanty. Nonetheless, several retinal transcription factors (TFs) relevant for cone and rod fate, as well as some circadian rhythm regulators, are regulated by sumoylation. Here we present a comprehensive survey of SUMO pathway gene expression in the murine retina by quantitative RT-qPCR and in situ hybridization (ISH). The mRNA expression levels were quantified in retinas obtained under four different light/dark conditions, revealing distinct levels of gene expression. In addition, a SUMO pathway retinal gene atlas based on the mRNA expression pattern was drawn. Although most genes are ubiquitously expressed, some patterns could be defined in a first step to determine its biological significance and interdependence. On the other hand, the orphan nuclear receptor NR2E3 is a retinal transcription factor (TF) that plays an essential role in the development and maintenance of rod photoreceptors by both activating rod-specific and repressing cone-specific genes. This repression is partially regulated by the E3 SUMO ligase PIAS3, which sumoylates NR2E3 in three specific –and evolutionarily conserved– lysine residues, turning it into a transcriptional repressor. However, PIAS3 is not the unique E3 SUMO ligase acting upon this TF. In this context, we aimed to find new interactions with other E3 SUMO ligases in order to elucidate the relevance of this post-translational modification in regulating the function of NR2E3. Two new interactions with the E3 SUMO ligase HDAC4 and CBX4 were discovered. In addition, the nonsumoylable mutant NR2E3 123 showed a diminished interaction with PIAS3 in comparison to the wild-type (and sumoylable) NR2E3 (WT). Moreover, we detected a new SUMO-conjugated NR2E3 form not detectable in NR2E3 123, although neither PIAS3, CBX4 nor HDAC4 seem to participate in this SUMO conjugation. Although the physiological role of these newly found interactions is still unknown, they provide new insights in the regulation of the transcriptional activity of this retinal TF by SUMO modification

SUMO a la retina: expressió gènica i rellevància en la modificació posttaducciontal del factor de transcripció NRE3

La sumoïlació és una modificació posttraduccional (PTM) que consisteix en la conjugació covalent i reversible d’una proteïna SUMO (Small ubiquitin-like modifier) al residu lisina d’una proteïna diana. Igual que la ubiquitina, el cicle metabòlic de SUMO consta d’una conjugació, on hi participen tres tipus diferents d’enzims (E1 activadors, E2 conjugadors i E3 lligases), i d’una deconjugació, que es dóna a través de l’acció de proteases. A nivell funcional, la sumoïlació regula processos cel·lulars tan diversos com la replicació, la reparació del DNA, la localització, activitat i degradació de proteïnes, o la transcripció, entre d’altres. Els estudis existents sobre la sumoïlació se centraven en la modificació de proteïnes diana concretes, pel que mancaven estudis de la interdependència dels elements de la maquinària de SUMO en la regulació i el manteniments de teixits especialitzats. Un d’aquests teixits és la retina, que recobreix l’ull per la seva cara interna i s’encarrega de la captació i el processat de l’estímul lumínic. Forma part del sistema nerviós central, pel que es tracta de teixit neuronal, format per tres capes nuclears de neurones i les seves connexions. Les cèl·lules encarregades d’iniciar la cadena de la fototransducció són els fotoreceptors: cons i bastons; que comparteixen una mateixa estructura, i que difereixen en els fotopigments que hi contenen, fet que els dóna diferents funcionalitats lumíniques. Per aquest motiu, un dels objectius d’aquesta tesi va ser l’estudi de l’expressió dels gens de la via de SUMO a la retina de ratolí adult, per mitjà de la seva quantificació per RT-qPCR i de la seva localització en talls de retina per hibridació in situ. Es van detectar l’expressió de tots els gens de la maquinària de SUMO, amb nivells variables en concordança amb la seva importància dins del cicle. . A més, atès que la llum és un dels marcadors externs que controlen els ritmes circadiaris de l’organisme, es va estudiar l’expressió dels gens de la via de SUMO en diferents condicions del cicle de llum/foscor, detectant alguns patrons diferencials d’entre el conjunt de gens estudiats. Pel que fa a la localització de l’expressió, els gens de la via de SUMO van donar diferents patrons de localització a les diverses capes de la retina, coincidint tots a la capa de fotoreceptors. A continuació, es va estudiar la sumoïlació del factor de transcripció (TF) de fotoreceptors NR2E3, que juga un paper central en el desenvolupament i manteniment dels bastons, activant els gens específics de bastons i silenciant els de cons. Aquesta repressió està regulada, en part, per la seva sumoïlació a través de la E3 lligasa PIAS3, tot i que aquesta no és la única que modificava el TF. En aquest context, es va plantejar l’estudi de noves interaccions del TF amb altres E3 lligases de SUMO. Per mitjà de la tècnica de BRET es va poder trobar una nova interacció amb HDAC4, posteriorment confirmada per CoIP. A més, l’estudi comparatiu de la forma humana d’NR2E3 WT amb el seu mutant no sumoïlable (123) va permetre detectar diferències d’interacció amb PIAS3, més baixa amb el TF mutant. D’altra banda, la tècnica del BioID va permetre el descobriment d’una segona interacció amb una E3 lligasa de SUMO: CBX4. Per últim, amb la comparació de les formes WT i no sumoïlable d’NR2E3 es va detectar una nova conjugació de SUMO amb el TF, que no es veia alterada per cap de les E3 lligases descrites com a interactores. En conjunt, aquest treball demostra la importància de la sumoïlació en la regulació i el manteniment de teixits i tipus cel·lulars, com ho són la retina i els fotoreceptors.Sumoylation is a reversible post-translational modification that regulates different cellular processes by conjugation/deconjugation of SUMO moieties to target proteins. Most work on the functional relevance of SUMO has focused on cell cycle, DNA repair and cancer in cultured cells, but data on the inter-dependence of separate components of the SUMO pathway in highly specialized tissues, such as the retina, is still scanty. Nonetheless, several retinal transcription factors (TFs) relevant for cone and rod fate, as well as some circadian rhythm regulators, are regulated by sumoylation. Here we present a comprehensive survey of SUMO pathway gene expression in the murine retina by quantitative RT-qPCR and in situ hybridization (ISH). The mRNA expression levels were quantified in retinas obtained under four different light/dark conditions, revealing distinct levels of gene expression. In addition, a SUMO pathway retinal gene atlas based on the mRNA expression pattern was drawn. Although most genes are ubiquitously expressed, some patterns could be defined in a first step to determine its biological significance and interdependence. On the other hand, the orphan nuclear receptor NR2E3 is a retinal transcription factor (TF) that plays an essential role in the development and maintenance of rod photoreceptors by both activating rod-specific and repressing cone-specific genes. This repression is partially regulated by the E3 SUMO ligase PIAS3, which sumoylates NR2E3 in three specific –and evolutionarily conserved– lysine residues, turning it into a transcriptional repressor. However, PIAS3 is not the unique E3 SUMO ligase acting upon this TF. In this context, we aimed to find new interactions with other E3 SUMO ligases in order to elucidate the relevance of this post-translational modification in regulating the function of NR2E3. Two new interactions with the E3 SUMO ligase HDAC4 and CBX4 were discovered. In addition, the nonsumoylable mutant NR2E3 123 showed a diminished interaction with PIAS3 in comparison to the wild-type (and sumoylable) NR2E3 (WT). Moreover, we detected a new SUMO-conjugated NR2E3 form not detectable in NR2E3 123, although neither PIAS3, CBX4 nor HDAC4 seem to participate in this SUMO conjugation. Although the physiological role of these newly found interactions is still unknown, they provide new insights in the regulation of the transcriptional activity of this retinal TF by SUMO modification

New Insights on the Genetic Basis Underlying SHILCA Syndrome: Characterization of the NMNAT1 Pathological Alterations Due to Compound Heterozygous Mutations and Identification of a Novel Alternative Isoform

This study aims to genetically characterize a two-year-old patient suffering from multiple systemic abnormalities, including skeletal, nervous and developmental involvements and Leber congenital amaurosis (LCA). Genetic screening by next-generation sequencing identified two heterozygous pathogenic variants in nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) as the molecular cause of the disease: c.439+5G&gt;T and c.299+526_*968dup.This splice variant has never been reported to date, whereas pathogenic duplication has recently been associated with cases displaying an autosomal recessive disorder that includes a severe form of spondylo-epiphyseal dysplasia, sensorineural hearing loss, intellectual disability and LCA (SHILCA), as well as some brain anomalies. Our patient presented clinical manifestations which correlated strongly with this reported syndrome. To further study the possible transcriptional alterations resulting from these mutations, mRNA expression assays were performed in the patient and her father. The obtained results detected aberrant alternative transcripts and unbalanced levels of expression, consistent with severe systemic involvement. Moreover, these analyses also detected a novel NMNAT1 isoform, which is variably expressed in healthy human tissues. Altogether, these findings represent new evidence of the correlation of NMNAT1 and SHILCA syndrome, and provide additional insights into the healthy and pathogenic expression of this gene

mRNA expression analysis of the SUMO pathway genes in the adult mouse retina

Sumoylation is a reversible post-translational modification that regulates different cellular processes by conjugation/deconjugation of SUMO moieties to target proteins. Most work on the functional relevance of SUMO has focused on cell cycle, DNA repair and cancer in cultured cells, but data on the inter-dependence of separate components of the SUMO pathway in highly specialized tissues, such as the retina, is still scanty. Nonetheless, several retinal transcription factors (TFs) relevant for cone and rod fate, as well as some circadian rhythm regulators, are regulated by sumoylation. Here we present a comprehensive survey of SUMO pathway gene expression in the murine retina by quantitative RT-PCR and in situ hybridization (ISH). The mRNA expression levels were quantified in retinas obtained under four different light/dark conditions, revealing distinct levels of gene expression. In addition, a SUMO pathway retinal gene atlas based on the mRNA expression pattern was drawn. Although most genes are ubiquitously expressed, some patterns could be defined in a first step to determine its biological significance and interdependence. The wide expression of the SUMO pathway genes, the transcriptional response under several light/dark conditions, and the diversity of expression patterns in different cell layers clearly support sumoylation as a relevant post-translational modification in the retina. This expression atlas intends to be a reference framework for retinal researchers and to depict a more comprehensive view of the SUMO-regulated processes in the retina

Synthesis of cobalt ferrite core/metallic shell nanoparticles for the development of a specific PNA/DNA biosensor

Controlled synthesis of cobalt ferrite superparamagnetic nanoparticles covered with a gold shell has been achieved by an affinity and trap strategy. Magnetic nanoparticles are functionalized with a mixture of amino and thiol groups that facilitate the electrostatic attraction and further chemisorption of gold nanoparticles, respectively. Using these nanoparticles as seeds, a complete coating shell is achieved by gold salt-iterative reduction leading to monodisperse water-soluble gold-covered magnetic nanoparticles, with an average diameter ranging from 21 to 29 nm. These constitute a versatile platform for immobilization of biomolecules via thiol chemistry, which is exemplified by the immobilization of peptide nucleic acid (PNA) oligomers that specifically hybridize with complementary DNA molecules in solution. Hybridation with DNA probes has been measured using Rhodamine 6G fluorescence marker and the detection of a single nucleotide mutation has been achieved. These results suggest the PNA-nanoparticles application as a biosensor for DNA genotyping avoiding commonly time-consuming procedures employed.This work has been funded by the Programa Intramural of CSIC (Ref. 200450F0340). M.P. thanks the Consejería de Educación de la Comunidad de Madrid (CM), and the European Social Funding (F.S.E.) for the fellowship. J.M.A. acknowledges a postdoctoral fellowship from Fundación Ramón Areces. C.V. thanks the FPU program of the Science and Education Ministry for the fellowship. Work carried out at CAB was supported by EU, INTA, MEC and CM.Peer reviewe

Comparison of mRNA and protein immunodetection of selected DUBs in mouse retinal cryosections.

<p>Most analyzed genes render a consistent expression pattern when comparing mRNA and protein localization in the wild type mouse retina. The merge immunohistochemistry show DUBs immunodetected in red, and nuclei counter-staining with DAPI (in blue). Details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150364#pone.0150364.s003" target="_blank">S3 Fig</a>. <b>RPE</b>- Retinal pigmented epithelium; <b>Phr</b>- Photoreceptor cell layer; <b>ONL</b>- Outer nuclear layer; <b>OPL</b>. Outer plexiform layer; <b>INL</b>- Inner nuclear layer, <b>IPL</b>- Inner plexiform layer; <b>GCL</b>- Ganglion cell layer.</p