Estudio de la interacción del receptor Htr6 de serotonina con las proteínas Cdk5 y TULP3

Los cilios primarios son prolongaciones de la membrana plasmática en cuya membrana hay una gran cantidad de canales iónicos y receptores como los GPCRs. En concreto, nuestro estudio lo centramos en el receptor ciliar Htr6 de serotonina, el cual es un GPCR de la familia de las rodopsinas, de gran importancia a nivel neuronal. Aunque sus mecanismos de señalización son poco conocidos todavía, se ha visto que este receptor interacciona mediante su extremo C-terminal con una gran cantidad de proteínas implicadas en diferentes vías de señalización. Mediante ensayos de coinmunoprecipitación en células HEK293T, hemos estudiado la interacción del receptor Htr6 con una proteína efectora denominada Cdk5, la cual fosforila dicho receptor. Sin embargo, no obtuvimos resultados que confirmen dicha unión. Por otro lado, el tráfico ciliar de las proteínas de membrana al cilio, incluido el receptor Htr6, es poco conocido por lo que en este estudio esclarecemos el mecanismo de transporte. Para ello, realizamos ensayos de interacción por biotinilación en células HEK293T para analizar la interacción del receptor Htr6 con la proteína TULP3, la cual actúa como proteína adaptadora para el tráfico ciliar de proteínas de membrana. En concreto, estudiamos la interacción de la proteína TULP3 completa, y de sus dominios N-terminal y C-terminal con la CLS del dominio C-terminal del receptor Htr6. De acuerdo con el modelo actual que explica el tráfico ciliar de proteínas de membrana, observamos la interacción entre el extremo C-terminal del receptor Htr6 con el dominio C-terminal de la proteína TULP3. Esto nos permitirá conocer el mecanismo de entrada de ciertas proteínas en el cili

The male mouse meiotic cilium emanates from the mother centriole at zygotene prior to centrosome duplication

Cilia are hair-like projections of the plasma membrane with an inner microtubule skeleton known as axoneme. Motile cilia and flagella beat to displace extracellular fluids, playing important roles in the airways and reproductive system. On the contrary, primary cilia function as cell-type-dependent sensory organelles, detecting chemical, mechanical, or optical signals from the extracellular environment. Cilia dysfunction is associated with genetic diseases called ciliopathies and with some types of cancer. Cilia have been recently identified in zebrafish gametogenesis as an important regulator of bouquet conformation and recombination. However, there is little information about the structure and functions of cilia in mammalian meiosis. Here we describe the presence of cilia in male mouse meiotic cells. These solitary cilia formed transiently in 20% of zygotene spermatocytes and reached considerable lengths (up to 15–23 µm). CEP164 and CETN3 localization studies indicated that these cilia emanate from the mother centriole prior to centrosome duplication. In addition, the study of telomeric TFR2 suggested that cilia are not directly related to the bouquet conformation during early male mouse meiosis. Instead, based on TEX14 labeling of intercellular bridges in spermatocyte cysts, we suggest that mouse meiotic cilia may have sensory roles affecting cyst function during prophase IThis work was funded by BIOUAM02-2020 to J.P. and R.G. and grants PID2019-104941RB-I00 from MCIN/AEI/10.13039/501100011033 and ACCI-2020 from CIBERER to F.G.

Comparison of postoperative pain after root canal treatment using reciprocating instruments based on operator?s experience : a prospective clinical study

The aim of the present study was to compare clinically the incidence of postoperative pain after endodontic treatment using the Reciproc System, taking into account the operator?s experience. One hundred patients scheduled for routine endodontic treatment were enrolled in this study. Endodontic treatment was carried out in a single visit by undergraduate and postgraduate students. The chemomechanical preparation of root canals was performed with Reciproc instruments. Pretreatment and postoperative pain was recorded using a visual analogue scale (VAS). Postoperative pain and the need for analgesic consumption were assessed at 4, 8, 16, 24, 48 and 72 hours post-treatment. The data were analyzed using the Mann?Whitney U and Chi-Square test, and the significance was set at P<0.05. The mean value of pain after root canal treatment was 1.13±1.94 and 1.91±2.07 on a VAS between 0 and 10 in treatments performed by undergraduate and postgraduate students, respectively. There was a significant difference in the incidence of postoperative pain between the two groups (P<0.05). The prevalence of postoperative pain was high in the treatments performed by postgraduate students in comparison with undergraduate students. This suggests that operator experience has an influence on the prevalence of postoperative pain after root canal treatment

Relationship of Gaming Disorder with parenting based on low affection-communication and personality trait of neuroticism in adolescents

Background: Gaming Disorder is increasingly common in adolescents. We aimed to evaluate the relationship between parenting, personality traits, and Gaming Disorder. Methods: An observational and cross-sectional study in six secondary schools of Castelló, obtaining a final sample of 397 students. Results: Adolescents with Gaming Disorder had lower scores in Adolescent Affection-Communication (F = 8.201; p < 0.001), Father’s Warmth (F = 3.459; p = 0.028), and Father’s Acceptance/Involvement (F = 5.467; p = 0.003), and higher scores in Mother’s Revoking Privileges (F = 4.277; p = 0.034) and Father’s Indifference (F = 7.868; p = 0.002) than healthy participants. Male sex was a risk factor for Gaming Disorder (OR = 12.221; p = 0.004), while Adolescent Affection-Communication (OR = 0.908; p = 0.001) and Agreeableness (OR = 0.903; p = 0.022) were protective factors. Data modeling described the protective effect that Adolescent Affection-Communication had on Gaming Disorder, which was both directly (B = -0.20; p < 0.001) and indirectly mediated by Neuroticism (B = -0.20; p < 0.001), while Neuroticism itself was a risk factor for Gaming Disorder (B = 0.50; p < 0.001). Conclusion: These results reflect that Parental style with low affection and communication was directly and indirectly related to the Gaming Disorder, as well as male sex and personality trait of Neuroticism

NRF2-dependent gene expression promotes ciliogenesis and Hedgehog signaling

The transcription factor NRF2 is a master regulator of cellular antioxidant and detoxification responses, but it also regulates other processes such as autophagy and pluripotency. In human embryonic stem cells (hESCs), NRF2 antagonizes neuroectoderm differentiation, which only occurs after NRF2 is repressed via a Primary Cilia-Autophagy-NRF2 (PAN) axis. However, the functional connections between NRF2 and primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae, remain poorly understood. For instance, nothing is known about whether NRF2 affects cilia, or whether cilia regulation of NRF2 extends beyond hESCs. Here, we show that NRF2 and primary cilia reciprocally regulate each other. First, we demonstrate that fibroblasts lacking primary cilia have higher NRF2 activity, which is rescued by autophagy-activating mTOR inhibitors, indicating that the PAN axis also operates in differentiated cells. Furthermore, NRF2 controls cilia formation and function. NRF2-null cells grow fewer and shorter cilia and display impaired Hedgehog signaling, a cilia-dependent pathway. These defects are not due to increased oxidative stress or ciliophagy, but rather to NRF2 promoting expression of multiple ciliogenic and Hedgehog pathway genes. Among these, we focused on GLI2 and GLI3, the transcription factors controlling Hh pathway output. Both their mRNA and protein levels are reduced in NRF2-null cells, consistent with their gene promoters containing consensus ARE sequences predicted to bind NRF2. Moreover, GLI2 and GLI3 fail to accumulate at the ciliary tip of NRF2-null cells upon Hh pathway activation. Given the importance of NRF2 and ciliary signaling in human disease, our data may have important biomedical implicationsThis work was supported by European Regional Development Fund (ERDF)-cofunded grants from the Spanish Ministry of Economy and Competitiveness (MINECO) to FRGG (SAF2015-66568-R and RYC2013-14887) and to A.C. and I.L.B. (SAF2016-76520-R

Multiple ciliary localization signals control INPP5E ciliary targeting

Primary cilia are sensory membrane protrusions whose dysfunction causes ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, but how it accumulates in cilia remains poorly understood. Herein, we show INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four conserved ciliary localization signals (CLSs): LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial CLS1-CLS4 redundancy explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 clarifies why CLS3-CLS4 are together insufficient for ciliary accumulation. Further-more, we reveal that some Joubert syndrome mutations perturb INPP5E ciliary targeting, and clarify how each CLS works: (i) CLS4 recruits PDE6D, RPGR and ARL13B, (ii) CLS2-CLS3 regulate association to TULP3, ARL13B, and CEP164, and (iii) CLS1 and CLS4 cooperate in ATG16L1 binding. Altogether, we shed light on the mechanisms of INPP5E ciliary targeting, revealing a complexity without known parallels among ciliary cargoesMinisterio de Ciencia e Innovación PID2019-104941RB-I00 Raquel Martin-Morales, Pablo Barbeito Francesc R Garcia-Gonzalo. Ministerio de Ciencia e Innovación PID2020-114148RB-I00 Manuel Izquierdo. Instituto de Salud Carlos III CIBERER-ACCI-2020 Francesc R Garcia-Gonzalo. National Institutes of Health R01HD099784 Sarah C Goetz. American Heart Association 20POST35220046 Abhijit Deb Roy. Comunidad de Madrid PEJD-2016/BMD-2341 Belen Sierra-Rodero Ministerio de Ciencia e Innovación BES2016-077828 Raquel Martin-Morale

Relationship of Gaming Disorder with parenting based on low affection-communication and personality trait of neuroticism in adolescents

BackgroundGaming Disorder is increasingly common in adolescents. We aimed to evaluate the relationship between parenting, personality traits, and Gaming Disorder.MethodsAn observational and cross-sectional study in six secondary schools of Castelló, obtaining a final sample of 397 students.ResultsAdolescents with Gaming Disorder had lower scores in Adolescent Affection-Communication (F = 8.201; p &lt; 0.001), Father’s Warmth (F = 3.459; p = 0.028), and Father’s Acceptance/Involvement (F = 5.467; p = 0.003), and higher scores in Mother’s Revoking Privileges (F = 4.277; p = 0.034) and Father’s Indifference (F = 7.868; p = 0.002) than healthy participants. Male sex was a risk factor for Gaming Disorder (OR = 12.221; p = 0.004), while Adolescent Affection-Communication (OR = 0.908; p = 0.001) and Agreeableness (OR = 0.903; p = 0.022) were protective factors. Data modeling described the protective effect that Adolescent Affection-Communication had on Gaming Disorder, which was both directly (B = -0.20; p &lt; 0.001) and indirectly mediated by Neuroticism (B = -0.20; p &lt; 0.001), while Neuroticism itself was a risk factor for Gaming Disorder (B = 0.50; p &lt; 0.001).ConclusionThese results reflect that Parental style with low affection and communication was directly and indirectly related to the Gaming Disorder, as well as male sex and personality trait of Neuroticism

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets. Graphical abstract: [Figure not available: see fulltext.].This work has been funded by Instituto de Salud Carlos III (PI15/00354, PI18/00804), MINECO (MTM2015-64465-C2-1R) and GRBIO (2014-SGR-464) and co-financed by the European Regional Development Fund (FEDER). Neurovascular Research Laboratory takes part in the Spanish stroke research network INVICTUS + (RD16/0019/0021). L.R is supported by a pre-doctoral fellowship from the Instituto de Salud Carlos III (IFI17/00012).Peer reviewe

Healthcare experience and their relationship with demographic, disease and healthcare-related variables: a cross-sectional survey of patients with chronic diseases using the IEXPAC scale.

[Abstract] BACKGROUND: Patient experience is acknowledged as a principal aspect of quality healthcare delivery, and it has implications with regard to outcomes. OBJECTIVES: Our objective was to evaluate the healthcare experience of patients with chronic diseases to identify patient-perceived healthcare gaps and to assess the influence of demographic and healthcare-related variables on patient experiences. METHODS: A cross-sectional survey was delivered to adult patients with chronic diseases: diabetes mellitus (DM), human immunodeficiency virus (HIV) infection, inflammatory bowel disease (IBD) or rheumatic diseases. Patient experiences were assessed with the Instrument for Evaluation of the Experience of Chronic Patients (IEXPAC) questionnaire, with possible scores ranging from 0 (worst) to 10 (best experience). RESULTS: Of the 2474 patients handed the survey, 1618 returned it (response rate 65.4%). Patients identified gaps in healthcare related mainly to access to reliable information and services, interaction with other patients and continuity of healthcare after hospital discharge. The mean ± standard deviation (SD) IEXPAC score was 6.0 ± 1.9 and was higher for patients with HIV (6.6 ± 1.7) than for those with rheumatic disease (5.5 ± 2.0), IBD (5.9 ± 2.0) or DM (5.9 ± 1.9) (p < 0.001). In multivariate models, better overall IEXPAC experience was associated with follow-up by the same physician, follow-up by a nurse, receiving healthcare support from others and treatment with subcutaneous or intravenous drugs. The multivariate model that confirmed patients with HIV or DM had better experience than did those with rheumatic diseases. CONCLUSIONS: Through IEXPAC, patients identified aspects for healthcare quality improvements and circumstances associated with better experience, which may permit greater redirection of healthcare toward patient-centered goals while facilitating improvements in social care and long-term healthcare quality