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Background: In March 2018, the European pregnancy prevention programme for oral retinoids was updated as part of risk minimisation measures (RMM), emphasising their contraindication in pregnant women.Objective: To measure the impact of the 2018 revision of the RMMs in Europe by assessing the utilisation patterns of isotretinoin, alitretinoin and acitretin, contraceptive measures, pregnancy testing, discontinuation, and pregnancy occurrence concomitantly with a retinoid prescription.Methods: An interrupted time series (ITS) analysis to compare level and trend changes after the risk minimisation measures implementation was conducted on a cohort of females of childbearing age (12–55 years of age) from January 2010 to December 2020, derived from six electronic health data sources in four countries: Denmark, Netherlands, Spain, and Italy. Monthly utilisation figures (incidence rates [IR], prevalence rates [PR] and proportions) of oral retinoids were calculated, as well as discontinuation rates, contraception coverage, pregnancy testing, and rates of exposed pregnancies to oral retinoids, before and after the 2018 RMMs.Results: From 10,714,182 females of child-bearing age, 88,992 used an oral retinoid at any point during the study period (mean age 18.9–22.2 years old). We found non-significant level and trend changes in incidence or prevalence of retinoid use in females of child-bearing age after the 2018 RMMs. The reason of discontinuation was unknown in >95% of cases. Contraception use showed a significant increase trend in Spain; for other databases this information was limited. Pregnancy testing was hardly recorded thus was not possible to model ITS analyses. After the 2018 RMM, rates of pregnancy occurrence during retinoid use, and start of a retinoid during a pregnancy varied from 0.0 to 0.4, and from 0.2 to 0.8, respectively.Conclusion: This study shows a limited impact of the 2018 RMMs on oral retinoids utilisation patterns among females of child-bearing age in four European countries. Pregnancies still occur during retinoid use, and oral retinoids are still prescribed to pregnant women. Contraception and pregnancy testing information was limited in most databases. Regulators, policymakers, prescribers, and researchers must rethink implementation strategies to avoid any pregnancy becoming temporarily related to retinoid use.

PurposeTo evaluate repeatability, reproducibility, and accordance between ocular surface measurements within three different imaging devices.MethodsWe performed an observational study on 66 healthy eyes. Tear meniscus height, non-invasive tear break-up time (NITBUT) and meibography were measured using three corneal imaging devices: Keratograph 5M (Oculus, Wetzlar, Germany), Antares (Lumenis, Sidney, Australia), and LacryDiag (Quantel Medical, Cournon d’Auvergne, France). One-way ANOVAs with post hoc analyses were used to calculate accordance between the tear meniscus and NITBUT. Reproducibility was assessed through coefficients of variation and repeatability with intraclass correlation coefficients (ICC). Reliability of meibography classification was analyzed by calculating Fleiss’ Kappa Index and presented in Venn diagrams.ResultsCoefficients of variation were high and differed greatly depending on the device and measurement. ICCs showed moderate reliability of NITBUT and tear meniscus height measurements. We observed discordance between measurements of tear meniscus height between the three devices, F2, 195 = 15.24, p < 0.01. Measurements performed with Antares were higher; 0.365 ± 0.0851, than those with Keratograph 5M and LacryDiag; 0.293 ± 0.0790 and 0.306 ± 0.0731. NITBUT also showed discordance between devices, F2, 111 = 13.152, p < 0.01. Measurements performed with LacryDiag were lower (10.4 ± 1.82) compared to those of Keratograph 5M (12.6 ± 4.01) and Antares (12.6 ± 4.21). Fleiss’ Kappa showed a value of -0.00487 for upper lid and 0.128 for inferior lid Meibography classification, suggesting discrete to poor agreement between measurements.ConclusionDepending on the device used and parameter analyzed, measurements varied between each other, showing a difference in image processing.

The immune response promoted by SARS-CoV-2 vaccination is relevant to develop novel vaccines and optimized prevention strategies. We analyzed the adaptive immunity in healthy donors (HD) and convalescent individuals (CD), before and after administering BNT162b2 vaccine. Our results revealed specific changes in CD4+ T cell reactivity profile in vaccinated HD and CD, with an increase in S1 and S2 positive individuals, proportionally higher for S2. On the contrary, NCAP reactivity observed in HD and CD patients was no longer detectable after vaccination. Despite the substantial antibody response in CD, MPro-derived peptides did not elicit CD4+ lymphocyte activation in our assay in either condition. HD presented an increment in anti-S and anti-RBD IgG after first dose vaccination, which increased after the second vaccination. Conversely, anti-S and anti-RBD IgG and IgA titers increased in already positive CD after first dose administration, remaining stable after second dose inoculation. Interestingly, we found a strong significant correlation between S1-induced CD4+ response and anti-S IgA pre-vaccination, which was lost after vaccine administration.

Objective: Despite the continuous update of clinical guidelines, little is known about the real-world management of patients with atrial fibrillation (AF) who survived a stroke. We aimed to assess patterns of therapeutic management of stroke survivors with AF and clinical outcomes using data from routine practice in a large population-based cohort.Methods: A population-based retrospective cohort study of all patients with AF who survived a stroke, from January 2010 to December 2017 in the Valencia region, Spain (n = 10,986), was carried out. Treatment strategies and mean time to treatment initiation are described. Temporal trends are shown by the management pattern during the study period. Factors associated with each pattern (including no treatment) vs. oral anticoagulant (OAC) treatment were identified using logistic multivariate regression models. Incidence rates of clinical outcomes (mortality, stroke/TIA, GI bleeding, and ACS) were also estimated by the management pattern.Results: Among stroke survivors with AF, 6% were non-treated, 23% were prescribed antiplatelets (APT), 54% were prescribed OAC, and 17% received OAC + APT at discharge. Time to treatment was 8.0 days (CI 7.6–8.4) for APT, 9.86 (CI 9.52–10.19) for OAC, and 16.47 (CI 15.86–17.09) for OAC + APT. Regarding temporal trends, management with OAC increased by 20%, with a decrease of 50% for APT during the study period. No treatment and OAC + APT remained relatively stable. The strongest predictor of no treatment and APT treatment was having the same management strategy pre-stroke. Those treated with APT had the highest rates of GI bleeding and recurrent stroke/TIA, and untreated patients showed the highest rates of mortality.Conclusion: In this large population-based cohort using real-world data, nearly 30% of AF patients who suffered a stroke were untreated or treated with APT, which overall is not recommended. Treatment was started within 2 weeks as recommended, except for OAC + APT, which was started later. The strong association of APT treatment or non-treatment with the same treatment strategy before stroke occurrence suggests a strong therapeutic inertia and opposes recommendations. Patients under these two strategies had the highest rates of adverse outcomes. An inadequate prescription poses a great risk on patients with AF and stroke; thus monitoring their management is necessary and should be setting-specific.

Mitochondria regulate multiple cell processes, including calcium signaling, apoptosis and cell metabolism. Mitochondria contain their own circular genome encoding selected subunits of the oxidative phosphorylation complexes. Recent findings reveal that, in addition to being maternally inherited, mitochondria can traverse cell boundaries and thus be horizontally transferred between cells. Although the physiological relevance of this phenomenon is still under debate, mitochondria uptake rescues mitochondrial respiration defects in recipient cells and regulates signaling, proliferation or chemotherapy resistance in vitro and in vivo. In this review, we outline the pathophysiological consequences of horizontal mitochondrial transfer and offer a perspective on the cellular and molecular mechanisms mediating their intercellular transmission, including tunneling nanotubes, extracellular vesicles, cellular fusion and GAP junctions. The physiological relevance of mitochondrial transfer and the potential therapeutic application of this exchange for treating mitochondrial-related diseases are discussed.

Sex inequities in liver transplantation (LT) have been documented in several, mostly US-based, studies. Our aim was to describe sex-related differences in access to LT in a system with short waiting times. All adult patients registered in the RETH-Spanish Liver Transplant Registry (2000–2022) for LT were included. Baseline demographics, presence of hepatocellular carcinoma, cause and severity of liver disease, time on the waiting list (WL), access to transplantation, and reasons for removal from the WL were assessed. 14,385 patients were analysed (77% men, 56.2 ± 8.7 years). Model for end-stage liver disease (MELD) score was reported for 5,475 patients (mean value: 16.6 ± 5.7). Women were less likely to receive a transplant than men (OR 0.78, 95% CI 0.63, 0.97) with a trend to a higher risk of exclusion for deterioration (HR 1.17, 95% CI 0.99, 1.38), despite similar disease severity. Women waited longer on the WL (198.6 ± 338.9 vs. 173.3 ± 285.5 days, p < 0.001). Recently, women’s risk of dropout has reduced, concomitantly with shorter WL times. Even in countries with short waiting times, women are disadvantaged in LT. Policies directed at optimizing the whole LT network should be encouraged to guarantee a fair and equal access of all patients to this life saving resource.

A twelve-year-old patient with a previous clinical diagnosis of spondylocostal skeletal dysplasia and moderate intellectual disability was genetically analyzed through next generation sequencing of a targeted gene panel of 179 genes associated to skeletal dysplasia and mucopolysaccharidosis in order to stablish a precision diagnosis. A homozygous nonsense [c.62C>G; p.(Ser21Ter)] mutation in DYM gene was identified in the patient. Null mutations in DYM have been associated to Dyggve-Melchior-Clausen syndrome, which is a rare autosomal-recessive disorder characterized by skeletal dysplasia and mental retardation, compatible with the patient´s phenotype. To confirm the pathogenicity of this mutation, a segregation analysis was carried out, revealing that the mutation p(Ser21Ter) was solely inherited from the father, who is a carrier of the mutation, while the mother does not carry the mutation. With the suspicion that a paternal disomy could be causing the disease, a series of microsatellite markers in chromosome 18, where the DYM gene is harbored, was analyzed in all the members of the family. Haplotype analysis provided strong evidence of paternal isodisomy and heterodisomy in that chromosome, confirming the pathological effect of this mutation. Furthermore, the patient may have a compromised expression of the ELOA3 gene due to modifications in the genomic imprinting that may potentially increase the risk of digestive cancer. All these results highlight the importance of obtaining a precision diagnosis in rare diseases.

Introduction: Europe has seen a steady increase in the use of prescription opioids, especially in non-cancer indications. Epidemiological data on the patterns of use of opioids is required to optimize prescription. We aim to describe the patterns of opioid therapy initiation for non-cancer pain and characteristics of patients treated in a region with five million inhabitants in the period 2012 to 2018.Methods: Population-based retrospective cohort study of all adult patients initiating opioid therapy for non-cancer pain in the region of Valencia. We described patient characteristics at baseline and the characteristics of baseline and subsequent treatment initiation. We used multinominal regression models to identify individual factors associated with initiation.Results: A total of 957,080 patients initiated 1,509,488 opioid treatments (957,080 baseline initiations, 552,408 subsequent initiations). For baseline initiations, 738,749 were with tramadol (77.19%), 157,098 with codeine (16.41%) 58,436 (6.11%) with long-acting opioids, 1,518 (0.16%) with short-acting opioids and 1,279 (0.13%) with ultrafast drugs. When compared to tramadol, patients initiating with short-acting, long-acting and ultrafast opioids were more likely to be older and had more comorbidities, whereas initiators with codeine were more prone to be healthier and younger. Treatments lasting less than 7 days accounted for 41.82% of initiations, and 11.89% lasted more than 30 days. 19.55% of initiators with ultrafast fentanyl received more than 120 daily Morphine Milligram Equivalents (MME), and 16.12% of patients initiating with long-acting opioids were prescribed more than 90 daily MME (p < 0.001). Musculoskeletal indications accounted for 65.05% of opioid use. Overlap with benzodiazepines was observed in 24.73% of initiations, overlap with gabapentinoids was present in 11.04% of initiations with long-acting opioids and 28.39% of initiators with short-acting opioids used antipsychotics concomitantly. In subsequent initiations, 55.48% of treatments included three or more prescriptions (vs. 17.60% in baseline initiations) and risk of overlap was also increased.Conclusion: Opioids are initiated for a vast array of non-oncological indications, and, despite clinical guidelines, short-acting opioids are used marginally, and a significant number of patients is exposed to potentially high-risk patterns of initiation, such as treatments lasting more than 14 days, treatments surpassing 50 daily MMEs, initiating with long-acting opioids, or hazardous overlapping with other therapies.

IntroductionThe COVID-19 pandemic has brought about important changes. On March 14, 2020, a strict home confinement was decreed in Spain. Children did not attend school and were not allowed to leave their homes. The aim of this study was to determine the emotional state of these children, as well as associated factors.Material and methodsA cross-sectional descriptive study was conducted using an online questionnaire sent by cell phone. This survey includes sociodemographic items and questions concerning the emotional impact of the lockdown. With the questions on emotions, two categories of emotional state were established with the variables fear, irritability, sadness and somatization: those who were less or more emotionally affected. A multivariate logistic model was used to estimate the associations between the variables.ResultsA total of 3,890 responses were obtained. The mean age of the children was 6.78 years (range 0 to 16). A score indicating poor emotional state was reported by 40.12%. The multivariate logistic model for poor emotional state was directly associated with having less appetite, sleep disturbances, and with parents' beliefs that their child will have difficulties returning to normal life after lockdown. A better emotional state was associated with being an only child, access to outdoor spaces at home, having pets, and parents informing their children about the pandemic using creative explanations.ConclusionsDuring strict home confinement, a considerable emotional impact was observed in children as described by their parents. Specific elements were associated with a better or poorer emotional state.

Communication through cell-cell contacts and extracellular vesicles (EVs) enables immune cells to coordinate their responses against diverse types of pathogens. The function exerted by EVs in this context depends on the proteins and nucleic acids loaded into EVs, which elicit specific responses involved in the resolution of infection. Several mechanisms control protein and nucleic acid loading into EVs; in this regard, acetylation has been described as a mechanism of cellular retention during protein sorting to exosomes. HDAC6 is a deacetylase involved in the control of cytoskeleton trafficking, organelle polarity and cell migration, defense against Listeria monocytogenes (Lm) infection and other immune related functions. Here, we show that the protein content of dendritic cells (DCs) and their secreted EVs (DEVs) vary during Lm infection, is enriched in proteins related to antiviral functions compared to non-infected cells and depends on HDAC6 expression. Analyses of the post-translational modifications revealed an alteration of the acetylation and ubiquitination profiles upon Lm infection both in DC lysates and DEVs. Functionally, EVs derived from infected DCs upregulate anti-pathogenic genes (e.g. inflammatory cytokines) in recipient immature DCs, which translated into protection from subsequent infection with vaccinia virus. Interestingly, absence of Listeriolysin O in Lm prevents DEVs from inducing this anti-viral state. In summary, these data underscore a new mechanism of communication between bacteria-infected DC during infection as they alert neighboring, uninfected DCs to promote antiviral responses.

BackgroundInterleukin 6 (IL6) levels and SARS-CoV-2 viremia have been correlated with COVID-19 severity. The association over time between them has not been assessed in a prospective cohort. Our aim was to evaluate the relationship between SARS-CoV-2 viremia and time evolution of IL6 levels in a COVID-19 prospective cohort.MethodsSecondary analysis from a prospective cohort including COVID-19 hospitalized patients from Hospital Universitario La Princesa between November 2020 and January 2021. Serial plasma samples were collected from admission until discharge. Viral load was quantified by Real-Time Polymerase Chain Reaction and IL6 levels with an enzyme immunoassay. To represent the evolution over time of both variables we used the graphic command twoway of Stata.ResultsA total of 57 patients were recruited, with median age of 63 years (IQR [53–81]), 61.4% male and 68.4% Caucasian. The peak of viremia appeared shortly after symptom onset in patients with persistent viremia (more than 1 sample with > 1.3 log10 copies/ml) and also in those with at least one IL6 > 30 pg/ml, followed by a progressive increase in IL6 around 10 days later. Persistent viremia in the first week of hospitalization was associated with higher levels of IL6. Both IL6 and SARS-CoV-2 viral load were higher in males, with a quicker increase with age.ConclusionIn those patients with worse outcomes, an early peak of SARS-CoV-2 viral load precedes an increase in IL6 levels. Monitoring SARS-CoV-2 viral load during the first week after symptom onset may be helpful to predict disease severity in COVID-19 patients.

The aim of this work is to present a reproducible methodology for the evaluation of total equivalent doses in organs during proton therapy facilities. The methodology is based on measuring the dose equivalent in representative locations inside an anthropomorphic phantom where photon and neutron dosimeters were inserted. The Monte Carlo simulation was needed for obtaining neutron energy distribution inside the phantom. The methodology was implemented for a head irradiation case in the passive proton beam of iThemba Labs (South Africa). Thermoluminescent dosimeter (TLD)-600 and TLD-700 pairs were used as dosimeters inside the phantom and GEANT code for simulations. In addition, Bonner sphere spectrometry was performed inside the treatment room to obtain the neutron spectra, some relevant neutron dosimetric quantities per treatment Gy, and a percentual distribution of neutron fluence and ambient dose equivalent in four energy groups, at two locations. The neutron spectrum at one of those locations was also simulated so that a reasonable agreement between simulation and measurement allowed a validation of the simulation. Results showed that the total out-of-field dose equivalent inside the phantom ranged from 1.4 to 0.28 mSv/Gy, mainly due to the neutron contribution and with a small contribution from photons, 10% on average. The order of magnitude of the equivalent dose in organs was similar, displaying a slow reduction in values as the organ is farther from the target volume. These values were in agreement with those found by other authors in other passive beam facilities under similar irradiation and measurement conditions.

AimAdherence to multiple medications recommended for secondary prevention of cardiovascular conditions represents a challenge. We aimed to identify patterns of concurrent adherence to combined therapy and assess their impact on clinical outcomes in a cohort of patients with acute coronary syndrome (ACS).MethodsPopulation-based retrospective cohort of all patients discharged after hospitalization for ACS (2009–2011), prescribed ≥3 therapeutic groups within the first month. We assessed monthly concurrent adherence (≥24 days of medication out of 30) to ≥3 medications during the first year, and patterns were identified through group-based trajectory models. A composite clinical outcome during the second year was constructed. The association between adherence patterns and traditional refill adherence metrics [e.g., the proportion of days covered (PDC)], and outcomes were assessed through a multivariable Cox proportional hazards model.ResultsAmong 15,797 patients discharged alive, 12,057 (76.32%) initiated treatment with ≥3 therapeutic groups after discharge. We identified seven adherence trajectories to ≥3 medications: Adherent (52.94% of patients); Early Gap (6.64%); Middle Gap (5.67%); Late Decline (10.93%); Occasional Users (5.45%); Early Decline (8.79%); Non-Adherent (9.58%). Compared to the Adherent group, patients belonging to Early Gap (HR:1.30, 95%CI 1.07;1.60), Late decline (hazards ratio (HR): 1.31, 95% CI 1.1; 1.56), and Non-Adherent trajectories (HR: 1.36, 95% CI 1.14; 1.63) had a greater risk of adverse clinical outcomes, which was also different to the risk ascertained through concurrent PDC < 80 (HR: 1.13, 95% CI 1.01; 1.27).ConclusionOverall, seven adherence trajectories to ≥3 drugs were identified, with three distinct adherence patterns being at higher risk of adverse outcomes. The identification of patterns of concurrent adherence, a more comprehensive approach than traditional measurements, may be useful to target interventions to improve adherence to multiple medications.

Background: The Spanish health authorities are concerned by the off-label use of immediate-release formulations of fentanyl (IRF) in noncancer pain and cancer pain in patients with no chronic pain therapy.Aim: To evaluate the impact of different interventions to improve appropriateness of IRF prescription on off-label prescription.Patients and methods: We used interrupted time series (ITS) to estimate immediate and trend changes of IRF prescription for noncancer pain (NCP) and breakthrough cancer pain (BCP) in patients with and without chronic cancer pain therapy associated with two medication reviews (I1 and I2) and the issue of a safety warning letter (I3) with data from a Spanish region with 5 million inhabitants, from 2015 to 2018.Results: The use of IRF for NCP in the region Valencia was reduced from about 1,800 prescriptions per week to around 1,400. The first medication review was followed by an immediate level change of −192.66 prescriptions per week (p < 0.001) and a downward trend change of −6.75 prescriptions/week (p < 0.001), resulting in a post-intervention trend of −1.99 (p < 0.001). I2 was associated with a trend change of -23.07 (p < 0.001) prescriptions/week. After I3, the trend changed markedly to 27.23 additional prescriptions/week, for a final post-intervention trend of 2.17 (p < 0.001). Controlled-ITS provided comparable results. For potentially inappropriate BCP use, the second medication review was followed by a downward, immediate level change of −10.10 prescriptions/week (p = 0.011) and a trend change of 2.31 additional prescriptions/week (p < 0.001) and the issue of the safety warning (I3) was followed by a downward trend change of −2.09 prescriptions/week (p = 0.007).Conclusion: Despite IRF prescription for NCP decreased, the interventions showed modest and temporary effect on off-label prescription. Our results call for a review of the design and implementation of safety interventions addressing inappropriate opioid use.

Coronavirus Disease 2019 (COVID-19) pneumonia is a life-threatening infectious disease, especially for elderly patients with multiple comorbidities. Despite enormous efforts to understand its underlying etiopathogenic mechanisms, most of them remain elusive. In this study, we compared differential plasma miRNAs and cytokines profiles between COVID-19 and other community-acquired pneumonias (CAP). A first screening and subsequent validation assays in an independent cohort of patients revealed a signature of 15 dysregulated miRNAs between COVID-19 and CAP patients. Additionally, multivariate analysis displayed a combination of 4 miRNAs (miR-106b-5p, miR-221-3p, miR-25-3p and miR-30a-5p) that significantly discriminated between both pathologies. Search for targets of these miRNAs, combined with plasma protein measurements, identified a differential cytokine signature between COVID-19 and CAP that included EGFR, CXCL12 and IL-10. Significant differences were also detected in plasma levels of CXCL12, IL-17, TIMP-2 and IL-21R between mild and severe COVID-19 patients. These findings provide new insights into the etiopathological mechanisms underlying COVID-19.

BackgroundAlthough proteomics has been employed in the study of several models of liver injury, proteomic methods have only recently been applied not only to biomarker discovery and validation but also to improve understanding of the molecular mechanisms involved in transplantation.MethodsThe study was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology and the guidelines for performing systematic literature reviews in bioinformatics (BiSLR). The PubMed, ScienceDirect, and Scopus databases were searched for publications through April 2020. Proteomics studies designed to understand liver transplant outcomes, including ischemia-reperfusion injury (IRI), rejection, or operational tolerance in human or rat samples that applied methodologies for differential expression analysis were considered.ResultsThe analysis included 22 studies after application of the inclusion and exclusion criteria. Among the 497 proteins annotated, 68 were shared between species and 10 were shared between sample sources. Among the types of studies analyzed, IRI and rejection shared a higher number of proteins. The most enriched pathway for liver biopsy samples, IRI, and rejection was metabolism, compared to cytokine-cytokine receptor interactions for tolerance.ConclusionsProteomics is a promising technique to detect large numbers of proteins. However, our study shows that several technical issues such as the identification of proteoforms or the dynamic range of protein concentration in clinical samples hinder the successful identification of biomarkers in liver transplantation. In addition, there is a need to minimize the experimental variability between studies, increase the sample size and remove high-abundance plasma proteins.

Introduction: Laparoscopic surgery is an increasingly used technique, but it requires a high degree of learning, and communication between the operating room crew is considerably difficult. The use of eye tracking has been proposed as a didactic and evaluation tool in several settings, including in laparoscopy in simulators.Objectives: This study aimed to evaluate the usefulness of the use of eye tracking systems (Tobii glasses 2) in laparoscopic surgery as a didactic and assessment tool to improve communication in the operating room and improve patients' security.Methodology: An anonymous survey was sent to the students and medical teachers of a faculty of medicine and practicing doctors and residents. The message contained an explanation about the use of the Tobii glasses, a link to watch the video showing its use in a laparoscopic surgery, and the survey to complete after watching the video.Results: The survey was answered by 113 participants (51.3% medical students, 27.4% medical teachers, 18.6% practicing doctors, and 2.7% medicine residents). Eighty-three percent agreed with the usefulness of the “Tobii glasses” in the operating room for improving communication between the main surgeon and the assistant, for learning complex surgery techniques, for obtaining didactic videos, and for indicating anatomical structures. The item scored worst was the price of the glasses.Conclusions: It is possible to record and project expert gaze patterns in the operating room in real time using the Tobii glasses. This device allows improving communication among the surgical crew and the learning of residents and also improving the security of surgical patients.

Lymphocytes rearrange their shape, membrane receptors and organelles during cognate contacts with antigen-presenting cells (APCs). Activation of T cells by APCs through pMHC-TCR/CD3 interaction (peptide-major histocompatibility complex-T cell receptor/CD3 complexes) involves different steps that lead to the reorganization of the cytoskeleton and organelles and, eventually, activation of nuclear factors allowing transcription and ultimately, replication and cell division. Both the positioning of the lymphocyte centrosome in close proximity to the APC and the nucleation of a dense microtubule network beneath the plasma membrane from the centrosome support the T cell’s intracellular polarity. Signaling from the TCR is facilitated by this traffic, which constitutes an important pathway for regulation of T cell activation. The coordinated enrichment upon T cell stimulation of the chaperonin CCT (chaperonin-containing tailless complex polypeptide 1; also termed TRiC) and tubulins at the centrosome area support polarized tubulin polymerization and T cell activation. The proteasome is also enriched in the centrosome of activated T cells, providing a mechanism to balance local protein synthesis and degradation. CCT assists the folding of proteins coming from de novo synthesis, therefore favoring mRNA translation. The functional role of this chaperonin in regulating cytoskeletal composition and dynamics at the immune synapse is discussed.

We describe the first application of a non-invasive and novel approach to estimate the growth rate of Asconema setubalense (Porifera, Hexactinellida) through the use of 3D photogrammetric methodology. Structure-from-Motion techniques (SfM) were applied to videos acquired with the Politolana ROTV in the El Cachucho Marine Protected Area (MPA) (Cantabrian Sea) on three different dates (2014, 2017, and 2019) over six years. With these data, a multi-temporal study was conducted within the framework of MPA monitoring. A complete 3D reconstruction of the deep-sea floor was achieved with Pix4D Mapper Pro software for each date. Having 3D point clouds of the study area enabled a series of measurements that were impossible to obtain in 2D images. In 3D space, the sizes (height, diameter, cup-perimeter, and cup-surface area) of several A. setubalense specimens were measured each year. The annual growth rates recorded ranged from zero (“no growth”) for a large size specimen, to an average of 2.2 cm year–1 in cup-diameter, and 2.5 cm year–1 in height for developing specimens. Von Bertalanffy growth parameters were estimated. Taking into account the size indicators used in this study and based on the von Bertalanffy growth model, this sponge reaches 95% maximum size at 98 years of age. During the MPA monitoring program, a high number of specimens disappeared. This raised suspicions of a phenomenon affecting the survival of this species in the area. This type of image-based methodology does not cause damage or alterations to benthic communities and should be employed in vulnerable ecosystem studies and MPA monitoring.

In the present work we focus on the distribution of two species of sponges. One of these is Asconema setubalense, a sponge found in rocky substrate that was sampled with a photogrammetric vehicle through georeferenced images. The other is Pheronema carpenteri, which inhabits soft bottoms and was sampled by beam trawl. For the spatial distribution modeling of both sponges, the geomorphological variables of depth, slope, broad and fine scale bathymetric position index (BPI), aspect, and types of bottoms were used, all with a resolution of 32 m. Additionally, layers of silicates and currents near the bottom were extracted from Copernicus Marine Environment Monitoring Service (CMEMS), with a resolution of ∼4 and ∼9 km, respectively. Due to the low resolution of the layers, it was considered necessary to validate their use by model comparison, where those that included these variables turned out to be more explanatory than the others. The models were developed in a complex continental break of the Central Cantabrian Sea, which comprises several submarine canyons and a seamount (Le Danois Bank). On the one hand, a very high resolution (32 m) spatial distribution model based on A. setubalense presence was developed using the MaxEnt maximum entropy model. On the other, depending on the availability of density data, generalized additive models (GAMs) were developed for P. carpenteri distribution, although in this case the sampler only allowed a maximum resolution of almost 1 Km. For the A. setubalense, the variables that best explained their distribution were ground types and depth, and for P. carpenteri, silicates, slope, northness, and eastward seawater velocity. The final model scores obtained were an AUC of 0.98 for the MaxEnt model, and an R squared of 0.87 for the GAM model.

This study analyzes the fauna composition of the community of brittle and sea stars associated with sponge aggregations located in Avilés Canyons System and El Cachucho, Marine Protected Area (MPA). Diverse sampling methods were used depending on bottom morphology, such as rock dredges and specific samplers for sedimentary bottoms, mainly beam trawl models. These banks are made up of sponge and coral species that build a very appropriate substrate for the proliferation of benthic species, which together create Vulnerable Marine Ecosystems that are highly relevant for management and conservation. Among these benthic species, echinoderms are of great interest due to their value as indicators of good habitat. In total, 1261 specimens were collected (934 brittle stars and 327 starfishes), belonging to 42 species (28 ophiuroids and 14 asteroids) from INDEMARES AVILÉS, ECOMARG, and SponGES project surveys. Specimens were distributed among four sponge aggregations (F: fields) that were considered according to the sponge records obtained in the same stations (36). These fields were defined and named based on the five most common sponge species: Aphrocallistes beatrix and Regadrella phoenix (F1: Avilés Canyon); Pheronema carpenteri (F2: Intraslope basin of Le Danois Bank); Asconema setubalense (F3: Le Danois Bank); and Neoschrammeniella aff. bowerbankii (F4: Corbiro Canyon). Faunistic results show that Ophiactis abyssicola (55.55% occurrence), Brisinga endecacnemos, Ophiolycus purpureus, and Peltaster placenta (33.33%) were the most frequently found species in F1; Psilaster andromeda (80%), Pseudarchaster parelii (60%), and Nymphaster arenatus (46.67%) in F2; Ophiura carnea (71.43%) and Ophiacantha smitti (42.86%) in F3; and Ophiacantha densa, and Henricia caudani (100%) in F4. The asteroid and ophiuroid species collected seem to be composed of four different communities that fit to areas with particular morphological and biological features, related to the presence of species specialized in the use of the resources they can find there. In general, the abiotic factor controlling this structure is depth. This assemblage structure, which favors the dissimilarity between the canyons and the bank, is not so clear, since the deepest stations are located on the intraslope basin of El Cachucho, Marine Protected Area (MPA), therefore, using it a priori could lead to misunderstandings. Once the structure of the echinoderm community was known, we compared the expected and obtained results to analyze evidence which should prove the existence of any association between echinoderms and sponges, which enabled us to refute the incongruous hypothesis.

Deep-sea sponge-dominated communities are complex habitats considered hotspots of biodiversity and ecosystem functioning. They are classified as Vulnerable Marine Ecosystem and are listed as threatened or declining as a result of anthropogenic activities. Yet, studies into the distribution, community structure and composition of these habitats are scarce, hampering the development of appropriate management measures to ensure their conservation. In this study we describe a diverse benthic community, dominated by a lithistid sponge, found in two geomorphological features of important conservation status —Le Danois Bank and El Corbiro Canyon— of the Cantabrian Sea. Based on the analyses of visual transects using a photogrammetric towed vehicle and samples collected by rock dredge, we characterize the habitat and the associated community in detail. This deep-sea sponge aggregation was found on bedrock. It is dominated by one lithistid sponge, Neoschrammeniella aff. bowerbankii (0.2 ind./m2) and further composed of various sponge species as well as of other benthic invertebrates such as cnidarians, bryozoans and crustaceans. Using a non-invasive methodology (SfM – Structure from Motion) and empirical relationships of individuals size and biomass/volume obtained in laboratory for N. aff. bowerbankii, we were able to estimate a total biomass of 41 kg and volume of 39 l of this species in the surveyed area. This approach allows a fine tune methodology for estimating biomass and volume by image-based-observed area avoiding destructive techniques for this species.

Accumulating evidence on the role of Thrombospondin-1 (TSP-1) in the immune response has emerged during the last years. In spite of the importance of TSP-1 not only as anti-angiogenic factor but also as an immunomodulatory molecule, studies on the role of TSP-1 in psoriasis have been neglected. TSP-1 and CD47 expression were analyzed in skin samples from psoriasis patients and control subjects using RT-PCR and immunofluorescence. Expression of these molecules was also evaluated in peripheral blood CD4+ T cells, moDCs, and circulating primary DCs. The functional role of TSP-1/CD47 signaling axis in psoriasis was assessed in Th17 and Treg differentiation assays. Additionally, small interfering RNA assays specific to TSP-1 were performed in CD4+ T cells and monocyte derived DC to specifically evaluate the function of this protein. Lesional skin of psoriasis patients expressed lower TSP-1 and CD47 mRNA levels compared to non-lesional skin or skin from controls. Immunofluorescence staining revealed decreased expression of CD47 in CD45+ dermal cells from psoriasis samples compared to control subjects. Peripheral CD4+ T cells and circulating primary DCs from psoriasis also expressed lower levels of CD47 compared to controls. Although no significant differences were detected in TSP-1 expression in CD4+ T cells and moDCs between patients and controls, TSP-1 expression in psoriasis patients inversely correlated with disease activity evaluated by the Psoriasis Area and Index Activity. Furthermore, exogenous TSP-1 inhibited Th17 differentiation and stimulated the differentiation of CD4+ T cells toward Treg cells. Furthermore, RNA interference specific for TSP-1 confirmed the role of this molecule as a negative regulator of T cell activation. Because of the impact of TSP-1/CD47 signaling axis in Th17 and Treg differentiation, a dysregulated expression of these molecules in the immune cells from psoriasis patients may favor the exacerbated inflammatory response in this disease.

ObjectivesThe present study explores the hypothesis that the anatomical bone structures of the oral cavity have probably evolved under the influence of language function. The possible changes have been evaluated by comparing two close species essentially differentiated from each other by spoken language.Materials and MethodsTwenty dry skulls and 20 mandibles of modern Caucasians were compared with 12 dry skulls and 12 mandibles of chimpanzees, with the analysis of 37 variables and the definition of new anatomical parameters.ResultsA number of highly significant differences were found between humans and chimpanzees. The human temporomandibular joint is comparatively less flat and has a more limited excursive movement range, with structural elements that seem to be lighter. A significant difference is noted in mandibular alveolar vergency and in the internal slope of the mandibular symphysis where the oral cavity’s morphology is modified, thereby increasing the free space for tongue movements in humans. The chin, which is unique to the human species, is quantified through the external slope of the mandibular symphysis with a lesser angle in humans.DiscussionIt is obvious that there are differences between humans and chimpanzees in the bone morphology of the oral cavity structures. This has been confirmed with the analysis of new variables. Together with other factors (bipedalism, habits, and genetics) speech in humans must have played an important role in the aforementioned differences between humans and chimpanzees. The number of mandibular movements involved in speech is far greater than those used in chewing, which must have conditioned the evolution of the oral structures implicated in the development of language. On average, humans weigh 70 kg and chimpanzees 44 kg. However, the majority of the variables studied in skulls and mandibles are greater in chimpanzees, which suggests that the evolution of the oral zone in humans has suffered a reduction in size with changes in shape. The refinement of the supralaryngeal vocal tract in the human species must have co-evolved with speech fairly recently. The human skull has temporomandibular joints that are comparatively less flat with a more limited movement. There is a greater lingual space and there is also a chin that suggests a muscular stimulant. This leads to the conclusion that, at least in part, speech is behind all these changes, although it is difficult to establish a cause-effect relationship.

Echocardiography is currently the main diagnostic technique in pediatric cardiology, but sometimes it is difficult to use in very young children, as a complete and accurate study depends on the patient's and family's cooperation. Children's behavior is one of the main problems for this procedure, and interventions like sedative medication have been used to facilitate its performance. The aim of this study was to analyze the effects of TV entertainment in infants and preschool children during echocardiography. We designed an experimental study in children with a heart murmur. An examination room was prepared with a TV on the ceiling, giving the children an unobstructed view during the echocardiography procedure. Fifty-eight patients were randomized into two groups: TV intervention vs. usual care (consisting of other distraction measures). The primary outcome was echocardiography time, but we also assessed blood pressure, quality of technique, child behavior, and parents' stress level. The TV group showed a statistically significant reduction in duration of the echocardiography and systolic and diastolic blood pressure, as well as better quality of technique and child behavior. Consequently, we recommend the use of a TV as a simple and useful distraction method for improving echocardiography in young children.

Myocardial ischemia-related disorders constitute a major health problem, being a leading cause of death in the world. Upon ischemia, tissue remodeling processes come into play, comprising a series of inter-dependent stages, including inflammation, cell proliferation and repair. Neovessel formation during late phases of remodeling provides oxygen supply, together with cellular and soluble components necessary for an efficient myocardial reconstruction. Immune system plays a central role in processes aimed at repairing ischemic myocardium, mainly in inflammatory and angiogenesis phases. In addition to cellular components and soluble mediators as chemokines and cytokines, the immune system acts in a paracrine fashion through small extracellular vesicles (EVs) release. These vesicular structures participate in multiple biological processes, and transmit information through bioactive cargoes from one cell to another. Cell therapy has been employed in an attempt to improve the outcome of these patients, through the promotion of tissue regeneration and angiogenesis. However, clinical trials have shown variable results, which put into question the actual applicability of cell-based therapies. Paracrine factors secreted by engrafted cells partially mediate tissue repair, and this knowledge has led to the hypothesis that small EVs may become a useful tool for cell-free myocardial infarction therapy. Current small EVs engineering strategies allow delivery of specific content to selected cell types, thus revealing the singular properties of these vesicles for myocardial ischemia treatment.

The regulatory role of most dual specific phosphatases during T cell activation remains unknown. Here, we have studied the expression and function of phosphatases of regenerating liver (PRLs: PRL-1, PRL-2, and PRL-3) during T cell activation, as well as, the dynamic delivery of PRL-1 to the Immunological Synapse (IS). We found that T cell activation downregulates the expression of PRL-2, resulting in an increased PRL-1/PRL-2 ratio. PRL-1 redistributed at the IS in two stages: Initially, it was transiently accumulated at scanning membranes enriched in CD3 and actin, and at later times, it was delivered at the contact site from pericentriolar, CD3ζ-containing, vesicles. Once at the established IS, PRL-1 distributed to LFA-1 and CD3ε sites. Remarkably, PRL-1 was found to regulate actin dynamics during IS assembly and the secretion of IL-2. Moreover, pharmacological inhibition of the catalytic activity of the three PRLs reduced the secretion of IL-2. These results provide evidence indicating a regulatory role of PRL-1 during IS assembly and highlight the involvement of PRLs in immune responses by mature T cells.

T cells form adhesive contacts with antigen-presenting cells (APCs) as part of the normal surveillance process that occurs in lymph nodes and other tissues. Most of these adhesive interactions are formed by integrins that interact with ligands expressed on the surface of the APC. The interactive strength of integrins depends on their degree of membrane proximity as well as intracellular signals that dictate the conformation of the integrin. Integrins appear in different conformations that endow them with different affinities for their ligand(s). Integrin conformation and thus adhesive strength between the T cell and the APC is tuned by intracellular signals that are turned on by ligation of the T cell receptor (TCR) and chemokine receptors. During the different stages of the process, integrins, the TCR and chemokine receptors may be interconnected by the actin cytoskeleton underneath the plasma membrane, forming a chemical and physical network that facilitates the spatiotemporal dynamics, positioning, and function of these receptors and supports cell-cell adhesion during T cell activation, allowing it to perform its effector function.

MiRNA targeting of key immunoregulatory molecules fine-tunes the immune response. This mechanism boosts or dampens immune functions to preserve homeostasis while supporting the full development of effector functions. MiRNA expression changes during T cell activation, highlighting that their function is constrained by a specific spatiotemporal frame related to the signals that induce T cell-based effector functions. Here, we update the state of the art regarding the miRNAs that are differentially expressed during T cell stimulation. We also revisit the existing data on miRNA function in T cell activation, with a special focus on the modulation of the most relevant immunoregulatory molecules.

The different cytoskeleton systems and their connecting molecular motors move vesicles and intracellular organelles to shape cells. Polarized cells with specialized functions display an exquisite spatio-temporal regulation of both cytoskeletal and organelle arrangements that support their specific tasks. In particular, T cells rapidly change their shape and cellular function through the establishment of cell surface and intracellular polarity in response to a variety of cues. This review focuses on the contribution of the microtubule-based dynein/dynactin motor complex, the tubulin and actin cytoskeletons, and different organelles to the formation of the antigen-driven immune synapse.

Professional antigen-presenting cells (APCs) include dendritic cells, monocytes, and B cells. APCs internalize and process antigens, producing immunogenic peptides that enable antigen presentation to T lymphocytes, which provide the signals that trigger T-cell activation, proliferation, and differentiation, and lead to adaptive immune responses. After detection of microbial antigens through pattern recognition receptors (PRRs), APCs migrate to secondary lymphoid organs where antigen presentation to T lymphocytes takes place. Tetraspanins are membrane proteins that organize specialized membrane platforms, called tetraspanin-enriched microdomains, which integrate membrane receptors, like PRR and major histocompatibility complex class II (MHC-II), adhesion proteins, and signaling molecules. Importantly, through the modulation of the function of their associated membrane partners, tetraspanins regulate different steps of the immune response. Several tetraspanins can positively or negatively regulate the activation threshold of immune receptors. They also play a role during migration of APCs by controlling the surface levels and spatial arrangement of adhesion molecules and their subsequent intracellular signaling. Finally, tetraspanins participate in antigen processing and are important for priming of naïve T cells through the control of T-cell co-stimulation and MHC-II-dependent antigen presentation. In this review, we discuss the role of tetraspanins in APC biology and their involvement in effective immune responses.

The complexity of the pathogenesis of inflammatory bowel disease (ulcerative colitis and Crohn’s disease) has led to the quest of empirically drug therapies, combining immunosuppressant agents, biological therapy and modulators of the microbiota. Helminth parasites have been proposed as an alternative treatment of these diseases based on the hygiene hypothesis, but ethical and medical problems arise. Recent reports have proved the utility of parasite materials, mainly excretory/secretory products as therapeutic agents. The identification of extracellular vesicles on those secreted products opens a new field of investigation, since they exert potent immunomodulating effects. To assess the effect of extracellular vesicles produced by helminth parasites to treat ulcerative colitis, we have analyzed whether extracellular vesicles produced by the parasitic helminth Fasciola hepatica can prevent colitis induced by chemical agents in a mouse model. Adult parasites were cultured in vitro and secreted extracellular vesicles were purified and used for immunizing both wild type C57BL/6 and RAG1-/- mice. Control and immunized mice groups were treated with dextran sulfate sodium 7 days after last immunization to promote experimental colitis. The severity of colitis was assessed by disease activity index and histopathological scores. Mucosal cytokine expression was evaluated by ELISA. The activation of NF-kB, COX-2, and MAPK were evaluated by immunoblotting. Administration of extracellular vesicles from F. hepatica ameliorates the pathological symptoms reducing the amount of pro-inflammatory cytokines and interfering with both MAPK and NF-kB pathways. Interestingly, the observed effects do not seem to be mediated by T-cells. Our results indicate that extracellular vesicles from parasitic helminths can modulate immune responses in dextran sulfate sodium (DSS)-induced colitis, exerting a protective effect that should be mediated by other cells distinct from B- and T-lymphocytes.

Tetraspanins are a family of proteins with four transmembrane domains that associate between themselves and cluster with other partner proteins, conforming a distinct class of membrane domains, the tetraspanin-enriched microdomains (TEMs). These TEMs constitute macromolecular signaling platforms that regulate key processes in several cellular settings controlling signaling thresholds and avidity of receptors. In this study, we investigated the role of CD9, a tetraspanin that regulates major biological processes such as cell migration and immunological responses, in two mouse models of colitis that have been used to study the pathogenesis of inflammatory bowel disease (IBD). Previous in vitro studies revealed an important role in the interaction of leukocytes with inflamed endothelium, but in vivo evidence of the involvement of CD9 in inflammatory diseases is scarce. Here, we studied the role of CD9 in the pathogenesis of colitis in vivo. Colitis was induced by administration of dextran sodium sulfate (DSS), a chemical colitogen that causes epithelial disruption and intestinal inflammation. CD9−/− mice showed less severe colitis than wild-type counterparts upon exposure to DSS (2% solution) and enhanced survival in response to a lethal DSS dose (4%). Decreased neutrophil and macrophage cell infiltration was observed in colonic tissue from CD9−/− animals, in accordance with their lower serum levels of TNF-α, IL-6, and other proinflammatory cytokines in the colon. The specific role of CD9 in IBD was further dissected by transfer of CD4+ CD45RBhi naive T cells into the Rag1−/− mouse colitis model. However, no significant differences were observed in these settings between both groups, ruling out a role for CD9 in IBD in the lymphoid compartment. Experiments with bone marrow chimeras revealed that CD9 in the non-hematopoietic compartment is involved in colon injury and limits the proliferation of epithelial cells. Our data indicate that CD9 in non-hematopoietic cells plays an important role in colitis by limiting epithelial cell proliferation. Future strategies to repress CD9 expression may be of therapeutic benefit in the treatment of IBD.

The widespread use of systemic neonicotinoid insecticides in agriculture results first in contamination of the soil of the treated crops, and secondly in the transfer of residues to the aquatic environment. The high toxicity of these insecticides to aquatic insects and other arthropods has been recognized, but there is little awareness of the impacts these chemicals have on aquatic environments and the ecosystem at large. Recent monitoring studies in several countries, however, have revealed a world-wide contamination of creeks, rivers and lakes with these insecticides, with residue levels in the low μg/L (ppb) range. The current extent of aquatic contamination by neonicotinoids is reviewed first, and the findings contrasted with the known acute and chronic toxicity of neonicotinoids to various aquatic organisms. Impacts on populations and aquatic communities, mostly using mesocosms, are reviewed next to identify the communities most at risk from those that undergo little or no impact. Finally, the ecological links between aquatic and terrestrial organisms are considered. The consequences for terrestrial vertebrate species that depend mainly on this food source are discussed together with impacts on ecosystem function. Gaps in knowledge stem from difficulties in obtaining long-term experimental data that relates the effects on individual organisms to impacts on populations and ecosystems. The paper concludes with a summary of findings and the implications they have for the larger ecosystem.

Gene duplications are a feature of bacterial genomes. In the present work we analyze the extent of gene duplications in the genomes of three microorganisms that belong to the Firmicutes phylum and that are etiologic agents of several nosocomial infections: Staphylococcus aureus, Enterococcus faecium, and Enterococcus faecalis. In all three groups, there is an irregular distribution of duplications in the genomes of the strains analyzed. Whereas in some of the strains duplications are scarce, hundreds of duplications are present in others. In all three species, mobile DNA accounts for a large percentage of the duplicated genes: phage DNA in S. aureus, and plasmid DNA in the enterococci. Duplicates also include core genes. In all three species, a reduced group of genes is duplicated in all strains analyzed. Duplication of the deoC and rpmG genes is a hallmark of S. aureus genomes. Duplication of the gene encoding the PTS IIB subunit is detected in all enterococci genomes. In E. faecalis it is remarkable that the genomes of some strains encode duplicates of the prgB and prgU genes. They belong to the prgABCU cluster, which responds to the presence of the peptide pheromone cCF10 by expressing the surface adhesins PrgA, PrgB, and PrgC.