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Introduction: Treatment of calcium (Ca) and vitamin D (VD) deficiency (VDD) is crucial for health, especially in bone conditions, such as low bone mineral density (BMD) and osteoporosis. Despite updates in clinical guideline recommendations, no studies have evaluated the efficacy and safety of administering 2000 IU of cholecalciferol combined with calcium. Thus, the main objective of this study was to evaluate VD levels following treatment with Ca 600 mg/ cholecalciferol 2000 IU in real-life clinical practice. Methods: This multicenter, retrospective, observational study included 302 adult patients receiving Ca 600 mg/D3 2000 IU orodispersible tablets, daily for ≥24 weeks. The primary outcome was 25-hydroxivitamin D [25(OH)D] serum levels following treatment. Key secondary outcomes included changes in serum 25(OH)D levels and other bone metabolism (BM) parameters, safety and tolerability. The protocol was approved by a Research Ethics Committee. Results: 285 patients were evaluated (mean age [SD]: 67.4 [12.6] years old; 88.4 % women; basal serum 25(OH)D: 20.0 [8.6] ng/mL); 80.7 % reported previous history of osteoporosis/low BMD (osteopenia) and 37.2 % had received other Ca/VD prior to start study treatment. Median treatment duration was 38.5 weeks [range 24.0–82.4]. Overall, 94.4 % of patients increased serum 25(OH)D following treatment to a mean of 36.3 [11.8] ng/mL (p

Abstract Background Alcohol use disorder (AUD) is one of the most common psychiatric disorders, with the consumption of alcohol considered a leading cause of preventable deaths worldwide. Lipids play a crucial functional role in cell membranes; however, we know little about the role of lipids in extracellular vesicles (EVs) as regulatory molecules and disease biomarkers. Methods We employed a sensitive lipidomic strategy to characterize lipid species from the plasma EVs of AUD patients to evaluate functional roles and enzymatic activity networks to improve the knowledge of lipid metabolism after alcohol consumption. We analyzed plasma EV lipids from AUD females and males and healthy individuals to highlight lipids with differential abundance and biologically interpreted lipidomics data using LINEX2, which evaluates enzymatic dysregulation using an enrichment algorithm. Results Our results show, for the first time, that AUD females exhibited more significant substrate-product changes in lysophosphatidylcholine/phosphatidylcholine lipids and phospholipase/acyltransferase activity, which are potentially linked to cancer progression and neuroinflammation. Conversely, AUD males suffer from dysregulated ceramide and sphingomyelin lipids involving sphingomyelinase, sphingomyelin phosphodiesterase, and sphingomyelin synthase activity, which relates to hepatotoxicity. Notably, the analysis of plasma EVs from AUD females and males demonstrates enrichment of lipid ontology terms associated with “negative intrinsic curvature” and “positive intrinsic curvature”, respectively. Conclusions Our methodological developments support an improved understanding of lipid metabolism and regulatory mechanisms, which contribute to the identification of novel lipid targets and the discovery of sex-specific clinical biomarkers in AUD.

Our previous studies have reported that activation of the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3)-inflammasome complex in ethanol-treated astrocytes and chronic alcohol-fed mice could be associated with neuroinflammation and brain damage. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been shown to restore the neuroinflammatory response, along with myelin and synaptic structural alterations in the prefrontal cortex, and alleviate cognitive and memory dysfunctions induced by binge-like ethanol treatment in adolescent mice. Considering the therapeutic role of the molecules contained in mesenchymal stem cell-derived extracellular vesicles, the present study analyzed whether the administration of mesenchymal stem cell-derived extracellular vesicles isolated from adipose tissue, which inhibited the activation of the NLRP3 inflammasome, was capable of reducing hippocampal neuroinflammation in adolescent mice treated with binge drinking. We demonstrated that the administration of mesenchymal stem cell-derived extracellular vesicles ameliorated the activation of the hippocampal NLRP3 inflammasome complex and other NLRs inflammasomes (e.g., pyrin domain-containing 1, caspase recruitment domain-containing 4, and absent in melanoma 2, as well as the alterations in inflammatory genes (interleukin-1β, interleukin-18, inducible nitric oxide synthase, nuclear factor-kappa B, monocyte chemoattractant protein-1, and C–X3–C motif chemokine ligand 1) and miRNAs (miR-21a-5p, miR-146a-5p, and miR-141-5p) induced by binge-like ethanol treatment in adolescent mice. Bioinformatic analysis further revealed the involvement of miR-21a-5p and miR-146a-5p with inflammatory target genes and NOD-like receptor signaling pathways. Taken together, these findings provide novel evidence of the therapeutic potential of MSC-derived EVs to ameliorate the hippocampal neuroinflammatory response associated with NLRP3 inflammasome activation induced by binge drinking in adolescence.

Stress is a critical factor in the development of mental disorders such as addiction, underscoring the importance of stress resilience strategies. While the ketogenic diet (KD) has shown efficacy in reducing alcohol consumption in male mice without cognitive impairment, its impact on the stress response and addiction development, especially in females, remains unclear. This study examined the KD’s effect on increasing ethanol intake due to vicarious social defeat (VSD) in female mice. Sixty-four female OF1 mice were divided into two dietary groups: standard diet (n = 32) and KD (n = 32). These were further split based on exposure to four VSD or exploration sessions, creating four groups: EXP-STD (n = 16), VSD-STD (n = 16), EXP-KD (n = 16), and VSD-KD (n = 16). KD-fed mice maintained ketosis from adolescence until the fourth VSD/EXP session, after which they switched to a standard diet. The Social Interaction Test was performed 24 h after the last VSD session. Three weeks post-VSD, the Drinking in the Dark test and Oral Ethanol Self-Administration assessed ethanol consumption. The results showed that the KD blocked the increase in ethanol consumption induced by VSD in females. Moreover, among other changes, the KD increased the expression of the ADORA1 and CNR1 genes, which are associated with mechanisms modulating neurotransmission. Our results point to the KD as a useful tool to increase resilience to social stress in female mice.

Oleoylethanolamide (OEA) is a lipid with anti-inflammatory activity that modulates multiple reward-related behaviors. Previous studies have shown that OEA treatment reduces alcohol self-administration (SA) while inhibiting alcohol-induced inflammatory signaling. Nevertheless, the specific mechanisms that OEA targets to achieve these effects have not been widely explored. Here, we tested the effects of OEA treatment during alcohol SA, extinction or previous to cue-induced reinstatement of alcohol seeking. In addition, we measured gene expression changes in the striatum and hippocampus of relevant receptors for alcohol consumption (Drd1, Drd2, Cnr1, Oprm) as well as immune-related proteins (Il-6, Il-1β, Tlr4) and the brain-derived neurotrophic factor (Bdnf). Our results confirmed that when administered contingently, systemic OEA administration reduced alcohol SA and attenuated cue-induced reinstatement. Interestingly, we also observed that OEA treatment reduced the number of sessions needed for the extinction of alcohol seeking. Biochemical analyses showed that OEA induced gene expression changes in dopamine and cannabinoid receptors in the striatum and hippocampus. In addition, OEA treatment modulated the long-term immune response and increased Bdnf expression. These results suggest that boosting OEA levels may be an effective strategy for reducing alcohol SA and preventing relapse.

Abstract The lipid oleoylethanolamide (OEA) has been shown to affect reward-related behavior. However, there is limited experimental evidence about the specific neurotransmission systems OEA may be affecting to exert this modulatory effect. The aim of this study was to evaluate the effects of OEA on the rewarding properties of cocaine and relapse-related gene expression in the striatum and hippocampus. For this purpose, we evaluated male OF1 mice on a cocaine-induced CPP procedure (10 mg/kg) and after the corresponding extinction sessions, we tested drug-induced reinstatement. The effects of OEA (10 mg/kg, i.p.) were evaluated at three different timepoints: (1) Before each cocaine conditioning session (OEA-C), (2) Before extinction sessions (OEA-EXT) and (3) Before the reinstatement test (OEA-REINST). Furthermore, gene expression changes in dopamine receptor D1 gene, dopamine receptor D2 gene, opioid receptor µ, cannabinoid receptor 1, in the striatum and hippocampus were analyzed by qRT-PCR. The results obtained in the study showed that OEA administration did not affect cocaine CPP acquisition. However, mice receiving different OEA treatment schedules (OEA-C, OEA-EXT and OEA-REINST) failed to display drug-induced reinstatement. Interestingly, the administration of OEA blocked the increase of dopamine receptor gene D1 in the striatum and hippocampus caused by cocaine exposure. In addition, OEA-treated mice exhibited reduced striatal dopamine receptor gene D2 and cannabinoid receptor 1. Together, these findings suggest that OEA may be a promising pharmacological agent in the treatment of cocaine use disorder.

Abstract Background Lipids represent essential components of extracellular vesicles (EVs), playing structural and regulatory functions during EV biogenesis, release, targeting, and cell uptake. Importantly, lipidic dysregulation has been linked to several disorders, including metabolic syndrome, inflammation, and neurological dysfunction. Our recent results demonstrated the involvement of plasma EV microRNAs as possible amplifiers and biomarkers of neuroinflammation and brain damage induced by ethanol intoxication during adolescence. Considering the possible role of plasma EV lipids as regulatory molecules and biomarkers, we evaluated how acute ethanol intoxication differentially affected the lipid composition of plasma EVs in male and female adolescents and explored the participation of the immune response. Methods Plasma EVs were extracted from humans and wild-type (WT) and Toll-like receptor 4 deficient (TLR4-KO) mice. Preprocessing and exploratory analyses were conducted after the extraction of EV lipids and data acquisition by mass spectrometry. Comparisons between ethanol-intoxicated and control human female and male individuals and ethanol-treated and untreated WT and TLR4-KO female and male mice were used to analyze the differential abundance of lipids. Annotation of lipids into their corresponding classes and a lipid set enrichment analysis were carried out to evaluate biological functions. Results We demonstrated, for the first time, that acute ethanol intoxication induced a higher enrichment of distinct plasma EV lipid species in human female adolescents than in males. We observed a higher content of the PA, LPC, unsaturated FA, and FAHFA lipid classes in females, whereas males showed enrichment in PI. These lipid classes participate in the formation, release, and uptake of EVs and the activation of the immune response. Moreover, we observed changes in EV lipid composition between ethanol-treated WT and TLR4-KO mice (e.g., enrichment of glycerophosphoinositols in ethanol-treated WT males), and the sex-based differences in lipid abundance are more notable in WT mice than in TLR4-KO mice. All data and results generated have been made openly available on a web-based platform ( http://bioinfo.cipf.es/sal ). Conclusions Our results suggest that binge ethanol drinking in human female adolescents leads to a higher content of plasma EV lipid species associated with EV biogenesis and the propagation of neuroinflammatory responses than in males. In addition, we discovered greater differences in lipid abundance between sexes in WT mice compared to TLR4-KO mice. Our findings also support the potential use of EV-enriched lipids as biomarkers of ethanol-induced neuroinflammation during adolescence.

Abstract Background Accessible and cost-effective diagnostic tools are urgently needed to accurately quantify blood biomarkers to support early diagnosis of Alzheimer’s disease (AD). In this study, we investigated the ability of plasma amyloid-beta (Aβ)42/Aβ40 ratio measured by an antibody-free mass-spectrometric (MS) method, ABtest-MS, to detect early pathological changes of AD. Methods This cohort study included data from the baseline and 2-year follow-up visits from the Fundació ACE Healthy Brain Initiative (FACEHBI) study. Plasma Aβ42/Aβ40 was measured with ABtest-MS and compared to 18F-Florbetaben PET as the reference standard (cutoff for early amyloid deposition of 13.5 centiloids). Cross-validation was performed in an independent DPUK-Korean cohort. Additionally, associations of plasma Aβ42/Aβ40 with episodic memory performance and brain atrophy were assessed. Results The FACEHBI cohort at baseline included 200 healthy individuals with subjective cognitive decline (SCD), of which 36 (18%) were Aβ-PET positive. Plasma Aβ42/Aβ40 levels were significantly lower in Aβ-PET positive individuals (median [interquartile range, IQR], 0.215 [0.203–0.236]) versus Aβ-PET negative subjects (median [IQR], 0.261 [0.244–0.279]) (P < .001). Plasma Aβ42/Aβ40 was significantly correlated with Aβ-PET levels (rho = −0.390; P < .001) and identified Aβ-PET status with an area under the receiver operating characteristic curve (AUC) of 0.87 (95% confidence interval [CI], 0.80–0.93). A cutoff for the Aβ42/Aβ40 ratio of 0.241 (maximum Youden index) yielded a sensitivity of 86.1% and a specificity of 80.5%. These findings were cross-validated in an independent DPUK-Korean cohort (AUC 0.86 [95% CI 0.77–0.95]). Lower plasma Aβ42/Aβ40 ratio was associated with worse episodic memory performance and increased brain atrophy. Plasma Aβ42/Aβ40 at baseline predicted clinical conversion to mild cognitive impairment and longitudinal changes in amyloid deposition and brain atrophy at 2-year follow-up. Conclusions This study suggests that plasma Aβ42/Aβ40, as determined by this MS-based assay, has potential value as an accurate and cost-effective tool to identify individuals in the earliest stages of AD, supporting its implementation in clinical trials, preventative strategies and clinical practice.

Abstract INTRODUCTION This study explored the ability of plasma amyloid beta (Aβ)42/Aβ40 to identify brain amyloid deposition in cognitively unimpaired (CU) individuals. METHODS Plasma Aβ was quantified with an antibody‐free high‐performance liquid chromatography tandem mass spectrometry method from Araclon Biotech (ABtest‐MS) in a subset of 731 CU individuals from the screening visit of the Anti‐Amyloid Treatment in Asymptomatic Alzheimer's (A4) Study, to assess associations of Aβ42/Aβ40 with Aβ positron emission tomography (PET). RESULTS A model including Aβ42/Aβ40, age, apolipoprotein E ε4, and recruitment site identified Aβ PET status with an area under the curve of 0.88 and an overall accuracy of 81%. A plasma‐based pre‐screening step could save up to 42% of the total number of Aβ PET scans. DISCUSSION ABtest‐MS accurately identified brain amyloid deposition in a population of CU individuals, supporting its implementation in AD secondary prevention trials to reduce recruitment time and costs. Although a certain degree of heterogeneity is inherent to large and multicentric trials, ABtest‐MS could be more robust to pre‐analytical bias compared to other immunoprecipitation mass spectrometry methods. HIGHLIGHTS Plasma amyloid beta (Aβ)42/Aβ40 accurately identified brain Aβ deposition in cognitively unimpaired individuals from the Anti‐Amyloid Treatment in Asymptomatic Alzheimer's (A4) Study. The inclusion of the recruitment site in the predictive models has a non‐negligible effect. A plasma biomarker‐based model could reduce recruitment costs in Alzheimer's disease secondary prevention trials. Antibody‐free liquid chromatography mass spectrometry methods may be more robust to pre‐analytical variability than other platforms.

BackgroundEarly detection of β-amyloid (Aβ) accumulation, a major biomarker for Alzheimer’s disease (AD), has become important. As fluid biomarkers, the accuracy of cerebrospinal fluid (CSF) Aβ for predicting Aβ deposition on positron emission tomography (PET) has been extensively studied, and the development of plasma Aβ is beginning to receive increased attention recently. In the present study, we aimed to determine whether APOE genotypes, age, and cognitive status increase the predictive performance of plasma Aβ and CSF Aβ levels for Aβ PET positivity.MethodsWe recruited 488 participants who underwent both plasma Aβ and Aβ PET studies (Cohort 1) and 217 participants who underwent both cerebrospinal fluid (CSF) Aβ and Aβ PET studies (Cohort 2). Plasma and CSF samples were analyzed using ABtest-MS, an antibody-free liquid chromatography-differential mobility spectrometry-triple quadrupole mass spectrometry method and INNOTEST enzyme-linked immunosorbent assay kits, respectively. To evaluate the predictive performance of plasma Aβ and CSF Aβ, respectively, logistic regression and receiver operating characteristic analyses were performed.ResultsWhen predicting Aβ PET status, both plasma Aβ42/40 ratio and CSF Aβ42 showed high accuracy (plasma Aβ area under the curve (AUC) 0.814; CSF Aβ AUC 0.848). In the plasma Aβ models, the AUC values were higher than plasma Aβ alone model, when the models were combined with either cognitive stage (p

In recent years, a type of extracellular vesicles named exosomes has emerged that play an important role in intercellular communication under physiological and pathological conditions. These nanovesicles (30–150 nm) contain proteins, RNAs and lipids, and their internalization by bystander cells could alter their normal functions. This review focuses on recent knowledge about exosomes as messengers of neuron-glia communication and their participation in the physiological and pathological functions in the central nervous system. Special emphasis is placed on the role of exosomes under toxic or pathological stimuli within the brain, in which the glial exosomes containing inflammatory molecules are able to communicate with neurons and contribute to the pathogenesis of neuroinflammation and neurodegenerative disorders. Given the small size and characteristics of exosomes, they can cross the blood-brain barrier and be used as biomarkers and diagnosis for brain disorders and neuropathologies. Finally, although the application potential of exosome is still limited, current studies indicate that exosomes represent a promising strategy to gain pathogenic information to identify therapeutically targets and biomarkers for neurological disorders and neuroinflammation.

Non-alcoholic fatty liver disease (NAFLD) includes a spectrum of liver alterations that can result in severe disease and even death. Consumption of ultra-processed foods (UPF) has been associated with obesity and related comorbidities. However, the link between UPF and NAFLD has not been sufficiently assessed. We aimed to investigate the prospective association between UPF consumption and liver health biomarkers. Methods: We followed for 1 year 5867 older participants with overweight/obesity and metabolic syndrome (MetS) from the PREDIMED-Plus trial. A validated 143-item semi-quantitative food frequency questionnaire was used to evaluate consumption of UPF at baseline, 6, and 12 months. The degree of processing for foods and beverages (g/day) was established according to the NOVA classification system. The non-invasive fatty liver index (FLI) and hepatic steatosis index (HSI) were used to evaluate liver health at three points in time. The associations between changes in UPF consumption (percentage of total daily dietary intake (g)) and liver biomarkers were assessed using mixed-effects linear models with repeated measurements. Results: In this cohort, UPF consumption at baseline was 8.19% (SD 6.95%) of total daily dietary intake in grams. In multivariable models, each 10% daily increment in UPF consumption in 1 year was associated with significantly greater FLI (β 1.60 points, 95% CI 1.24;1.96 points) and HSI (0.43, 0.29; 0.57) scores (all p-values < 0.001). These associations persisted statistically significant after adjusting for potential dietary confounders and NAFLD risk factors. Conclusions: A higher UPF consumption was associated with higher levels of NAFLD-related biomarkers in older adults with overweight/obesity and MetS.

Abstract Background Current evidence indicates that extracellular vesicles (EVs) participate in intercellular signaling, and in the regulation and amplification of neuroinflammation. We have previously shown that ethanol activates glial cells through Toll-like receptor 4 (TLR4) by triggering neuroinflammation. Here, we evaluate if ethanol and the TLR4 response change the release and inflammatory content of astrocyte-derived EVs, and whether these vesicles are capable of communicating with neurons by spreading neuroinflammation. Methods Cortical neurons and astrocytes in culture were used. EVs were isolated from the extracellular medium of the primary culture of the WT and TLR4-KO astrocytes treated with or without ethanol (40 mM) for 24 h. Flow cytometry, nanoparticle tracking analysis technology, combined with exosomal molecular markers (tetraspanins) along with electron microscopy, were used to characterize and quantify EVs. The content of EVs in inflammatory proteins, mRNA, and miRNAs was analyzed by Western blot and RT-PCR in both astrocyte-derived EVs and the neurons incubated or not with these EVs. Functional analyses of miRNAs were also performed. Results We show that ethanol increases the number of secreted nanovesicles and their content by raising the levels of both inflammatory-related proteins (TLR4, NFκB-p65, IL-1R, caspase-1, NLRP3) and by changing miRNAs (mir-146a, mir-182, and mir-200b) in the EVs from the WT-astrocytes compared with those from the untreated WT cells. No changes were observed in either the number of isolated EVs or their content between the untreated and ethanol-treated TLR4-KO astrocytes. We also show that astrocyte-derived EVs could be internalized by naïve cortical neurons to increase the neuronal levels of inflammatory protein (COX-2) and miRNAs (e.g., mir-146a) and to compromise their survival. The functional analysis of miRNAs revealed the regulatory role of the expressed miRNAs in some genes involved in several inflammatory pathways. Conclusions These results suggest that astrocyte-derived EVs could act as cellular transmitters of inflammation signaling by spreading and amplifying the neuroinflammatory response induced by ethanol through TLR4 activation.

Una de las reticencias sociales más extendidas acerca del uso de combustibles de biomasa son las emisiones que producen. En el caso de la biomasa forestal, son puntos conflictivos para el sector ambientalista el recuento y la gestión del carbono absorbido, los posibles daños a la biodiversidad y a la calidad de los aprovechamientos forestales, y el posible aumento de emisiones. Por el contrario, el sector técnico apuesta por una gestión forestal sostenible, mediante el uso de técnicas avanzadas que permitan conocer cómo funcionan los mecanismos de los bosques como sumideros de carbono. Los riesgos ambientales del uso energético de los biocombustibles pueden ser revertidos mediante el empleo de parámetros técnicos. La normativa europea establece criterios de sostenibilidad para la biomasa con destino energético, pero no fija parámetros acerca de la sostenibilidad del resto de fuentes renovables. Este trabajo se basa en la necesaria imbricación entre la técnica normativa consagrada el programa europeo “Legislar mejor para obtener mejores resultados - Un programa de la UE” y la ciencia, por una mayor sostenibilidad del uso de fuentes de energía renovables.

Verapamil is a phenylalkylamine class calcium channel blocker that for half a century has been used for the treatment of cardiovascular diseases. Nowadays, verapamil is also considered as a drug option for the treatment of several neurological and psychiatric disorders, such as cluster headache, bipolar disorders, epilepsy, and neurodegenerative diseases. Here, we review insights into the potential preventive and therapeutic role of verapamil on Alzheimer’s disease (AD) based on limited experimental and clinical data. Pharmacological studies have shown that verapamil has a wide therapeutic spectrum, including antihypertensive, anti-inflammatory, and antioxidative effects, regulation of the blood-brain barrier function, due to its effect on P-glycoprotein, as well as adjustment of cellular calcium homeostasis, which may result in the delay of AD onset or ameliorate the symptoms of patients. However, the majority of the AD individuals are on polypharmacotherapy, and the interactions between verapamil and other drugs need to be considered. Therefore, for an appropriate and successful AD treatment, a personalized approach is more than necessary. A well-known narrow pharmacological window of verapamil efficacy may hinder this approach. It is therefore important to note that the verapamil efficacy may be conditioned by different factors. The onset, grade, and brain distribution of AD pathological hallmarks, the time-sequential appearances of AD-related cognitive and behavioral dysfunction, the chronobiologic and gender impact on calcium homeostasis and AD pathogenesis may somehow be influencing that success. In the future, such insights will be crucial for testing the validity of verapamil treatment on animal models of AD and clinical approaches.

Abstract Background Immunotherapy targeting the amyloid-β (Aβ) peptide is a promising strategy for the treatment of Alzheimer’s disease (AD); however, none of the active or passive vaccines tested have been demonstrated to be effective to date. We have developed the first active vaccine against the C-terminal end of Aβ40, ABvac40, and assessed its safety and tolerability in a phase I clinical trial. Methods A randomised, double-blind, placebo-controlled, parallel-group, phase I study of ABvac40 was conducted with patients aged 50–85 years with mild to moderate AD. Participants were entered into three separate groups according to time of study entry and were randomly allocated to receive ABvac40 or placebo (overall ratio 2:1). The first group received two half-doses of ABvac40 or placebo, whereas the second and third groups received two and three full doses, respectively. All treatments were administered subcutaneously at 4-week intervals. Patients, carers and investigators were blind to treatment allocation throughout the study. The primary objective was to assess the safety and tolerability of ABvac40 by registering all adverse events (AEs). All patients who received at least one dose of treatment were included in the safety analysis. The secondary objective was to evaluate the immunogenicity of ABvac40 by titration of specific anti-Aβ40 antibodies in plasma. Results Twenty-four patients were randomly allocated: 16 patients to the ABvac40 group and 8 patients to the placebo group. All randomised patients completed the study, therefore the intention-to-treat and safety populations were identical. Overall, 71 AEs affecting 18 patients were recorded: 11 (69%) in the ABvac40 group and 7 (88%) in the placebo group (p = 0.6214). Neither incident vasogenic oedema nor sulcal effusion (amyloid-related imaging abnormalities corresponding to vasogenic oedema and sulcal effusions) nor microhaemorrhages (amyloid-related imaging abnormalities corresponding to microhaemorrhages and hemosiderin deposits) were detected throughout the study period in the ABvac40-treated patients. Eleven of 12 (~92%) individuals receiving three injections of ABvac40 developed specific anti-Aβ40 antibodies. Conclusions ABvac40 showed a favourable safety and tolerability profile while eliciting a consistent and specific immune response. An ongoing phase II clinical trial is needed to confirm these results and to explore the clinical efficacy of ABvac40. Trial registration ClinicalTrials.gov, NCT03113812. Retrospectively registered on 14 April 2017.

Chronic alcohol abuse causes an inflammatory response in the intestinal tract with damage to the integrity of the mucosa and epithelium, as well as dysbiosis in the gut microbiome. However, the role of gut bacteria in ethanol effects and how these microorganisms interact with the immune system are not well understood. The aim of the present study was to evaluate if TLR4 alters the ethanol-induced intestinal inflammatory response, and whether the response of this receptor affects the gut microbiota profile. We analyzed the 16S rRNA sequence of the fecal samples from wild-type (WT) and TLR4-knockout (TLR4-KO) mice with and without ethanol intake for 3 months. The results demonstrated that chronic ethanol consumption reduces microbiota diversity and causes dysbiosis in WT mice. Likewise, ethanol upregulates several inflammatory genes (IL-1β, iNOS, TNF-α) and miRNAs (miR-155-5p, miR-146a-5p) and alters structural and permeability genes (INTL1, CDH1, CFTR) in the colon of WT mice. Our results further demonstrated that TLR4-KO mice exhibit a different microbiota that can protect against the ethanol-induced activation of the immune system and colon integrity dysfunctions. In short, our results reveal that TLR4 is a key factor for determining the gut microbiota, which can participate in dysbiosis and the inflammatory response induced by alcohol consumption.

Recent evidence pinpoints extracellular vesicles (EVs) as key players in intercellular communication. Given the importance of cholesterol and sphingomyelin in EV biology, and the relevance of mitochondria-associated endoplasmic reticulum membranes (MAMs) in cholesterol/sphingomyelin homeostasis, we evaluated if MAMs and sphingomyelinases (SMases) could participate in ethanol-induced EV release. EVs were isolated from the extracellular medium of BV2 microglia treated or not with ethanol (50 and 100 mM). Radioactive metabolic tracers combined with thin layer chromatography were used as quantitative methods to assay phospholipid transfer, SMase activity and cholesterol uptake/esterification. Inhibitors of SMase (desipramine and GW4869) and MAM (cyclosporin A) activities were also utilized. Our data show that ethanol increases the secretion and inflammatory molecule concentration of EVs. Ethanol also upregulates MAM activity and alters lipid metabolism by increasing cholesterol uptake, cholesterol esterification and SMase activity in microglia. Notably, the inhibition of either SMase or MAM activity prevented the ethanol-induced increase in EV secretion. Collectively, these results strongly support a lipid-driven mechanism, specifically via SMases and MAM, to explain the effect of ethanol on EV secretion in glial cells.

The classic ketogenic diet is a diet high in fat, low in carbohydrates, and well-adjusted proteins. The reduction in glucose levels induces changes in the body’s metabolism, since the main energy source happens to be ketone bodies. Recent studies have suggested that nutritional interventions may modulate drug addiction. The present work aimed to study the potential effects of a classic ketogenic diet in modulating alcohol consumption and its rewarding effects. Two groups of adult male mice were employed in this study, one exposed to a standard diet (SD, n = 15) and the other to a ketogenic diet (KD, n = 16). When a ketotic state was stable for 7 days, animals were exposed to the oral self-administration paradigm to evaluate the reinforcing and motivating effects of ethanol. Rt-PCR analyses were performed evaluating dopamine, adenosine, CB1, and Oprm gene expression. Our results showed that animals in a ketotic state displayed an overall decrease in ethanol consumption without changes in their motivation to drink. Gene expression analyses point to several alterations in the dopamine, adenosine, and cannabinoid systems. Our results suggest that nutritional interventions may be a useful complementary tool in treating alcohol-use disorders.

Abstract Background Inflammation during brain development participates in the pathogenesis of early brain injury and cognitive dysfunctions. Prenatal ethanol exposure affects the developing brain and causes neural impairment, cognitive and behavioral effects, collectively known as fetal alcohol spectrum disorders (FASD). Our previous studies demonstrate that ethanol activates the innate immune response and TLR4 receptor and causes neuroinflammation, brain damage, and cognitive defects in the developmental brain stage of adolescents. We hypothesize that by activating the TLR4 response, maternal alcohol consumption during pregnancy triggers the release of cytokines and chemokines in both the maternal sera and brains of fetuses/offspring, which impairs brain ontogeny and causes cognitive dysfunction. Methods WT and TLR4-KO female mice treated with or without 10% ethanol in the drinking water during gestation and lactation were used. Cytokine/chemokine levels were determined by ELISA in the amniotic fluid, maternal serum, and cerebral cortex, as well as in the offspring cerebral cortex. Microglial and neuronal markers (evaluated by western blotting), myelin proteins (immunohistochemical and western blotting) and synaptic parameters (western blotting and electron microscopy) were assessed in the cortices of the WT and TLR4-KO pups on PND 0, 20, and 66. Behavioral tests (elevated plus maze and passive avoidance) were performed in the WT and TLR4-KO mice on PND 66 exposed or not to ethanol. Results We show that alcohol intake during gestation and lactation increases the levels of several cytokines/chemokines (IL-1β, IL-17, MIP-1α, and fractalkine) in the maternal sera, amniotic fluid, and brains of fetuses and offspring. The upregulation of cytokines/chemokines is associated with an increase in activated microglia markers (CD11b and MHC-II), and with a reduction in some synaptic (synaptotagmin, synapsin IIa) and myelin (MBP, PLP) proteins in the brains of offspring on days 0, 20, and 66 (long-term effects). These changes are associated with long-term behavioral impairments, in the 66-day-old alcohol-exposed pups. TLR4-deficient mice are protected against ethanol-induced cytokine/chemokine production in alcohol-treated dams and offspring, along with synaptic and myelin alterations, and the log-term behavioral dysfunction induced by ethanol in offspring. Conclusions These results suggest that the immune system activation, through the TLR4 response, might play an important role in the neurodevelopmental defects in FASD.

The alarming threat of the spread of multidrug resistant bacteria currently leaves clinicians with very limited options to combat infections, especially those from Gram-negative bacteria. Hence, innovative strategies to deliver the next generation of antibacterials are urgently needed. Penicillin binding proteins (PBPs) are proven targets inhibited by β-lactam antibiotics. To discover novel, non-β-lactam inhibitors against PBP3 of Pseudomonas aeruginosa, we optimised a fluorescence assay based on a well-known thioester artificial substrate and performed a target screening using a focused protease-targeted library of 2455 compounds, which led to the identification of pyrrolidine-2,3-dione as a potential scaffold to inhibit the PBP3 target. Further chemical optimisation using a one-pot three-component reaction protocol delivered compounds with excellent target inhibition, initial antibacterial activities against P. aeruginosa and no apparent cytotoxicity. Our investigation revealed the key structural features; for instance, 3-hydroxyl group (R2) and a heteroaryl group (R1) appended to the N-pyrroldine-2,3-dione via methylene linker required for target inhibition. Overall, the discovery of the pyrrolidine-2,3-dione class of inhibitors of PBP3 brings opportunities to target multidrug-resistant bacterial strains and calls for further optimisation to improve antibacterial activity against P. aeruginosa.

Human intestinal microbiota comprise of a dynamic population of bacterial species and other microorganisms with the capacity to interact with the rest of the organism and strongly influence the host during homeostasis and disease. Commensal and pathogenic bacteria coexist in homeostasis with the intestinal epithelium and the gastrointestinal tract’s immune system, or GALT (gut-associated lymphoid tissue), of the host. However, a disruption to this homeostasis or dysbiosis by different factors (e.g., stress, diet, use of antibiotics, age, inflammatory processes) can cause brain dysfunction given the communication between the gut and brain. Recently, extracellular vesicles (EVs) derived from bacteria have emerged as possible carriers in gut-brain communication through the interaction of their vesicle components with immune receptors, which lead to neuroinflammatory immune response activation. This review discusses the critical role of bacterial EVs from the gut in the neuropathology of brain dysfunctions by modulating the immune response. These vesicles, which contain harmful bacterial EV contents such as lipopolysaccharide (LPS), peptidoglycans, toxins and nucleic acids, are capable of crossing tissue barriers including the blood-brain barrier and interacting with the immune receptors of glial cells (e.g., Toll-like receptors) to lead to the production of cytokines and inflammatory mediators, which can cause brain impairment and behavioral dysfunctions.

Current studies evidence the role of miRNAs in extracellular vesicles (EVs) as key regulators of pathological processes, including neuroinflammation and neurodegeneration. As EVs can cross the blood–brain barrier, and EV miRNAs are very stable in peripheral circulation, we evaluated the potential gender differences in inflammatory-regulated miRNAs levels in human and murine plasma EVs derived from alcohol-intoxicated female and male adolescents, and whether these miRNAs could be used as biomarkers of neuroinflammation. We demonstrated that while alcohol intoxication lowers anti-inflammatory miRNA (mir-146a-5p, mir-21-5p, mir-182-5p) levels in plasma EVs from human and mice female adolescents, these EV miRNAs increased in males. In mice brain cortices, ethanol treatment lowers mir-146a-5p and mir-21-5p levels, while triggering a higher expression of inflammatory target genes (Traf6, Stat3, and Camk2a) in adolescent female mice. These results indicate, for the first time, that female and male adolescents differ as regards the ethanol effects associated with the inflammatory-related plasma miRNAs EVs profile, and suggest that female adolescents are more vulnerable than males to the inflammatory effects of binge alcohol drinking. These findings also support the view that circulating miRNAs in EVs could be useful biomarkers for screening ethanol-induced neuroinflammation and brain damage in adolescence.

Ethanol induces brain damage and neurodegeneration by triggering inflammatory processes in glial cells through activation of Toll-like receptor 4 (TLR4) signaling. Recent evidence indicates the role of protein degradation pathways in neurodegeneration and alcoholic liver disease, but how these processes affect the brain remains elusive. We have demonstrated that chronic ethanol consumption impairs proteolytic pathways in mouse brain, and the immune response mediated by TLR4 receptors participates in these dysfunctions. We evaluate the in vitro effects of an acute ethanol dose on the autophagy-lysosome pathway (ALP) on WT and TLR4-/- mouse astrocytes and neurons in primary culture, and how these changes affect cell survival. Our results show that ethanol induces overexpression of several autophagy markers (ATG12, LC3-II, CTSB), and increases the number of lysosomes in WT astrocytes, effects accompanied by a basification of lysosomal pH and by lowered phosphorylation levels of autophagy inhibitor mTOR, along with activation of complexes beclin-1 and ULK1. Notably, we found only minor changes between control and ethanol-treated TLR4-/- mouse astroglial cells. Ethanol also triggers the expression of the inflammatory mediators iNOS and COX-2, but induces astroglial death only slightly. Blocking autophagy by using specific inhibitors increases both inflammation and cell death. Conversely, in neurons, ethanol down-regulates the autophagy pathway and triggers cell death, which is partially recovered by using autophagy enhancers. These results support the protective role of the ALP against ethanol-induced astroglial cell damage in a TLR4-dependent manner, and provide new insight into the mechanisms that underlie ethanol-induced brain damage and are neuronal sensitive to the ethanol effects.

Este artículo es una reflexión sobre el Plan Bolonia, el Espacio Europeo de Educación Superior y las implicaciones que tienen en su implementación real en las aulas. El objetivo primordial de este artículo es compartir la experiencia de la implantación del nuevo Grado en Educación en la E.U. de Educación de Soria partiendo del nuevo panorama educativo al que nos enfrentamos y concretando cuáles han sido las acciones específicas desarrolladas en una asignatura particular, Inglés como lengua extranjera.

El pronunciamiento de autos resuelve la impugnación de la sentencia del Juzgado de lo Contencioso-administrativo n° 3 de Oviedo, desestimatoria del recurso de una particular contra la Orden del Ayuntamiento de Pola de Siero, de cesar una actividad de refugio o acogida de animales en su vivienda por no disponer de la licencia requerida, al exceder el número permitido de perros según el PGO de Pola de Siero. La apelante alega los siguientes motivos de impugnación: i) que la sentencia interpreta de forma errónea el artículo 4.30.3 del PGO de Siero, al considerar que se excede el número permitido de perros, dado que la propiedad está compuesta por dos fincas registradas separadamente; además, sostiene que la actividad no puede calificarse como "pequeña explotación ganadera" o "tenencia de perros de cría o guarda", ya que los animales están esterilizados y en acogida temporal bajo la custodia de una asociación protectora; ii) Rechaza que se haya vulnerado el procedimiento administrativo al modificar la calificación de la actividad, generando indefensión; iii) Finalmente, alega que exigir licencia de actividad para una casa de acogida de animales, como la suya, contravendría el artículo 3.l) de la Ley 7/2023 y dejaría sin efecto esta figura legal. Por su parte, el Ayuntamiento de Siero defiende la correcta aplicación de la normativa urbanística y el procedimiento administrativo, alegando que la actividad desarrollada por la apelante excede la tenencia simple de animales de compañía, requiriendo licencias de actividad según el informe técnico y la Ley 1/2023 de Calidad Ambiental. El Tribunal resuelve como sigue: i) En relación con la alegación de que la actividad se lleva a cabo en dos fincas registradas separadamente, invalidando el argumento de exceso de perros, la sentencia rechaza esta interpretación, afirmando que, independientemente de la división registral, las fincas constituyen una unidad física. ii) Sobre la aplicación del artículo 4.30.3 del PGMO de Siero, la Sala concluye que, incluso si se tratara de una actividad de guardería de perros, es necesaria una declaración responsable o una licencia de actividad, debido a los posibles impactos ambientales y molestias a los vecinos, según la Ley 1/2023 de Calidad Ambiental y el Reglamento de Actividades Molestas. iii) Respecto a la declaración responsable presentada el 28 de abril de 2023, el pronunciamiento explica que dicha declaración refuerza la necesidad de cumplir con la exigencia administrativa previa para desarrollar la actividad de refugio de animales. En conclusión, la sentencia ratifica que la actividad de la apelante requiere autorizaciones administrativas tanto desde la perspectiva urbanística como ambiental, y que el Ayuntamiento actuó correctamente dentro de sus competencias. [ABSTRACT FROM AUTHOR]

El pronunciamiento que hoy analizamos resuelve el recurso de apelación interpuesto por la Federación Provincial de Ecologistas en Acción contra la sentencia dictada por el Juzgado de lo Contencioso-Administrativo de Almería. La sentencia de instancia versa sobre la desestimación presunta por silencio administrativo de la solicitud de licencia de obra y la calificación ambiental para la construcción de una nave ganadera y una explotación porcina de cebo por parte de una mercantil. Los motivos de apelación se centran en la suspensión de las licencias debido a modificaciones del PGOU de Huércal Overa, la falta de respuesta motivada por parte de la Administración a las alegaciones presentadas durante la exposición pública y la carencia de una adecuada valoración de los efectos medioambientales de la explotación, particularmente sobre el acuífero local y los recursos hídricos. En primer lugar, la Sala recuerda que el recurso procesal de apelación no permite introducir cuestiones nuevas que no fueron planteadas en primera instancia. Su objetivo es revisar la sentencia impugnada, señalando errores en la aplicación de la ley o en la valoración de la prueba, pero no se pueden introducir argumentos nuevos que no fueron presentados en la fase inicial. De ahí que el Tribunal no se pronuncie sobre algunos de los motivos de interés ambiental planteados por la apelante. A los anteriores efectos, la Sala examina si se vulneraron el artículo 27 de la Ley de Ordenación Urbanística de Andalucía (LOUA) y los artículos 21 a 24 de la Ley 39/2015 (Ley de Procedimiento Administrativo Común). El Tribunal se remite a la sentencia de instancia y a la jurisprudencia que cita la misma. De este modo, concluye que, si bien las administraciones competentes pueden suspender temporalmente la concesión de aprobaciones y licencias urbanísticas por un plazo de hasta un año en casos donde se esté elaborando o modificando instrumentos de planeamiento, dicha suspensión no puede aplicarse retroactivamente a solicitudes anteriores a la adopción del acuerdo de suspensión, como es el presente caso. Otra de las cuestiones que analiza el pronunciamiento es la obligación de la Administración de resolver expresamente en un plazo determinado, en relación con el silencio administrativo aplicable a las solicitudes para el ejercicio de actividades que puedan dañar el medio ambiente. En el supuesto que traemos a colación, se aplicó artículo 24.1 de la Ley 39/2015, que establece que el silencio administrativo tiene efecto desestimatorio para las meritadas solicitudes. No obstante, la sentencia de autos, en línea con la instancia, considera que la desestimación tácita no es conforme a derecho en tanto no existe ningún motivo impeditivo de una resolución expresa estimatoria. Lo anterior, porque la recurrente había cumplido con todos los requerimientos. Por todo lo anterior, el Tribunal desestima el recurso de la Federación Provincial de Ecologistas en Acción. [ABSTRACT FROM AUTHOR]

La sentencia que traemos a colación resuelve la impugnación de la Resolución de la Confederación Hidrográfica del Guadalquivir, que impuso una multa y medidas compensatorias por la extracción de agua subterránea sin la correspondiente autorización a un particular y a la Consejería de Medio Ambiente a una sanción por la extracción de aguas sin título legal. Asimismo, a meritada Consejería se le impuso la obligación de inutilizar los pozos, y al particular, el cese de inmediato en la derivación hasta la obtención de los títulos habilitantes precisos. El recurrente argumenta que los hechos sancionados ya fueron objeto de proceso penal, del cual fue absuelto. Alegó la existencia de prejudicialidad penal, al haberse declarado en sede penal la falta de dolo en la conducta. La Confederación Hidrográfica del Guadalquivir, sin embargo, se opone, argumentando que los hechos sancionados en el expediente administrativo son diferentes a los del proceso penal, por estar relacionados con diferentes períodos de extracción. El Tribunal suspendió el procedimiento contencioso-administrativo hasta la resolución del proceso penal, que exoneró a los acusados de delitos contra los recursos naturales y el medio ambiente por extracción ilegal de aguas. La sentencia penal concluyó que los agricultores actuaron bajo la creencia de estar amparados por convenios suscritos con la Junta de Andalucía y el Ayuntamiento de Almonte, y, por ende, convencidos en la legalidad en sus actividades. La Sala considera que, a pesar de no darse en el caso se autos identidad de sujetos entre el procedimiento penal y el sancionador (ya que el recurrente no fue parte en el proceso penal), los hechos probados en aquel son vinculantes para la Administración (art. 77.4 de la Ley 39/2015), e influyen directamente sobre la valoración de los hechos en el procedimiento sancionador. Consecuentemente, anula la resolución sancionadora y estima el recurso del recurrente, sin imposición de costas dada la complejidad del asunto. [ABSTRACT FROM AUTHOR]

Abstract The demersal fish orange roughy (Hoplostethus atlanticus) can live for up to 250 years, twenty times more than its congener silver roughy (Hoplostethus mediterraneus). Studies of Hoplostethus have focused mainly on its ecology and conservation due to its vulnerability to commercial fishing. In this work, we present the de novo genomes of orange and silver roughies and explore the genomic mechanisms that could contribute to such differential longevities. Using comparative genomics on a list of more than 400 genes, we identified gene candidates with differential residue changes in Hoplostethus that are related to genomic instability, disabled macroautophagy and intercellular communication. We hypothesized that these mechanisms could have been selected as adaptations to the deep environment and, as an epiphenomenon of these mechanisms, may have contributed to an extension of the lifespan of H. atlanticus.