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Abstract Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disorder of the neuromuscular junction. A small subset of patients (

Abstract Background Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. Methods A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. Results Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. Conclusion Our results demonstrate a crucial role of CERTL in regulating ceramide levels in the brain, in amyloid plaque formation and neuroinflammation, thereby opening research avenues for therapeutic targets of AD and other neurodegenerative diseases.

Abstract Background Telomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients. Methods This article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes. Results Forty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening. Conclusion Novel variants in telomere, DNA repair and replication genes are described that might indicate the contribution of variants in these genes to the development of telomeropathies. Patients carrying variants in telomere-related genes presented worse evolution after diagnosis than the rest of patients analyzed.

Abstract Background Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease dementia (PDD), and frontotemporal lobar dementia (FTLD) are characterized by progressive neuronal loss but differ in their underlying pathological mechanisms. However, neuroinflammation is commonly observed within these different forms of dementia. Recently, it has been suggested that an altered sphingolipid metabolism may contribute to the pathogenesis of a variety of neurodegenerative conditions. Especially ceramide, the precursor of all complex sphingolipids, is thought to be associated with pro-apoptotic cellular processes, thereby propagating neurodegeneration and neuroinflammation, although it remains unclear to what extent. The current pathological study therefore investigates whether increased levels of ceramide are associated with the degree of neuroinflammation in various neurodegenerative disorders. Methods Immunohistochemistry was performed on human post-mortem tissue of PDD and FTLD Pick’s disease cases, which are well-characterized cases of dementia subtypes differing in their neuroinflammatory status, to assess the expression and localization of ceramide, acid sphingomyelinase, and ceramide synthase 2 and 5. In addition, we determined the concentration of sphingosine, sphingosine-1-phosphate (S1P), and ceramide species differing in their chain-length in brain homogenates of the post-mortem tissue using HPLC-MS/MS. Results Our immunohistochemical analysis reveals that neuroinflammation is associated with increased ceramide levels in astrocytes in FTLD Pick’s disease. Moreover, the observed increase in ceramide in astrocytes correlates with the expression of ceramide synthase 5. In addition, HPLC-MS/MS analysis shows a shift in ceramide species under neuroinflammatory conditions, favoring pro-apoptotic ceramide. Conclusions Together, these findings suggest that detected increased levels of pro-apoptotic ceramide might be a common denominator of neuroinflammation in different neurodegenerative diseases.

Abstract Background Couple relationship distress is common and associated with poor physical, psychological, and relational outcomes for both partners. Emotionally Focused Therapy for couples (EFT) is a short-term structured approach based on attachment theory that integrates a humanistic, experiential approach to restructuring emotional experience and a systemic structural approach to restructuring interactions. This model has been shown to be an effective treatment for couple distress. The supporting research, however, has only been conducted with English-speaking couples. Despite Spanish being the second-most spoken language and meaningful cultural differences between English- and Spanish-speaking countries, the efficacy of EFT has not been examined in this cultural context. This study will examine the efficacy of EFT in this particular context and advance the understanding of potential mechanisms of change. Methods We will use a multicenter randomized wait-list controlled design to examine the efficacy of EFT in a Spanish-speaking sample of moderately distressed couples. One hundred forty individuals in 70 couples in Argentina, Costa Rica, Guatemala, Mexico, and Spain will be randomly assigned to receive 19–21 sessions of EFT or be placed on a waitlist. Outcomes on a range of relational and individual mental health variables will be assessed prior to random assignment, throughout treatment, and at the conclusion of treatment. Primary outcomes will include dyadic adjustment, couple satisfaction, and attachment. Secondary variables, such as loneliness, parenting, affective communication, and sexual satisfaction, will be included as potential mediators of the treatment effect. Couples in the treatment group will also be assessed at 3-, 6-, 12-, 18-, and 24-month follow-ups. Process variables such as the therapeutic alliance will also be assessed routinely in couples assigned to the treatment group. Couples in the waitlist will receive a psycho-educational program based on EFT after completing the study. Discussion This study will be the first RCT of Emotionally Focused Therapy in a Spanish-speaking context. The results of the study will inform researchers interested in whether treatments developed and tested in the USA and Canada can be effective in differing cultural contexts. It may also point researchers and clinicians to areas where cultural adaptation is needed to improve efficacy. Trial registration ClinicalTrials.gov NCT04277325. Registered on February 20, 2020

Abstract Background Patients with cancer have been shown to have a higher risk of clinical severity and mortality compared to non-cancer patients with COVID-19. Patients with hematologic malignancies typically are known to have higher levels of immunosuppression and may develop more severe respiratory viral infections than patients with solid tumors. Data on COVID-19 in patients with hematologic malignancies are limited. Here we characterize disease severity and mortality and evaluate potential prognostic factors for mortality. Methods In this population-based registry study, we collected de-identified data on clinical characteristics, treatment and outcomes in adult patients with hematologic malignancies and confirmed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection within the Madrid region of Spain. Our case series included all patients admitted to 22 regional health service hospitals and 5 private healthcare centers between February 28 and May 25, 2020. The primary study outcome was all-cause mortality. We assessed the association between mortality and potential prognostic factors using Cox regression analyses adjusted for age, sex, comorbidities, hematologic malignancy and recent active cancer therapy. Results Of 833 patients reported, 697 were included in the analyses. Median age was 72 years (IQR 60–79), 413 (60%) patients were male and 479 (69%) and 218 (31%) had lymphoid and myeloid malignancies, respectively. Clinical severity of COVID-19 was severe/critical in 429 (62%) patients. At data cutoff, 230 (33%) patients had died. Age ≥ 60 years (hazard ratios 3.17–10.1 vs 2 comorbidities (1.41 vs ≤ 2), acute myeloid leukemia (2.22 vs non-Hodgkin lymphoma) and active antineoplastic treatment with monoclonal antibodies (2·02) were associated with increased mortality; conventional chemotherapy showed borderline significance (1.50 vs no active therapy). Conversely, Ph-negative myeloproliferative neoplasms (0.33) and active treatment with hypomethylating agents (0.47) were associated with lower mortality. Overall, 574 (82%) patients received antiviral therapy. Mortality with severe/critical COVID-19 was higher with no therapy vs any antiviral combination therapy (2.20). Conclusions In this series of patients with hematologic malignancies and COVID-19, mortality was associated with higher age, more comorbidities, type of hematological malignancy and type of antineoplastic therapy. Further studies and long-term follow-up are required to validate these criteria for risk stratification.

Abstract Background Xenobiotics are primarily metabolized by hepatocytes in the liver, and primary human hepatocytes are the gold standard model for the assessment of drug efficacy, safety, and toxicity in the early phases of drug development. Recent advances in single-cell genomics demonstrate liver zonation and ploidy as main drivers of cellular heterogeneity. However, little is known about the impact of hepatocyte specialization on liver function upon metabolic challenge, including hepatic metabolism, detoxification, and protein synthesis. Results Here, we investigate the metabolic capacity of individual human hepatocytes in vitro. We assess how chronic accumulation of lipids enhances cellular heterogeneity and impairs the metabolisms of drugs. Using a phenotyping five-probe cocktail, we identify four functional subgroups of hepatocytes responding differently to drug challenge and fatty acid accumulation. These four subgroups display differential gene expression profiles upon cocktail treatment and xenobiotic metabolism-related specialization. Notably, intracellular fat accumulation leads to increased transcriptional variability and diminishes the drug-related metabolic capacity of hepatocytes. Conclusions Our results demonstrate that, upon a metabolic challenge such as exposure to drugs or intracellular fat accumulation, hepatocyte subgroups display different and heterogeneous transcriptional responses.

Abstract Background The Ibizan Hound is a canine breed native to the Mediterranean region, where leishmaniasis is an endemic zoonosis. Several studies indicate a low prevalence of this disease in Ibizan Hound dogs, whereas other canine breeds present a high prevalence. However, the underlying molecular mechanisms still remain unknown. The aim of this work is to analyse the relationship between serum levels of cytokines and the genomic profiles in two canine breeds, Ibizan Hound (resistant canine breed model) and Boxer (susceptible canine breed model). Methods In this study, we analyse the haplotypes of genes encoding cytokines related to immune response of Leishmania infantum infection in twenty-four Boxers and twenty-eight Ibizan Hounds apparently healthy using CanineHD DNA Analysis BeadChip including 165,480 mapped positions. The haplo.glm extension of haplo.score was used to perform a General Linear Model (GLM) regression to estimate the magnitude of individual haplotype effects within each cytokine. Results Mean levels of interferon gamma (IFN-γ), interleukin 2 (IL-2) and IL-18 in Boxer dogs were 0.19 ± 0.05 ng/ml, 46.70 ± 4.54 ng/ml, and 36.37 ± 30.59 pg/ml, whereas Ibizan Hound dogs present 0.49 ± 0.05 ng/ml, 64.55 ± 4.54 ng/ml, and 492.10 ± 31.18 pg/ml, respectively. The GLM regression shows fifteen haplotypes with statistically significant effect on the cytokine serum levels (P

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already caused 6 million deaths worldwide. While asymptomatic individuals are responsible of many potential transmissions, the difficulty to identify and isolate them at the high peak of infection constitutes still a real challenge. Moreover, SARS-CoV-2 provokes severe vascular damage and thromboembolic events in critical COVID-19 patients, deriving in many related deaths and long-hauler symptoms. Understanding how these processes are triggered as well as the potential long-term sequelae, even in asymptomatic individuals, becomes essential. Methods We have evaluated, by application of a proteomics-based quantitative approach, the effect of serum from COVID-19 asymptomatic individuals over circulating angiogenic cells (CACs). Healthy CACs were incubated ex-vivo with the serum of either COVID-19 negative (PCR −/IgG −, n:8) or COVID-19 positive asymptomatic donors, at different infective stages: PCR +/IgG − (n:8) and PCR −/IgG + (n:8). Also, a label free quantitative approach was applied to identify and quantify protein differences between these serums. Finally, machine learning algorithms were applied to validate the differential protein patterns in CACs. Results Our results confirmed that SARS-CoV-2 promotes changes at the protein level in the serum of infected asymptomatic individuals, mainly correlated with altered coagulation and inflammatory processes (Fibrinogen, Von Willebrand Factor, Thrombospondin-1). At the cellular level, proteins like ICAM-1, TLR2 or Ezrin/Radixin were only up-regulated in CACs treated with the serum of asymptomatic patients at the highest peak of infection (PCR + /IgG −), but not with the serum of PCR −/IgG + individuals. Several proteins stood out as significantly discriminating markers in CACs in response to PCR or IgG + serums. Many of these proteins particiArticle title: Kindly check and confirm the edit made in the article title.pate in the initial endothelial response against the virus. Conclusions The ex vivo incubation of CACs with the serum of asymptomatic COVID-19 donors at different stages of infection promoted protein changes representative of the endothelial dysfunction and inflammatory response after viral infection, together with activation of the coagulation process. The current approach constitutes an optimal model to study the response of vascular cells to SARS-CoV-2 infection, and an alternative platform to test potential inhibitors targeting either the virus entry pathway or the immune responses following SARS-CoV-2 infection.

Abstract Background Exposure to bisphenols may affect fetal growth and development. The trimester-specific effects of bisphenols on repeated measures of fetal growth remain unknown. Our objective was to assess the associations of maternal bisphenol urine concentrations with fetal growth measures and birth outcomes and identify potential critical exposure periods. Methods In a population-based prospective cohort study among 1379 pregnant women, we measured maternal bisphenol A, S and F urine concentrations in the first, second and third trimester. Fetal head circumference, length and weight were measured in the second and third trimester by ultrasound and at birth. Results An interquartile range increase in maternal pregnancy-averaged bisphenol S concentrations was associated with larger fetal head circumference (difference 0.18 (95% confidence interval (CI) 0.01 to 0.34) standard deviation scores (SDS), p-value