N-terminal palmitoylation within the appropriate amino acid environment conveys on NOS2 the ability to progress along the intracellular sorting pathways.

We have analysed the mechanism by which palmitoylation permits the progression of nitric oxide synthase 2 (NOS2) along the ER-Golgi-TGN pathway. Introduction of an additional myristoylation site at the N-terminus of NOS2 resulted in a chimera that displayed an enhanced association with the particulate fraction and with the plasma membrane but did not display increased enzymatic activity. In the absence of palmitoylation, introduction of a surrogate myristoylation site resulted in a mutant NOS2 with only 25% activity compared with the wild-type enzyme. Hence, the novel surrogate myristoyl moiety not only failed to increase NOS2 activity when introduced in a wild-type sequence environment, but was also unable to rescue the inactive phenotype of the Cys3Ser mutant. Introduction of an additional palmitoylatable Cys at position 2 of the wild-type sequence resulted in a chimera that associated to a larger degree with membranes and displayed decreased activity. Our data indicate that palmitoylation of inducible NOS at position 3 exquisitely determines its transit along the secretory pathway following a route that cannot be mimicked by a surrogate myristoylation or by a palmitate at position 2. In addition, the exit of NOS2 from the TGN and the accumulation in the cellular plasma membrane per se did not correlate with increased .NO synthesis.S

Distance-dependent cellular palmitoylation of de-novo-designed sequences and their translocation to plasma membrane subdomains.

Using recursive PCR, we created an artificial protein sequence that consists of a consensus myristoylation motif (MGCTLS) followed by the triplet AGS repeated nine times and fused to the GFP reporter. This linker-GFP sequence was utilized as a base to produce multiple mutants that were used to transfect COS-7 cells. Constructs where a 'palmitoylable' cysteine residue was progressively moved apart from the myristoylation site to positions 3, 9, 15 and 21 of the protein sequence were made, and these mutants were used to investigate the effect of protein myristoylation on subsequent palmitoylation, subcellular localization, membrane association and caveolin-1 colocalization. In all cases, dual acylation of the GFP chimeras correlated with translocation to Triton X-100-insoluble cholesterol/sphingomyelin-enriched subdomains. Whereas a strong Golgi labeling was observed in all the myristoylated chimeras, association with the plasma membrane was only observed in the dually acylated constructs. Taking into account the conflicting data regarding the existence and specificity of cellular palmitoyl-transferases, our results provide evidence that de-novo-designed sequences can be efficiently S-acylated with palmitic acid in vivo, strongly supporting the hypothesis that non-enzymatic protein palmitoylation can occur within mammalian cells. Additionally, this palmitoylation results in the translocation of the recombinant construct to low-fluidity domains in a myristate-palmitate distance-dependent manner.S

The proteome of large or small extracellular vesicles in pig seminal plasma differs, defining sources and biological functions

Seminal plasma contains many morphologically heterogeneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis and accessory sex glands, and involved in male and female reproductive processes. This study aimed to in-depth define sEV-subsets isolated by ultrafiltration and size-exclusion chromatography (SEC), decode their proteomic profiles using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to quantify identified proteins using Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS). The sEV-subsets defined Large (L-EVs) or Small (S-EVs) by their protein concentration, morphology, size distribution and EV-specific protein markers and purity. LC-MS/MS identified a total of 988 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs and non-EVs-enriched samples (18-20 SEC-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV-subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs vs non-EVs-enriched samples, respectively. The gene ontology (GO) enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating of subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV-subsets,indicating different sources and biological functions for the sEVs

Discrepancies Between Nitroglycerin and NO-Releasing Drugs on Mitochondrial Oxygen Consumption, Vasoactivity, and the Release of NO

It has been generally acknowledged that the actions of glyceryl trinitrate (GTN) are a result of its bioconversion into NO. However, recent observations have thrown this idea into doubt, with many studies demonstrating that NO is present only when there are high concentrations of GTN. We have explored this discrepancy by developing a new approach that uses confocal microscopy to directly detect NO. Intracellular levels of NO in the rat aortic vascular wall have been compared with those present after incubation with 3 different NO donors (DETA-NO, 3 morpholinosydnonimine, and S-nitroso-N-acetylpenicillamine), endothelial activation with acetylcholine, or administration of GTN. We have also evaluated the relaxant effects of these treatments on isolated rings of aorta following activation of the enzyme soluble guanylyl cyclase and their inhibitory action on mitochondrial respiration, which is an index of the interaction of NO with the enzyme of the electron transport chain cytochrome C oxidase. In the case of the various NO donors and acetylcholine, we detected a concentration-dependent relationship in the intensity of vascular relaxation and degree of NO fluorescence and an increase in the Michaelis constant (Km) for O2. GTN did not produce similar effects, and although clinically relevant concentrations of this compound caused clear, concentration-related relaxations, there was neither any increase in NO-related fluorescence nor an augmented Km for O2. The nature of these differences suggests that these concentrations of GTN do not release free NO but probably a different species that, although it interacts with soluble guanylyl cyclase in vascular smooth muscle, does not inhibit O2 consumption by vascular mitochondria.Victor Gonzalez, Victor Manuel, [email protected] ; Esplugues Mota, Juan Vicente, [email protected] ; d'Ocon Navaza, Maria Pilar, [email protected]

Cell Fusion Reprogramming Leads to a Specific Hepatic Expression Pattern during Mouse Bone Marrow Derived Hepatocyte Formation In Vivo

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation

The Dioxin receptor modulates Caveolin-1 mobilization during directional migration: role of cholesterol

Background: Adhesion and migration are relevant physiological functions that must be regulated by the cell under both normal and pathological conditions. The dioxin receptor (AhR) has emerged as a transcription factor regulating both processes in mesenchymal, epithelial and endothelial cells. Indirect results suggest that AhR could cooperate not only with additional transcription factors but also with membrane-associated proteins to drive such processes. Results: In this study, we have used immortalized and primary dermal fibroblasts from wild type (AhR+/+) and AhR-null (AhR-/-) mice to show that AhR modulates membrane distribution and mobilization of caveolin-1 (Cav-1) during directional cell migration. AhR co-immunoprecipitated with Cav-1 and a fraction of both proteins co-localized to detergent-resistant membrane microdomains (DRM). Consistent with a role of AhR in the process, AhR-/-cells had a significant reduction in Cav-1 in DRMs. Moreover, high cell density reduced AhR nuclear levels and moved Cav-1 from DRMs to the soluble membrane in AhR+/+ but not in AhR-/-cells. Tyrosine-14 phosphorylation had a complex role in the mechanism since its upregulation reduced Cav-1 in DRMs in both AhR+/+ and AhR-/-cells, despite the lower basal levels of Y-14-Cav-1 in the null cells. Fluorescence recovery after photobleaching revealed that AhR knock-down blocked Cav-1 transport to the plasma membrane, a deficit possibly influencing its depleted levels in DRMs. Membrane distribution of Cav-1 in AhR-null fibroblasts correlated with higher levels of cholesterol and with disrupted membrane microdomains, whereas addition of exogenous cholesterol changed the Cav-1 distribution of AhR+/+ cells to the null phenotype. Consistently, higher cholesterol levels enhanced caveolae-dependent endocytosis in AhR-null cells. Conclusions: These results suggest that AhR modulates Cav-1 distribution in migrating cells through the control of cholesterol-enriched membrane microdomains. Our study also supports the likely possibility of membrane-related, transcription factor independent, functions of AhR.This work was supported by grants to P. M. F-S. from the Spanish Ministry of Science and Innovation (SAF2008-00462 and BFU2011-22678) and from the Junta de Extremadura (GR10008). Research at P. M. F-S laboratory is also funded by the Red Tematica de Investigacion Cooperativa en Cancer (RTICC), Fondo de Investigaciones Sanitarias (FIS), Carlos III Institute, Spanish Ministry of Health (RD12/0036/0032). J.R.B. was a F.P.U. program fellow from the Spanish Ministry of Education and Sciences. All Spanish funding is co-sponsored by the European Union FEDER program. The support and help of the Servicio de Tecnicas Aplicadas a las Biociencia (STAB) of the Universidad de Extremadura is greatly acknowledged. We are very grateful to Dr. Lisardo Bosca (Instituto de Investigaciones Biomedicas, Madrid, Spain) for providing the Cav-1-GFP and the Cav-1Y14F-GFP expression vectors and to Dr. Miguel A. Alonso Lebrero (Centro de Biologia Molecular Severo Ochoa, Madrid, Spain) for assistance with the sucrose density gradient method. The technical support of Eva Barrasa is greatly appreciated.S

Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor

Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.Trabajo financiado por: Ministerio de Economía y Competitividad. Proyectos BFU2011-22678, SAF2014-51813-R (I+D+i) para Pedro María Fernández Salguero Ministerio de Economía y Competitividad, Instituto Carlos III, Red Temática de Investigación Cooperativa en Cáncer Junta de Extremadura. Ayudas GR10008, GR15008 CICE-FEDER-P09-CTS-4980, CICE-FEDERP12-CTS-2256, Plan Nacional de I+D+I 2008–2011 y 2013–2016 (FIS-FEDER-PI11/01489, FIS-FEDERPI14/02152), PCIN-2014-115-ERA-NET NEURON II, para José Luis García Pérez European Research Council ERC-Consolidator ERC-STG-2012-233764 International Early Career Scientist Beca de la Howard Hughes Medical Institute IECS-55007420 Programa Unión Europea de Fondos FEDERpeerReviewe

May Measurement Month 2018: a pragmatic global screening campaign to raise awareness of blood pressure by the International Society of Hypertension

Aims Raised blood pressure (BP) is the biggest contributor to mortality and disease burden worldwide and fewer than half of those with hypertension are aware of it. May Measurement Month (MMM) is a global campaign set up in 2017, to raise awareness of high BP and as a pragmatic solution to a lack of formal screening worldwide. The 2018 campaign was expanded, aiming to include more participants and countries. Methods and results Eighty-nine countries participated in MMM 2018. Volunteers (≥18 years) were recruited through opportunistic sampling at a variety of screening sites. Each participant had three BP measurements and completed a questionnaire on demographic, lifestyle, and environmental factors. Hypertension was defined as a systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg, or taking antihypertensive medication. In total, 74.9% of screenees provided three BP readings. Multiple imputation using chained equations was used to impute missing readings. 1 504 963 individuals (mean age 45.3 years; 52.4% female) were screened. After multiple imputation, 502 079 (33.4%) individuals had hypertension, of whom 59.5% were aware of their diagnosis and 55.3% were taking antihypertensive medication. Of those on medication, 60.0% were controlled and of all hypertensives, 33.2% were controlled. We detected 224 285 individuals with untreated hypertension and 111 214 individuals with inadequately treated (systolic BP ≥ 140 mmHg or diastolic BP ≥ 90 mmHg) hypertension. Conclusion May Measurement Month expanded significantly compared with 2017, including more participants in more countries. The campaign identified over 335 000 adults with untreated or inadequately treated hypertension. In the absence of systematic screening programmes, MMM was effective at raising awareness at least among these individuals at risk

Healthcare Access and Quality Index based on mortality from causes amenable to personal health care in 195 countries and territories, 1990-2015 : a novel analysis from the Global Burden of Disease Study 2015

Background National levels of personal health-care access and quality can be approximated by measuring mortality rates from causes that should not be fatal in the presence of effective medical care (ie, amenable mortality). Previous analyses of mortality amenable to health care only focused on high-income countries and faced several methodological challenges. In the present analysis, we use the highly standardised cause of death and risk factor estimates generated through the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) to improve and expand the quantification of personal health-care access and quality for 195 countries and territories from 1990 to 2015. Methods We mapped the most widely used list of causes amenable to personal health care developed by Nolte and McKee to 32 GBD causes. We accounted for variations in cause of death certification and misclassifications through the extensive data standardisation processes and redistribution algorithms developed for GBD. To isolate the effects of personal health-care access and quality, we risk-standardised cause-specific mortality rates for each geography-year by removing the joint effects of local environmental and behavioural risks, and adding back the global levels of risk exposure as estimated for GBD 2015. We employed principal component analysis to create a single, interpretable summary measure-the Healthcare Quality and Access (HAQ) Index-on a scale of 0 to 100. The HAQ Index showed strong convergence validity as compared with other health-system indicators, including health expenditure per capita (r= 0.88), an index of 11 universal health coverage interventions (r= 0.83), and human resources for health per 1000 (r= 0.77). We used free disposal hull analysis with bootstrapping to produce a frontier based on the relationship between the HAQ Index and the Socio-demographic Index (SDI), a measure of overall development consisting of income per capita, average years of education, and total fertility rates. This frontier allowed us to better quantify the maximum levels of personal health-care access and quality achieved across the development spectrum, and pinpoint geographies where gaps between observed and potential levels have narrowed or widened over time. Findings Between 1990 and 2015, nearly all countries and territories saw their HAQ Index values improve; nonetheless, the difference between the highest and lowest observed HAQ Index was larger in 2015 than in 1990, ranging from 28.6 to 94.6. Of 195 geographies, 167 had statistically significant increases in HAQ Index levels since 1990, with South Korea, Turkey, Peru, China, and the Maldives recording among the largest gains by 2015. Performance on the HAQ Index and individual causes showed distinct patterns by region and level of development, yet substantial heterogeneities emerged for several causes, including cancers in highest-SDI countries; chronic kidney disease, diabetes, diarrhoeal diseases, and lower respiratory infections among middle-SDI countries; and measles and tetanus among lowest-SDI countries. While the global HAQ Index average rose from 40.7 (95% uncertainty interval, 39.0-42.8) in 1990 to 53.7 (52.2-55.4) in 2015, far less progress occurred in narrowing the gap between observed HAQ Index values and maximum levels achieved; at the global level, the difference between the observed and frontier HAQ Index only decreased from 21.2 in 1990 to 20.1 in 2015. If every country and territory had achieved the highest observed HAQ Index by their corresponding level of SDI, the global average would have been 73.8 in 2015. Several countries, particularly in eastern and western sub-Saharan Africa, reached HAQ Index values similar to or beyond their development levels, whereas others, namely in southern sub-Saharan Africa, the Middle East, and south Asia, lagged behind what geographies of similar development attained between 1990 and 2015. Interpretation This novel extension of the GBD Study shows the untapped potential for personal health-care access and quality improvement across the development spectrum. Amid substantive advances in personal health care at the national level, heterogeneous patterns for individual causes in given countries or territories suggest that few places have consistently achieved optimal health-care access and quality across health-system functions and therapeutic areas. This is especially evident in middle-SDI countries, many of which have recently undergone or are currently experiencing epidemiological transitions. The HAQ Index, if paired with other measures of health-systemcharacteristics such as intervention coverage, could provide a robust avenue for tracking progress on universal health coverage and identifying local priorities for strengthening personal health-care quality and access throughout the world. Copyright (C) The Author(s). Published by Elsevier Ltd.Peer reviewe