Upregulation of Orai Channels Contributes to Aging-Related Vascular Alterations in Rat Coronary Arteries.

Vascular territories display heterogeneous sensitivity to the impacts of aging. The relevance of the STIM/Orai system to vascular function depends on the vascular bed. We aimed to evaluate the contribution of the STIM/Orai system to aging-related vascular dysfunction in rat coronary circulation. Vascular function was evaluated according to myography in coronary arteries from young (three-month-old) and older (twenty-month-old) rats. The effects of aging and STIM/Orai inhibition on the contraction and relaxation of the coronary arteries and on the protein expression of STIM-1, Orai1, and Orai3 in these vessels were determined. Aging-related hypercontractility to serotonin and endothelin-1 in arteries from male rats was reversed by STIM/Orai inhibition with YM-58483 or by specifically blocking the Orai1 channel with Synta66. The inhibitory effects of Synta66 on coronary vasoconstriction were also observed in older female rats. YM-58483 relaxed serotonin- but not KCl-contracted arteries from males. STIM/Orai inhibition improved defective endothelial vasodilations in aged arteries, even in the presence of NO synthase and cyclooxygenase inhibitors, but not in KCl-contracted segments. YM-58483 significantly enhanced relaxations to calcium-activated potassium channel stimulation in aged vessels. Increased protein expression of Orai1 and Orai3 was detected in arterial homogenates and sections from older rats. Upregulation of the Orai channel contributes to aging-related coronary dysfunction, revealing a potential target in reducing CVD risk.

Health-related quality of life and radiological and functional lung changes of patients with COVID-19 Pneumonia 3 and 10 months after discharge.

BACKGROUND: Few studies have evaluated the long-term impact on health-related quality of life (HRQoL) in patients who have been hospitalized for COVID-19 pneumonia. Specific follow-up should be carried out to detect and treat possible pulmonary abnormalities, and the worsening of HRQoL should be estimated to target necessary resources for care of these patients after acute phase. The objective was to know the impact on HRQoL of patients who have been admitted for COVID-19 pneumonia, and to evaluate the clinical-radiological and functional changes of patients who have overcome COVID-19 pneumonia at 3 and 10 months of follow-up. METHODS: Prospective observational study of patients who required hospitalization for COVID-19 pneumonia between April and December 2020. All patients filled out the EuroQol five-dimension (EQ-5D) questionnaire with the EuroQol Visual Analogue Scale (E-VAS) for self-assessment of health status. Respiratory function tests and chest X-ray were carried out at 3 and 10 months of follow-up. RESULTS: 61 patients were included in the study. The need for ventilatory support was associated with anxiety/depression on the EQ-5D scale, as well as patients admitted to the intensive care unit (ICU). The mean EQ-5D and E-VAS index scores decreased with hospitalization time, the number of days spent in intermediate respiratory care unit (IRCU) and the level of dyspnoea at the beginning of the hospitalization period. Pulmonary sequelae were observed in 25 patients (41%) at 3 months and 17 (27.9%) at 10 months. Patients improve their forced vital capacity (FVC) by 196 ml (p = 0.001) at 10 months as well as 9% in diffusing capacity of lung for carbon monoxide (DLCO) (p = 0.001) at 10 months. DLCO was found to be correlated to lymphopenia and time spent in IRCU. Low FVC values were detected 10 months after discharge for subjects exhibiting high levels of dyspnoea at 3 months after discharge. CONCLUSIONS: Hospitalization for COVID-19 pneumonia affects the HRQoL of patients, with greater anxiety/depression in those who were more serious affected and are younger. A significant percentage of patients present fibrotic abnormalities and lung function impairment at the first and second follow-up after discharge.

PLATERO: A calibration protocol for plate reader green fluorescence measurements.

One of the most common sources of information in Synthetic Biology is the data coming from plate reader fluorescence measurements. These experiments provide a measure of the light emitted by a certain fluorescent molecule, such as the Green Fluorescent Protein (GFP). However, these measurements are generally expressed in arbitrary units and are affected by the measurement device gain. This limits the range of measurements in a single experiment and hampers the comparison of results among experiments. In this work, we describe PLATERO, a calibration protocol to express fluorescence measures in concentration units of a reference fluorophore. The protocol removes the gain effect of the measurement device on the acquired data. In addition, the fluorescence intensity values are transformed into units of concentration using a Fluorescein calibration model. Both steps are expressed in a single mathematical expression that returns normalized, gain-independent, and comparable data, even if the acquisition was done at different device gain levels. Most important, the PLATERO embeds a Linearity and Bias Analysis that provides an assessment of the uncertainty of the model estimations, and a Reproducibility and Repeatability analysis that evaluates the sources of variability originating from the measurements and the equipment. All the functions used to build the model, exploit it with new data, and perform the uncertainty and variability assessment are available in an open access repository.

Functional Role of STIM-1 and Orai1 in Human Microvascular Aging.

The impact of aging on vascular function is heterogeneous depending on the vascular territories. Calcium regulation plays a key role in vascular function and has been implicated in aging-related hypercontractility of corpus cavernosum. We aimed to evaluate stromal interaction molecule (STIM)/Orai system involvement in aging-related vascular alterations in the human macro and microvasculature. Aortae specimens and mesenteric arteries (MA), obtained from 45 organ donors, were functionally evaluated in organ chambers and wire myographs. Subjects were divided into groups either younger or older than 65-years old. The expressions of STIM-1, Orai1, and Orai3 were determined by immunofluorescence in the aorta and MA, and by Western blot in the aorta homogenates. The inhibition of STIM/Orai with YM-58483 (20 µM) reversed adrenergic hypercontractility in MA from older subjects but did not modify aging-related hypercontractility in the aortic strips. Aging was related to an increased expression of Orai1 in human aorta, while Orai1 and STIM-1 were upregulated in MA. STIM-1 and Orai1 protein expressions were inversely correlated to endothelial function in MA. Circulating levels of Orai1 were correlated with the inflammatory factor TNF-α and with the endothelial dysfunction marker asymmetric dimethylarginine. Aging is associated with an increased expression of the STIM/Orai system in human vessels with functional relevance only in the microvascular territory, suggesting its role in aging-related microvascular dysfunction.

Machine-learning-derived predictive score for early estimation of COVID-19 mortality risk in hospitalized patients.

The clinical course of COVID-19 is highly variable. It is therefore essential to predict as early and accurately as possible the severity level of the disease in a COVID-19 patient who is admitted to the hospital. This means identifying the contributing factors of mortality and developing an easy-to-use score that could enable a fast assessment of the mortality risk using only information recorded at the hospitalization. A large database of adult patients with a confirmed diagnosis of COVID-19 (n = 15,628; with 2,846 deceased) admitted to Spanish hospitals between December 2019 and July 2020 was analyzed. By means of multiple machine learning algorithms, we developed models that could accurately predict their mortality. We used the information about classifiers' performance metrics and about importance and coherence among the predictors to define a mortality score that can be easily calculated using a minimal number of mortality predictors and yielded accurate estimates of the patient severity status. The optimal predictive model encompassed five predictors (age, oxygen saturation, platelets, lactate dehydrogenase, and creatinine) and yielded a satisfactory classification of survived and deceased patients (area under the curve: 0.8454 with validation set). These five predictors were additionally used to define a mortality score for COVID-19 patients at their hospitalization. This score is not only easy to calculate but also to interpret since it ranges from zero to eight, along with a linear increase in the mortality risk from 0% to 80%. A simple risk score based on five commonly available clinical variables of adult COVID-19 patients admitted to hospital is able to accurately discriminate their mortality probability, and its interpretation is straightforward and useful.

New bladder cancer non-invasive surveillance method based on voltammetric electronic tongue measurement of urine.

Bladder cancer (BC) is the sixth leading cause of death by cancer. Depending on the invasiveness of tumors, patients with BC will undergo surgery and surveillance lifelong, owing the high rate of recurrence and progression. In this context, the development of strategies to support non-invasive BC diagnosis is focusing attention. Voltammetric electronic tongue (VET) has been demonstrated to be of use in the analysis of biofluids. Here, we present the implementation of a VET to study 207 urines to discriminate BC and non-BC for diagnosis and surveillance to detect recurrences. Special attention has been paid to the experimental setup to improve reproducibility in the measurements. PLSDA analysis together with variable selection provided a model with high sensitivity, specificity, and area under the ROC curve AUC (0.844, 0.882, and 0.917, respectively). These results pave the way for the development of non-invasive low-cost and easy-to-use strategies to support BC diagnosis and follow-up.

Diagnosis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome With Partial Least Squares Discriminant Analysis: Relevance of Blood Extracellular Vesicles.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), a chronic disease characterized by long-lasting persistent debilitating widespread fatigue and post-exertional malaise, remains diagnosed by clinical criteria. Our group and others have identified differentially expressed miRNA profiles in the blood of patients. However, their diagnostic power individually or in combinations seems limited. A Partial Least Squares-Discriminant Analysis (PLS-DA) model initially based on 817 variables: two demographic, 34 blood analytic, 136 PBMC miRNAs, 639 Extracellular Vesicle (EV) miRNAs, and six EV features, selected an optimal number of five components, and a subset of 32 regressors showing statistically significant discriminant power. The presence of four EV-features (size and z-values of EVs prepared with or without proteinase K treatment) among the 32 regressors, suggested that blood vesicles carry relevant disease information. To further explore the features of ME/CFS EVs, we subjected them to Raman micro-spectroscopic analysis, identifying carotenoid peaks as ME/CFS fingerprints, possibly due to erythrocyte deficiencies. Although PLS-DA analysis showed limited capacity of Raman fingerprints for diagnosis (AUC = 0.7067), Raman data served to refine the number of PBMC miRNAs from our previous model still ensuring a perfect classification of subjects (AUC=1). Further investigations to evaluate model performance in extended cohorts of patients, to identify the precise ME/CFS EV components detected by Raman and to reveal their functional significance in the disease are warranted.

MultiBaC: an R package to remove batch effects in multi-omic experiments.

MOTIVATION: Batch effects in omics datasets are usually a source of technical noise that masks the biological signal and hampers data analysis. Batch effect removal has been widely addressed for individual omics technologies. However, multi-omic datasets may combine data obtained in different batches where omics type and batch are often confounded. Moreover, systematic biases may be introduced without notice during data acquisition, which creates a hidden batch effect. Current methods fail to address batch effect correction in these cases. RESULTS: In this article, we introduce the MultiBaC R package, a tool for batch effect removal in multi-omics and hidden batch effect scenarios. The package includes a diversity of graphical outputs for model validation and assessment of the batch effect correction. AVAILABILITY AND IMPLEMENTATION: MultiBaC package is available on Bioconductor (https://www.bioconductor.org/packages/release/bioc/html/MultiBaC.html) and GitHub (https://github.com/ConesaLab/MultiBaC.git). The data underlying this article are available in Gene Expression Omnibus repository (accession numbers GSE11521, GSE1002, GSE56622 and GSE43747). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Early manifestation of aging-related vascular dysfunction in human penile vasculature-A potential explanation for the role of erectile dysfunction as a harbinger of systemic vascular disease.

Advanced age is related to functional alterations of human vasculature, but erectile dysfunction precedes systemic manifestations of vascular disease. The current study aimed to simultaneously evaluate the influence of aging on vascular function (relaxation and contraction responses) in systemic human vascular territories: aorta (HA) and resistance mesenteric arteries (HMA) and human corpus cavernosum (HCC) and penile resistance arteries (HPRA). Associations of oxidative stress and inflammation circulating biomarkers with age and functional responses were also determined. Vascular specimens were obtained from 76 organ donors (age range 18-87). Four age-groups were established: < 40, 40-55, 56-65 and > 65 years old. Increasing age was associated with a decline in endothelium-dependent relaxation induced by BK in HMA (r = -0.597, p = 0.0001), or by ACh in HCC (r = -0.505, p = 0.0022), and HPRA (r = -0.601, p = 0.0012). Significant impairment was detected at > 65 years old in HMA but earlier in penile vasculature (> 55 years old). Age-related reduction to H2O2-vasodilatory response started before in HCC (56-65 years old) than in HA (> 65 years old). In contrast to relaxation responses, aging-related hypercontractility to adrenergic stimulation was homogeneous: contractions significantly increased in subjects > 55 years old in all tested vessels. Although not significantly age related, circulating levels of ADMA (r = -0.681, p = 0.0052) and TNF-α (r = -0.537, p = 0.0385) were negatively correlated with endothelial vasodilation in HMA but not in HCC or HPRA. Penile vasculature exhibits an early impairment of endothelium-dependent and H2O2-induced vasodilations when compared to mesenteric microcirculation and aorta. Therefore, functional susceptibility of penile vasculature to the aging process may account for anticipation of erectile dysfunction to systemic manifestations of vascular disease.

Comprehensive functional core microbiome comparison in genetically obese and lean hosts under the same environment.

Our study provides an exhaustive comparison of the microbiome core functionalities (captured by 3,936 microbial gene abundances) between hosts with divergent genotypes for intramuscular lipid deposition. After 10 generations of divergent selection for intramuscular fat in rabbits and 4.14 phenotypic standard deviations (SD) of selection response, we applied a combination of compositional and multivariate statistical techniques to identify 122 cecum microbial genes with differential abundances between the lines (ranging from -0.75 to +0.73 SD). This work elucidates that microbial biosynthesis lipopolysaccharides, peptidoglycans, lipoproteins, mucin components, and NADH reductases, amongst others, are influenced by the host genetic determination for lipid accretion in muscle. We also differentiated between host-genetically influenced microbial mechanisms regulating lipid deposition in body or intramuscular reservoirs, with only 28 out of 122 MGs commonly contributing to both. Importantly, the results of this study are of relevant interest for the efficient development of strategies fighting obesity.

Ageing-induced hypercontractility is related to functional enhancement of STIM/Orai and upregulation of Orai 3 in rat and human penile tissue.

The role of STIM/Orai calcium entry system on vascular ageing has not been elucidated. We aimed to evaluate the influence of ageing on STIM/Orai signalling and its role on ageing-induced alterations of contractile function in rat corpus cavernosum (RCC) and human penile resistance arteries (HPRA) and corpus cavernosum (HCC). RCC was obtained from 3 months-old and 20 months-old animals. HPRA and HCC were obtained from organ donors of varied ages without history of erectile dysfunction. Aging was associated with enhanced norepinephrine (NE)- and thromboxane analogue (U46619)-induced contractions in RCC which were significantly inhibited by the STIM/Orai inhibitor, YM-58483 (20 µM). Other STIM/Orai inhibitor, 2-aminoethyldiphenylborate also reduced NE-induced contractions in RCC from aged rats. YM-58483 significantly reduced neurogenic contractions and potentiated neurogenic relaxations in RCC from aged rats. In HCC and HPRA, NE-induced contractions were significantly enhanced in older subjects (>65 years-old) but YM-58483 completely reversed ageing-related hypercontractility. Ageing did not modify STIM-1 and Orai1 protein expressions but Orai3 was significantly overexpressed in cavernosal tissue from old rats and older subjects. Contribution of STIM/Orai to cavernosal contraction increases with ageing together with increased expression of Orai3. Orai inhibition could be a potential therapeutic strategy to reduce ageing-related impact on vascular/erectile function.

MultiBaC: A strategy to remove batch effects between different omic data types.

Diversity of omic technologies has expanded in the last years together with the number of omic data integration strategies. However, multiomic data generation is costly, and many research groups cannot afford research projects where many different omic techniques are generated, at least at the same time. As most researchers share their data in public repositories, different omic datasets of the same biological system obtained at different labs can be combined to construct a multiomic study. However, data obtained at different labs or moments in time are typically subjected to batch effects that need to be removed for successful data integration. While there are methods to correct batch effects on the same data types obtained in different studies, they cannot be applied to correct lab or batch effects across omics. This impairs multiomic meta-analysis. Fortunately, in many cases, at least one omics platform-i.e. gene expression- is repeatedly measured across labs, together with the additional omic modalities that are specific to each study. This creates an opportunity for batch analysis. We have developed MultiBaC (multiomic Multiomics Batch-effect Correction correction), a strategy to correct batch effects from multiomic datasets distributed across different labs or data acquisition events. Our strategy is based on the existence of at least one shared data type which allows data prediction across omics. We validate this approach both on simulated data and on a case where the multiomic design is fully shared by two labs, hence batch effect correction within the same omic modality using traditional methods can be compared with the MultiBaC correction across data types. Finally, we apply MultiBaC to a true multiomic data integration problem to show that we are able to improve the detection of meaningful biological effects.

A multi-omics dataset of heat-shock response in the yeast RNA binding protein Mip6.

Gene expression is a biological process regulated at different molecular levels, including chromatin accessibility, transcription, and RNA maturation and transport. In addition, these regulatory mechanisms have strong links with cellular metabolism. Here we present a multi-omics dataset that captures different aspects of this multi-layered process in yeast. We obtained RNA-seq, metabolomics, and H4K12ac ChIP-seq data for wild-type and mip6Δ strains during a heat-shock time course. Mip6 is an RNA-binding protein that contributes to RNA export during environmental stress and is informative of the contribution of post-transcriptional regulation to control cellular adaptations to environmental changes. The experiment was performed in quadruplicate, and the different omics measurements were obtained from the same biological samples, which facilitates the integration and analysis of data using covariance-based methods. We validate our dataset by showing that ChIP-seq, RNA-seq and metabolomics signals recapitulate existing knowledge about the response of ribosomal genes and the contribution of trehalose metabolism to heat stress. Raw data, processed data and preprocessing scripts are made available.

Short-term pharmacological activation of Nrf2 ameliorates vascular dysfunction in aged rats and in pathological human vasculature. A potential target for therapeutic intervention.

Oxidative stress contributes to endothelial dysfunction, a key step in cardiovascular disease development. Ageing-related vascular dysfunction involves defective antioxidant response. Nuclear factor erythroid 2-like-2 (Nrf2), orchestrates cellular response to oxidative stress. We evaluated the impact of Nrf2-activation on endothelium-dependent and H2O2-mediated vasodilations in: aorta (RA), mesenteric artery (RMA), coronary artery (RCA) and corpus cavernosum (RCC) from ageing rats and in human penile arteries (HPRA) and corpus cavernosum (HCC) from erectile dysfunction (ED) patients. Relaxant responses were evaluated in organ chambers and wire myographs. Nrf2 content and heme oxygenase-1 (HO-1) were determined by ELISA. Superoxide and Nrf2 were detected by immunofluorescence. Pharmacological activation of Nrf2 with sulforaphane (SFN) improved NO- and endothelium-derived hyperpolarizing factor-mediated endothelium-dependent vasodilation and H2O2-induced relaxation in vascular beds from aging rats. SFN-induced effects were associated with increased Nrf2 (RMA, RCA) and reduced superoxide detection in RCA. Improvement of vascular function was confirmed in HPRA and HCC from ED patients and mimicked by another Nrf2 activator, oltipraz. Nrf2 increase and superoxide reduction together with HO-1 increase by Nrf2 activation was evidenced in HCC from ED patients. PDE5 inhibitor-induced relaxations of HPRA and HCC from ED patients were enhanced by SFN. Nrf2 short-term pharmacological activation attenuates age-related impairment of endothelium-dependent and reactive oxygen species (ROS)-induced vasodilation in different rat and human vascular territories by upregulation of Nrf2-related signaling and decreased oxidative stress. In ED patients target tissues, Nrf2 potentiates the functional effect of ED conventional pharmacological therapy suggesting potential therapeutic implication.

Validación del "Cuestionario de creencias de personalidad versión breve" (PBQ-SF) con muestra clínica / Validation of the Personality Belief Questionnaire Short-Form (PBQ-SF) with a clinical sample

El objetivo de este trabajo fue validar el "Cuestionario de creencias de personalidad, versión breve" (PBQ-SF), utilizando una muestra clínica (n= 343) y otra no clínica (n= 355), de entre 18 y 60 años. Mediante un análisis factorial confirmatorio se probó la estructura factorial del modelo de nueve factores (esquizoide, paranoide, antisocial, narcisista, histriónico, evitativo, dependiente, obsesivo compulsivo y pasivo agresivo) y la estructura del modelo del de siete factores (dependiente/evitativo, obsesivo compulsivo, narcisista, autónomo, paranoide, histriónico y esquizoide). El modelo de medida del factor de creencias asociadas a personalidad límite se evaluó separadamente dado que se compone de ítems incluidos en los otros factores. Los mejores indicadores de ajuste fueron los del modelo de nueve factores. La consistencia interna de todas las escalas del PBQ-SF fue buena (entre 0,71 y 0,90). La muestra clínica obtuvo puntuaciones más altas que la no clínica en todas las creencias disfuncionales, excepto para las escalas antisocial y narcisista. Los hombres puntuaron más alto en las escalas paranoide, antisocial y narcisista

The purpose of this project was to validate the "Personality Belief Questionnaire, Short Form" (PBQ-SF), using a clinical sample (n= 343) and a nonclinical sample (n= 355), with participants between 18 and 60 years of age. Through a confirmatory factorial analysis the factor structure of the nine factor model (schizoid, paranoid, antisocial, narcissistic, histrionic, avoidant, dependent, obsessive compulsive, and aggressive passive), and the structure of the seven factors model (dependent/avoidant, obsessive compulsive, narcissistic, autonomous, paranoid, histrionic, and schizoid) was tested. The model for measuring the belief factor associated with borderline personality was evaluated separately, since it is composed of items included in the other factors. The best adjustment indicators were those in the nine factor model. The internal consistency of all PBQ-SF scales was good (between. 71 and .90). The clinical sample obtained higher scores than the nonclinical sample in all dysfunctional beliefs, except for the antisocial and narcissistic scales. Men scored higher on the paranoid, antisocial, and narcissistic scales

Dynamic elementary mode modelling of non-steady state flux data.

BACKGROUND: A novel framework is proposed to analyse metabolic fluxes in non-steady state conditions, based on the new concept of dynamic elementary mode (dynEM): an elementary mode activated partially depending on the time point of the experiment. RESULTS: Two methods are introduced here: dynamic elementary mode analysis (dynEMA) and dynamic elementary mode regression discriminant analysis (dynEMR-DA). The former is an extension of the recently proposed principal elementary mode analysis (PEMA) method from steady state to non-steady state scenarios. The latter is a discriminant model that permits to identify which dynEMs behave strongly different depending on the experimental conditions. Two case studies of Saccharomyces cerevisiae, with fluxes derived from simulated and real concentration data sets, are presented to highlight the benefits of this dynamic modelling. CONCLUSIONS: This methodology permits to analyse metabolic fluxes at early stages with the aim of i) creating reduced dynamic models of flux data, ii) combining many experiments in a single biologically meaningful model, and iii) identifying the metabolic pathways that drive the organism from one state to another when changing the environmental conditions.

Multivessel analysis of progressive vascular aging in the rat: Asynchronous vulnerability among vascular territories.

Aging induces vascular dysfunction, representing the major risk factor for cardiovascular disease. Our aim was to ascertain specific vulnerability of vascular territories to aging by evaluating the progressive impact of aging on vascular function in four different vascular beds: aorta, mesenteric artery (MA), coronary artery (CA), and penile corpus cavernosum (CC) from 3, 6, 9, 12, 20 or 24 months-old male rats. Contractile/relaxant responses were evaluated in organ chambers (A/CC) and wire myographs (MA/CA). Relationships of systemic biomarkers with endothelial function impairment were also determined. Although all vessels manifested aging-related impairment in endothelial vasodilation, CA was the most impacted by aging considering the onset (at 6 months) and magnitude of endothelial dysfunction (reduction by 1.5 log units in the concentration required for 50% of maximal relaxation for acetylcholine). H2O2-induced vasodilations were progressively reduced by aging in aorta, CC and CA while NO-donor-induced vasodilation was impaired by aging only in CA. Serum asymmetric dimethylarginine significantly correlated to endothelial decline in aorta, MA, and CC, while HOMA-IR was significantly associated with endothelial dysfunction in CA and MA. CA are especially vulnerable to aging-related vascular dysfunction. Correlations of vascular dysfunction with systemic biomarkers differ among vessels, further suggesting heterogeneity in aging-induced vascular impact.

Haemostatic agent etamsylate in vitro and in vivo antagonizes anti-coagulant activity of heparin.

Etamsylate is indicated for several anti-hemorrhagic indications in human and veterinary medicine. However, etamsylate has been shown to be effective only in specific hemorrhagic situations. Furthermore, mechanism of action of etamsylate is not known but recent research has shown its ability to inhibit heparin binding to several growth factors. We have evaluated the ability of etamsylate to interfere with the activities of heparin. Effects of etamsylate on vasodilatory activity of heparin were evaluated in rat aortic segments. Influence of etamsylate on anticoagulant activity of heparin was evaluated in vitro by determining prothrombin (PT) time and activated partial thromboplastin time (aPTT) in dog blood and in vivo by determining the interference of systemic and topical etamsylate on heparin-induced extension in bleeding time (BT) in rats. Despite failing to inhibit heparin-induced vasodilation of rat aorta, etamsylate significantly reduced the increase in aPTT caused by heparin (+30.4 ±â€¯6.7% vs. +15.0 ±â€¯2.8% for etamsylate at 100 µM, P < 0.05). Etamsylate also antagonized the anticoagulant effects driven by heparin in vivo since prevented the heparin-induced increase in BT when systemically (i.p.) administered (+94.6 ±â€¯7.5% vs. +57.9 ±â€¯9.2% at 10 mg/kg, P < 0.05, vs. +22.2 ±â€¯16.8% at 30 mg/kg, P < 0.01). Additionally, topically applied etamsylate (125 mg/ml) significantly reduced heparin-induced BT increase (+102.5 ±â€¯3.2% vs. +54.0 ±â€¯5.8%, P < 0.01). These evidences show a pharmacological interference by etamsylate on heparin activities antagonizing pro-hemorrhagic effects of heparin in vitro and in vivo without inhibiting its vasodilatory properties. This ability could help to explain pharmacological effects of etamsylate and proposes its role for reversing pro-hemorrhagic states.

NIR hyperspectral imaging to evaluate degradation in captopril commercial tablets.

Pharmaceutical quality control is important for improving the effectiveness, purity and safety of drugs, as well as for the prevention or control of drug degradation. In the present work, near infrared hyperspectral images (HSI-NIR) of tablets with different expiration dates were employed to evaluate the degradation of captopril into captopril disulfide in different layers, on the top and on the bottom surfaces of the tablets. Multivariate curve resolution (MCR) models were used to extract the concentration distribution maps from the hyperspectral images. Afterward, multivariate image techniques were applied to the concentration distribution maps (CDMs), to extract features and build models relating the main characteristics of the images to their corresponding manufacturing dates. Resolution methods followed by extracting features were able to estimate the tablet manufacture date with a prediction error of 120days. The model developed could be useful to evaluate whether a sample shows a degradation pattern consistent with the date of manufacturing or to detect abnormal behaviors in the natural degradation process of the sample. The information provided by the HIS-NIR is important for the development of the process (QbD), looking inside the formulation, revealing the behavior of the active pharmaceutical ingredient (API) during the product's shelf life.

Asymmetric dimethylarginine (ADMA) elevation and arginase up-regulation contribute to endothelial dysfunction related to insulin resistance in rats and morbidly obese humans.

KEY POINTS: The presence of insulin resistance (IR) is determinant for endothelial dysfunction associated with obesity. Although recent studies have implicated the involvement of mitochondrial superoxide and inflammation in the defective nitric oxide (NO)-mediated responses and subsequent endothelial dysfunction in IR, other mechanisms could compromise this pathway. In the present study, we assessed the role of asymmetric dimethylarginine (ADMA) and arginase with respect to IR-induced impairment of endothelium-dependent vasodilatation in human morbid obesity and in a non-obese rat model of IR. We show that both increased ADMA and up-regulated arginase are determinant factors in the alteration of the l-arginine/NO pathway associated with IR in both models and also that acute treatment of arteries with arginase inhibitor or with l-arginine significantly alleviate endothelial dysfunction. These results help to expand our knowledge regarding the mechanisms of endothelial dysfunction that are related to obesity and IR and establish potential therapeutic targets for intervention. ABSTRACT: Insulin resistance (IR) is determinant for endothelial dysfunction in human obesity. Although we have previously reported the involvement of mitochondrial superoxide and inflammation, other mechanisms could compromise NO-mediated responses in IR. We evaluated the role of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) and arginase with respect to IR-induced impairment of l-arginine/NO-mediated vasodilatation in human morbid obesity and in a non-obese rat model of IR. Bradykinin-induced vasodilatation was evaluated in microarteries derived from insulin-resistant morbidly obese (IR-MO) and non-insulin-resistant MO (NIR-MO) subjects. Defective endothelial vasodilatation in IR-MO was improved by l-arginine supplementation. Increased levels of ADMA were detected in serum and adipose tissue from IR-MO. Serum ADMA positively correlated with IR score and negatively with pD2 for bradykinin. Gene expression determination by RT-PCR revealed not only the decreased expression of ADMA degrading enzyme dimethylarginine dimethylaminohydrolase (DDAH)1/2 in IR-MO microarteries, but also increased expression of arginase-2. Arginase inhibition improved endothelial vasodilatation in IR-MO. Analysis of endothelial vasodilatation in a non-obese IR model (fructose-fed rat) confirmed an elevation of circulating and aortic ADMA concentrations, as well as reduced DDAH aortic content and increased aortic arginase activity in IR. Improvement of endothelial vasodilatation in IR rats by l-arginine supplementation and arginase inhibition provided functional corroboration. These results demonstrate that increased ADMA and up-regulated arginase contribute to endothelial dysfunction as determined by the presence of IR in human obesity, most probably by compromising arginine availability. The results provide novel insights regarding the mechanisms of endothelial dysfunction related to obesity and IR and establish potential therapeutic targets for intervention.