Genome-Wide RNAi Screening Identifies Novel Pathways/Genes Involved in Oxidative Stress and Repurposable Drugs to Preserve Cystic Fibrosis Airway Epithelial Cell Integrity

Recurrent infection-inflammation cycles in cystic fibrosis (CF) patients generate a highly oxidative environment, leading to progressive destruction of the airway epithelia. The identification of novel modifier genes involved in oxidative stress susceptibility in the CF airways might contribute to devise new therapeutic approaches. We performed an unbiased genome-wide RNAi screen using a randomized siRNA library to identify oxidative stress modulators in CF airway epithelial cells. We monitored changes in cell viability after a lethal dose of hydrogen peroxide. Local similarity and protein-protein interaction network analyses uncovered siRNA target genes/pathways involved in oxidative stress. Further mining against public drug databases allowed identifying and validating commercially available drugs conferring oxidative stress resistance. Accordingly, a catalog of 167 siRNAs able to confer oxidative stress resistance in CF submucosal gland cells targeted 444 host genes and multiple circuitries involved in oxidative stress. The most significant processes were related to alternative splicing and cell communication, motility, and remodeling (impacting cilia structure/function, and cell guidance complexes). Other relevant pathways included DNA repair and PI3K/AKT/mTOR signaling. The mTOR inhibitor everolimus, the α1-adrenergic receptor antagonist doxazosin, and the Syk inhibitor fostamatinib significantly increased the viability of CF submucosal gland cells under strong oxidative stress pressure. Thus, novel therapeutic strategies to preserve airway cell integrity from the harsh oxidative milieu of CF airways could stem from a deep understanding of the complex consequences of oxidative stress at the molecular level, followed by a rational repurposing of existing "protective" drugs. This approach could also prove useful to other respiratory pathologies

Insights from behavioral economics to characterize substance use involvement in adolescents: a cluster analysis

Reinforcement pathology (RP), a framework rooted in behavioral economics, has contributed to advances in the etiology and treatment of substance use. Drug demand and delay discounting (DD) have gained considerable interest, as they inform on the risk for escalation to substance use as well as treatment-specific targets. No prior study conducted in Spain has explored the interplay of demand and DD in adolescents. This study was aimed to: 1) identify whether DD and alcohol demand can yield empirically driven subgroups, and 2) examine differences in substance use involvement. The sample comprised 107 (% females = 54.2) adolescents (M=15.46, SD=1.25) from a high school in Asturias (Spain). Participants filled out an ad-hoc survey on substance use over the prior 30 days and one year. A 20-item alcohol purchase task (APT) was used to assess the reinforcing value of alcohol. The 21-item Monetary Choice Questionnaire evaluated impulsive choice. Two subgroups emerged: Cluster 1 (n = 72) and Cluster 2 (n = 35). Participants in C2 consistently showed higher impulsivity and demand for alcohol, signifying lower responsiveness to alcohol pricing. As compared to C1, those in C2 had a higher prevalence of past-month substance use [C1: 26/72 (36.1%) vs. C2: 33/35 (94.3%), p <.001], and a greater frequency of drunkenness [p <.001] and binge drinking episodes [p <.001]. RP differentiate between subgroups of adolescent substance users with patterns of more versus less substance use involvement. The existence of specific drug use subpopulations should be considered when designing environmental preventive policiesLa economía conductual para caracterizar el uso de drogas en adolescentes: análisis de cluster. La patología del refuerzo (PR), basada en la economía conductual, ha contribuido al avance de la etiología y el tratamiento de drogas. La demanda y el descuento por demora (DD) informan sobre el incremento en el consumo y los objetivos de tratamiento. Ningún estudio realizado en España ha examinado la interacción entre ambas variables en adolescentes. Los objetivos fueron: 1) identificar si el DD y la demanda de alcohol pueden identificar distintos subgrupos y, 2) examinar sus diferencias en el consumo de sustancias. La muestra incluyó 107 (% mujeres = 54,2) adolescentes (M =15,46, DT =1,25) de enseñanza obligatoria en Asturias (España). Se evaluó el consumo de drogas en los últimos 30 días y el último año. El poder reforzante del alcohol se evaluó mediante una tarea de compra de 20 ítems (APT). La toma de decisiones impulsiva se evaluó mediante el Cuestionario de Elección Monetaria. Se identificaron dos subgrupos: Grupo 1 (n = 72) y Grupo 2 (n = 35). El Grupo 2 mostró una demanda e impulsividad más elevada, indicando menor sensibilidad a incrementos en el precio. Comparado con el Grupo 1, el 2 obtuvo una prevalencia más elevada de consumo en el último mes [G1: 26/72 (36,1%) vs. G2: 33/35 (94,3%), p <,001], mayor frecuencia de borracheras (p <,001) y episodios de consumo intensivo de alcohol (p <,001). La PR permite caracterizar e identificar grupos con distintos patrones de consumo de sustancias. La existencia de distintos perfiles debería ser considerada a la hora de desarrollar políticas preventiva

Engraftment Potential of Adipose Tissue-Derived Human Mesenchymal Stem Cells After Transplantation in the Fetal Rabbit

Due to their favorable intrinsic features, including engraftment, differentiation, and immunomodulatory potential, adult mesenchymal stem cells (MSCs) have been proposed for therapeutic in utero intervention. Further improvement of such attributes for particular diseases might merely be achieved by ex vivo MSC genetic engineering previous to transplantation. Here, we evaluated for the first time the feasibility, biodistribution, long-term engraftment, and transgenic enhanced green fluorescent protein (EGFP) expression of genetically engineered human adipose tissue-derived MSCs (EGFP+-ASCs) after intra-amniotic xenotransplantation at E17 of gestation into our validated pregnant rabbit model. Overall, the procedure was safe (86.4% survival rate; absence of anatomical defects). Stable, low-level engraftment of EGFP+-ASCs was confirmed by assessing the presence of the pWT-EGFP lentiviral provirus in the young transplanted rabbit tissues. Accordingly, similar frequencies of provirus-positive animals were found at both 8 weeks (60%) and 16 weeks (66.7%) after in utero intervention. The presence of EGFP+-ASCs was more frequent in respiratory epithelia (lung and trachea), according to the route of administration. However, we were unable to detect EGFP expression, neither by real-time polymerase chain reaction nor by immunohistochemistry, in the provirus-positive tissues, suggesting EGFP transgene silencing mediated by epigenetic events. Moreover, we noticed lack of both host cellular immune responses against xenogeneic ASCs and humoral immune responses against transgenic EGFP. Therefore, the fetal microchimerism achieved by the EGFP+-ASCs in the young rabbit hosts indicates induction of donor-specific tolerance after fetal rabbit xenotransplantation, which should boost postnatal transplantation for the early treatment/prevention of many devastating congenital disorders

Compelling new electrocardiographic markers for automatic diagnosis

Producción CientíficaBackground and Objective: The automatic diagnosis of heart diseases from the electrocardiogram (ECG) signal is crucial in clinical decision-making. However, the use of computer-based decision rules in clinical practice is still deficient, mainly due to their complexity and a lack of medical interpretation. The objetive of this research is to address these issues by providing valuable diagnostic rules that can be easily implemented in clinical practice. In this research, efficient diagnostic rules friendly in clinical practice are provided. Methods: In this paper, interesting parameters obtained from the ECG signals analysis are presented and two simple rules for automatic diagnosis of Bundle Branch Blocks are defined using new markers derived from the so-called FMM delineator. The main advantages of these markers are the good statistical properties and their clear interpretation in clinically meaningful terms. Results: High sensitivity and specificity values have been obtained using the proposed rules with data from more than 35000 patients from well known benchmarking databases. In particular, to identify Complete Left Bundle Branch Blocks and differentiate this condition from subjects without heart diseases, sensitivity and specificity values ranging from 93% to 99% and from 96% to 99%, respectively. The new markers and the automatic diagnosis are easily available at https://fmmmodel.shinyapps.io/fmmEcg/, an app specifically developed for any given ECG signal. Conclusions: The proposal is different from others in the literature and it is compelling for three main reasons. On the one hand, the markers have a concise electrocardiographic interpretation. On the other hand, the diagnosis rules have a very high accuracy. Finally, the markers can be provided by any device that registers the ECG signal and the automatic diagnosis is made straightforwardly, in contrast to the black-box and deep learning algorithms.Ministerio de Ciencia, Innovación y Universidades (grant PID2019-106363RB-I00

Effect of Biofumigation on Typical Weeds of Strawberry Fields

This research was conducted to evaluate the effect of biofumigation using fresh organic matter on typical weeds present in strawberry fields of southern Spain. Field experiments consisted of biofumigation (BF) treatment over 45 days, at two locations, with hen droppings or horse manure at a dose of 3 kg m-2, alone or in combination with solarization (BF+S) in an experimental randomized block design. Results showed good control of the main weed species, Poa annua, Portulaca oleracea and Lolium rigidum, with comparative populations of the three species under BF/BF+S of 42/4/8, 18/9/12, and 15/0/1 plants m-2 respectively. In addition, 20 seeds each of several weeds were sown inside cloth bags and were submitted to the treatment. The efficiency of biofumigation combined with solarization in the control of P. oleracea, Cynodon dactilon and Echinochloa crus-galli was 77/0, 50/15, and 30/2, as a percentage of germination of control/BF+S. Laboratory experiments consisted of simulating field conditions using a mix of soil, water and different fresh organic matter in a plastic bag, into which different seeds were sown and kept under controlled conditions over 30 days at 27ºC. Fresh organic matter used was whole strawberry plants, horse manure, hen droppings, and combinations of these. Best results were obtained with hen droppings, which showed effective control of Malva parviflora, Medicago sp., E. crus-galli, Amaranthus retroflesuxus, Chenopodium album and P. oleracea, while strawberry residues and horse manure were not useful as herbicides, sometimes even less so than the solarization treatment. Biofumigation with fresh hen droppings and solarization showed promising results in preliminary experiments

Biomarkers in ocular chronic graft versus host disease: tear cytokine- and chemokine-based predictive model.

Producción CientíficaPurpose: To develop a tear molecule level-based predictive model based on a panel of tear cytokines and their correlation with clinical features in ocular chronic graft versus host disease (cGVHD). Methods: Twenty-two ocular cGVHD patients and 21 healthy subjects were evaluated in a controlled environmental research laboratory (CERLab). Clinical parameters were recorded, and tears were collected. Levels of 15 molecules (epidermal growth factor [EGF], IL receptor antagonist [IL-1Ra], IL-1β, IL-2, IL-6, IL-8/CXCL8, IL-10, IL-12p70, IL-17A, interferon inducible protein [IP]-10/CXCL10, IFN-γ, VEGF, TNF-α, eotaxin 1, and regulated on activation normal T cell expressed and secreted [RANTES]) were measured by multiplex-bead assay and correlated with clinical parameters. Logistic regression was used to develop a predictive model. Leave-one-out cross-validation was applied. Classification capacity was evaluated in a cohort of individuals with dry eye (DE) of other etiologies different from GVHD. Results: Epidermal growth factor and IP-10/CXCL10 levels were significantly decreased in ocular cGVHD, positively correlating with tear production and stability and negatively correlating with symptoms, hyperemia, and vital staining. Interleukin-1Ra, IL-8/CXCL8, and IL-10 were significantly increased in ocular cGVHD, and the first two correlated positively with symptoms, hyperemia, and ocular surface integrity while negatively correlating with tear production and stability. Predictive models were generated, and the best panel was based on IL-8/CXCL8 and IP-10/CXCL10 tear levels along with age and sex, with an area under the receiving operating curve of 0.9004, sensitivity of 86.36%, and specificity of 95.24%. Conclusions: A predictive model based on tear levels of IL-8/CXCL8 and IP-10/CXCL10 resulted in optimal sensitivity and specificity. These results add further knowledge to the search for potential biomarkers in this devastating ocular inflammatory disease.Ministry of Economy and Competitiveness, Madrid, Spain, SAF-2010 15631 (AES)

Human Mesenchymal Stem Cells Resolve Airway Inflammation, Hyperreactivity, And Histopathology In A Mouse Model Of Occupational Asthma

Occupational asthma (OA) is characterized by allergic airway inflammation and hyperresponsiveness, leading to progressive airway remodeling and a concomitant decline in lung function. The management of OA remains suboptimal in clinical practice. Thus, establishing effective therapies might overcome the natural history of the disease. We evaluated the ability of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or engineered to secrete the IL-33 decoy receptor sST2, to attenuate the inflammatory and respiratory symptoms in a previously validated mouse model of OA to ammonium persulfate (AP). Twenty-four hours after a dermal AP sensitization and intranasal challenge regimen, the animals received intravenously 1 x 10(6) cells (either hASCs or hASCs overexpressing sST2) or saline and were analyzed at 1, 3, and 6 days after treatment. The infused hASCs induced an anti-inflammatory and restorative program upon reaching the AP-injured, asthmatic lungs, leading to early reduction of neutrophilic inflammation and total IgE production, preserved alveolar architecture with nearly absent lymphoplasmacytic infiltrates, negligible smooth muscle hyperplasia/hypertrophy in the peribronchiolar areas, and baseline airway hyperreactivity (AHR) to methacholine. Local sST2 overexpression barely increased the substantial efficacy displayed by unmodified hASCs. Thus, hASCs may represent a viable multiaction therapeutic capable to adequately respond to the AP-injured lung environment by resolving inflammation, tissue remodeling, and bronchial hyperresponsiveness typical of OA

Derivation and validation of a clinical prediction rule for sleep apnoea syndrome for use in primary care

Background: Several clinical prediction rules (CPRs) are available for sleep apnoea-hypopnoea syndrome (OSAH), but they are difficult to apply in primary care (PC). Aim: Derivation and validation of a CPR using simple measurements available in PC. Design & setting: A prospective study conducted in health centres from the area of influence of three Spanish hospitals. Method: Patients (aged 18-70 years) who attended for any reason; who presented with at least one of the three key symptoms for OSAH (snoring, breathing pauses while sleeping, and daytime sleepiness); and who were not undergoing non-invasive ventilation or prior treatment with continuous positive airway pressure (CPAP) were included. Anthropometric data, smoking habit, comorbidities, and Epworth test were collected. Patients were subsequently referred to the sleep unit (SU), where the decision was taken whether or not to instigate treatment. A multivariate logistic model was constructed using a sub-sample and scores assigned based on the regression coefficients; the CPR was validated with the remaining sample. Both receiver operating characteristic (ROC) curves were plotted and the sensitivity, specificity, and predictive values calculated. Results: The derivation sample comprised 352 patients, with 260 in the validation sample. The final factors (arterial hypertension [AHT], age, body mass index [BMI], and sex) were used to develop a rule with scores ranging from 0.00-5.50. The cut-off point that optimises the area under the curve (AUC) is ?2.50 points (AUC = 0.78; sensitivity = 86%; specificity = 54%; positive predictive value [PPV] = 45%; negative predictive value [NPV] = 90%; likelihood ratio [LR] = 0.26). The properties for the validation sample with this cut-off point are as follows: AUC = 0.68; sensitivity = 81%; specificity = 43%; PPV = 61%; NPV = 68%; LR = 0.44. Conclusion: As in similar cases, the specificity is low, meaning that healthy people are referred to a specialist. A negative result rules out the disease in most cases

Efecto de la biofumigación sobre flora arvense del cultivo del fresón

Debido a la biodescomposición de la materia orgánica, se producen una serie de compuestos capaces de controlar plagas, patógenos y flora arvense. Este proceso ha sido definido como biofumigación (BF). El objetivo de esta investigación fue evaluar el efecto de la biofumigación con materia orgánica fresca sobre flora arvense típica de los cultivos de fresón. Los experimentos de campo consistieron en el tratamiento con gallinaza o estiércol de caballo a la dosis de 3 kg m-2 durante 45 días. La biofumigación se combinó también con solarización (BF+S), en un diseño experimental de bloques al azar. Los resultados con gallinaza mostraron un buen control de las principales adventicias Poa annua, Portulaca oleracea y Lolium rigidum con resultados de control/BF/BF+S de 42/4/8, 18/9/12, y 15/0/1 plants m-2 respectivamente. Por otra parte, 20 semillas de varias arvenses se introdujeron en bolsas de tela y fueron sometidas a biofumigación con solarización. La eficacia del tratamiento BF+S fue claro en el control de P. oleracea, Cynodon dactilon y Echinochloa crus-galli con un porcentaje de germinación control/BF+S de 77/0, 50/15 y 30/2. Los experimentos de laboratorio consistieron en simular las condiciones de campo usando una mezcla de suelo, agua y diferentes materias orgánicas frescas. Las materias orgánicas utilizadas fueron restos de fresa, estiércol de caballo y gallinaza. Los mejores resultados se obtuvieron con gallinaza que mostró un control eficaz de Malva parviflora, Medicago sp., E. crus-galli, Amaranthus retroflexus, Chenopodium album y P. oleracea mientras que los residuos de fresa y el estiércol no mostraron inhibición de la germinación. En conclusión, los resultados obtenidos con los tratamientos de gallinaza se encuentran bajo investigación con resultados prometedores en los experimentos realizados hasta la fech

Antimicrobial Susceptibility and Characterization of Resistance Mechanisms of Corynebacterium urealyticum Clinical Isolates

Corynebacterium urealyticum is a non-diphtherial urease-producing clinically relevant corynebacterial, most frequently involved in urinary tract infections. Most of the C. urealyticum clinical isolates are frequently resistant to several antibiotics. We investigated the susceptibility of 40 C. urealyticum isolated in our institution during the period 2005-2017 to eight compounds representative of the main clinically relevant classes of antimicrobial agents. Antimicrobial susceptibility was determined by the Epsilometer test. Resistance genes were searched by PCR. All strains were susceptible to vancomycin whereas linezolid and rifampicin also showed good activity (MICs90 = 1 and 0.4 mg/L, respectively). Almost all isolates (39/40, 97.5%) were multidrug resistant. The highest resistance rate was observed for ampicillin (100%), followed by erythromycin (95%) and levofloxacin (95%). Ampicillin resistance was associated with the presence of the blaA gene, encoding a class A ?-lactamase. The two rifampicin-resistant strains showed point mutations driving amino acid replacements in conserved residues of RNA polymerase subunit ? (RpoB). Tetracycline resistance was due to an efflux-mediated mechanism. Thirty-nine PFGE patterns were identified among the 40 C. urealyticum, indicating that they were not clonally related, but producing sporadic infections. These findings raise the need of maintaining surveillance strategies among this multidrug resistant pathogen.This research was funded by Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI) D16/0016/0007 and RD16/0016/0008), and co-financed by European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 2014-2020