Urinary extracellular vesicles. A promising shortcut to novel biomarker discoveries

Proteomic and genomic techniques have reached full maturity and are providing unforeseen details for the comprehensive understanding of disease pathologies at a fraction of previous costs. However, for kidney diseases, many gaps in such information remain to inhibit major advances in the prevention, treatment and diagnostics of these devastating diseases, which have enormous global impact. The discovery of ubiquitous extracellular vesicles (EV) in all bodily fluids is rapidly increasing the fundamental knowledge of disease mechanisms and the ways in which cells communicate with distant locations in processes of cancer spread, immunological regulation, barrier functions and general modulation of cellular activity. In this review, we describe some of the most prominent research streams and findings utilizing urinary extracellular vesicles as highly versatile and dynamic tools with their extraordinary protein and small regulatory RNA species. While being a highly promising approach, the relatively young field of EV research suffers from a lack of adherence to strict standardization and carefully scrutinized methods for obtaining fully reproducible results. With the appropriate guidelines and standardization achieved, urine is foreseen as forming a unique, robust and easy route for determining accurate and personalized disease signatures and as providing highly useful early biomarkers of the disease pathology of the kidney and beyond.Peer reviewe

Novel Locally Acting Dual Antiplatelet and Anticoagulant (APAC) Targets Multiple Sites of Vascular Injury in an Experimental Porcine Model

Objectives: Vascular binding of dual antiplatelet and anticoagulant (APAC) was assessed in surgically created femoral arteriovenous fistula (AVF) and iliac and carotid artery injury in porcine models. Methods: Three models of collagen exposing injury were used: 1) femoral AVF, 2) in vivo iliac and carotid artery balloon angioplasty injury, and 3) in vitro femoral artery endothelial denudation injury. Biotinylated APAC (0.5 mg/mL) was incubated with the injury site before releasing blood flow. APAC, von Willebrand factor (vWF), laminin, platelet endothelial cell adhesion molecule 1 (PECAM-1), and podocalyxin were detected in histological sections using immunofluorescence and confocal microscopy and Manders' co-localisation coefficient (M1). Results: APAC bound to AVF at anastomosis and to both in vivo and in vitro injured arteries. APAC co-localised with matrix vWF (M1 >= 0.66) and laminin (M1 >= 0.60), but less so if endothelial PECAM-1 or podocalyxin was present (M1 Conclusions: APAC, compatible with its high negative charge, rapidly targets injured vessels co-localizing with matrix vWF and laminin, but not with endothelial PECAM-1 and podocalyxin. This localising feature may have potential antithrombotic implications for vascular interventions.Peer reviewe

Factores de inicio y mantenimiento del consumo de SPA y relación con las conductas antisociales y delictivas

Graficas, Encuestas, cuestinario,A nivel regional el consumo de Sustancias Psicoactivas SPA crece de forma inminente frente a una sociedad que no se inmuta ante este fenómeno que acaba con el ser humano sin discriminación alguna. Es por ello que nace la necesidad de realizar una investigación cuyo objetivo es dar a conocer los factores que inciden en el inicio y el mantenimiento del consumo de SPA y la relación de este con las conductas antisociales y delictivas en población privada de su libertad en el Centro Transitorio de Internamiento Preventivo “Nuevos Días” y en las Fundaciones de Rehabilitación: Emmanuel, Ríos de Agua Viva y Manantial de Vida de la ciudad de Florencia Caquetá. Partiendo de este planteamiento esta investigación se lleva a cabo bajo un enfoque cuanti-cualitativo y siguiendo el diseño de campo que nos permite acceder a la información directamente de la población a trabajar sin espacios a distorsionar los datos obtenidos. El tipo de investigación es descriptiva correlacional y a la población mencionada se le aplicaron dos instrumentos los cuales son la prueba psicológica A-D y un cuestionario elaborado para este fin. Dichos instrumentos fueron aplicados de forma individual y confidencial para crear un ambiente de confianza entre el investigador y los entrevistados y así generar las respuestas precisas a los interrogantes propuestos. Como resultados se encontró que los factores de inicio de consumo de SPA se convierten en factores de mantenimiento a medida que este aumenta, estos factores son de tipo familiar, socio-demográficos y académicos. La relación entre el consumo de SPA y las conductas antisociales y delictivas se da en el momento en que se crea la necesidad económica para suplir la ansiedad de consumir. Con el fin de mejorar los programas de rehabilitación estos resultados se dieron a conocer en las instituciones que participaron en esta investigación.Regionally Psychoactive Substances consumption grows SPA imminently facing a society that does not flinch at this phenomenon which finishes with a human being without discrimination. That is why we created the need for an investigation whose aim is to understand the factors that influence the initiation and maintenance of drug misuse and the relationship of this with antisocial and criminal behavior in population deprived of their freedom in the Transient Preventive Detention center "New Day " and the Foundations of Rehabilitation: Emmanuel Rios de Agua Viva and Manantial de Vida city of Florence Caquetá. Using this approach the research is carried out under a quantitative and qualitative approach and following the design field that allows us to access information directly from the people to work without spaces to distort the data. The research is correlational descriptive and people mentioned were applied two instruments which are the psychological test AD and a questionnaire developed for this purpose. These instruments were applied individually and confidentially to create an atmosphere of trust between the researcher and the respondents and thus generate precise answers to the proposed questions. As a result it was found that the factors of onset of SPA become maintenance factors as it increases, these factors are demographic socio- familial, and academics. The relationship between drug misuse and antisocial and criminal behavior occurs in the time of economic necessity is created to meet consuming anxiety. In order to improve rehabilitation programs these results were announced in the institutions that participated in this research

De Valencia a Querétaro. A devoción xacobea a través dunha estampa de Vicente Capilla Gil

Nunha estampa do gravador valenciano Vicente Capilla Gil, realizada en 1792, conmemórase a fundación da cidade de Querétaro (México), acontecida o 25 de xullo de 1531, día da festividade de Santiago. A coincidencia destes dous feitos propiciou un suceso milagroso rexistrado no devandito gravado: a aparición no ceo da Santa Cruz e da imaxe do Apóstolo Patrón das Españas. Este feito propiciou a extensión do culto a Santiago por todo o Vicerreinado da Nova España e a devoción á Santa Cruz, que daría nome a un dos principais colexios de misións: o da Santa Cruz de Querétaro. o labor realizado polos frades franciscanos no contexto das misións queda resaltado na estampa de forma exemplarizante. Por medio do gravado búscase salientar as virtudes ou actos dos frades Fr. Antonio Llinaz e Fr. Antonio Marfil, de orixe española, como fundadores do colexio e evanxelizadores e apóstolos de Cristo

From Valencia to Querétaro. Devotion to Saint James through an illustration by Vicente Capilla Gil

The conquest of the city of Querétaro (Mexico), on 25 July 1531, on the feast day of Saint James, is commemorated in an illustration by a Valencian engraver, Vicente Capilla Gil, produced in 1792. The coincidence in the date of the two events led to a miraculous happening being recorded in the illustration: the appearance of the Holy Cross in the sky together with the Apostle, Patron Saint of Spain. This in turn led to the extension of the cult of Saint James and devotion to the Holy Cross throughout the ViceRoyalty of New Spain, and one of the main mission schools is named after it: Santa Cruz de Querétaro. The work done by the Franciscan Brothers in the context of missions is highlighted in an exemplary way in the illustration. The virtues and deeds of Brother Antonio Llinaz and Brother Antonio Marfil, of Spanish origin, as founders of the school, evangelists and apostle of Christ are underlined in the illustration

AFM investigation of APAC (antiplatelet and anticoagulant heparin proteoglycan)

Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm(3), and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method.Peer reviewe

An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles

Extracellular vesicles (EV) are membranous particles secreted by all cells and found in body fluids. Established EV contents include a variety of RNA species, proteins, lipids and metabolites that are considered to reflect the physiological status of their parental cells. However, to date, little is known about cell-type enriched EV cargo in complex EV mixtures, especially in urine. To test whether EV secretion from distinct human kidney cells in culture differ and can recapitulate findings in normal urine, we comprehensively analysed EV components, (particularly miRNAs, long RNAs and protein) from conditionally immortalised human kidney cell lines (podocyte, glomerular endothelial, mesangial and proximal tubular cells) and compared to EV secreted in human urine. EV from cell culture media derived from immortalised kidney cells were isolated by hydrostatic filtration dialysis (HFD) and characterised by electron microscopy (EM), nanoparticle tracking analysis (NTA) and Western blotting (WB). RNA was isolated from EV and subjected to miRNA and RNA sequencing and proteins were profiled by tandem mass tag proteomics. Representative sets of EV miRNAs, RNAs and proteins were detected in each cell type and compared to human urinary EV isolates (uEV), EV cargo database, kidney biopsy bulk RNA sequencing and proteomics, and single-cell transcriptomics. This revealed that a high proportion of the in vitro EV signatures were also found in in vivo datasets. Thus, highlighting the robustness of our in vitro model and showing that this approach enables the dissection of cell type specific EV cargo in biofluids and the potential identification of cell-type specific EV biomarkers of kidney disease.Peer reviewe

Management of Tamm-Horsfall Protein for Reliable Urinary Analytics

Purpose Urinary extracellular vesicles (uEVs) are a novel source of biomarkers. However, urinary Tamm-Horsfall Protein (THP; uromodulin) interferes with all vesicle isolation attempts, precipitates with normal urinary proteins, thus, representing an unwanted "contaminant" in urinary assays. Thus, the aim is to develop a simple method to manage THP efficiently. Experimental design The uEVs are isolated by hydrostatic filtration dialysis (HFD) and treated with a defined solution of urea to optimize release of uEVs from sample. Presence of uEVs is confirmed by transmission electron microscopy, Western blotting, and proteomic profiling in MS. Results Using HFD with urea treatment for uEV isolation reduces sample complexity to a great extent. The novel simplified uEV isolation protocol allows comprehensive vesicle proteomics analysis and should be part of any urine analytics to release all sample constituents from THP trap. Conclusions and clinical relevance The method brings a quick and easy protocol for THP management during uEV isolation, providing major benefits for comprehensive sample analytics.Peer reviewe

Estreptococos beta-hemolítico en tilapias del Nilo (Oreochromis niloticus) cultivadas en Sullana, Piura - Perú

Objective. This investigation aimed to study the presence of Streptococcus spp. in tilapia (Oreochromis niloticus) from fish farm located in Sullana-Piura, Peru. Materials and methods. 150 fish with clinical signs of streptococcal disease were sampled, and the bacterium isolation was performed on blood agar, correlated to histopathological lesions description and molecular confirmation by real-time PCR. Results. The necropsy revealed exophthalmia, hyphema, congestion and/or haemorrhagic meninges, ascites, splenomegaly, hepatomegaly and diffuse haemorrhagic zones throughout the body. 102 isolated positives (54 tilapias) to Streptococcus spp. were identified in the microbiological analysis (prevalence of 26%), the brain was the organ with the highest percentage of this bacteria (34.31%), and 19 isolates were beta-haemolytic (18.63%) with prevalence of 10.12%. Fish beta-haemolytic streptococci presented epicarditis, perisplenitis and chronic meningitis, panophthalmitis, coagulative necrosis of skeletal muscle and granulomas formation. In the confirmatory test by real-time PCR, any positive tilapia to S. iniae was obtained. The results were analysed using a stochastic simulation of beta distribution using @Risk program uncertainty, reporting an average prevalence of 0.66% in sick tilapias. Conclusions. The analysed fishes were positive to bacteria of the genus Streptococcus, which confirms its presence in the fish farm. However, 19 isolates were beta-haemolytic, and the presence of S. iniae was not positive to the limit prevalence of 2.7% in real-time PCR.Objetivo. Esta investigación objetivó estudiar la presencia de Streptococcus spp. en tilapia (Oreochromis niloticus) de la piscifactoría localizada en Sullana-Piura, Perú. Materiales y Métodos. 150 peces con signos clínicos de la enfermedad estreptocócica fueron analizados, el aislamiento bacteriano se realizó en agar sangre, en correlación con las lesiones histopatológicas y diagnóstico molecular mediante PCR en tiempo real. Resultados. A la necropsia se observó exoftalmia, hifema, congestión y/o meninges hemorrágicas, ascitis, esplenomegalia, hepatomegalia y zonas hemorrágicas difusas en todo el cuerpo. 102 aislados positivos (54) tilapias para Streptococcus spp. fueron identificados en el análisis microbiológico (prevalencia del 26%), el cerebro fue el órgano con el más alto porcentaje del género de esta bacteria (34.31%), 19 aislados fueron beta-hemolítico (18.63%), con prevalencia de 10.12%. Peces con Streptococcus beta-hemolíticos presentaron epicarditis, perisplenitis y meningitis crónica, panoftalmitis, necrosis coagulativa del músculo esquelético y formación de granulomas. En el análisis de PCR en tiempo real, no se obtuvo ninguna tilapia positiva para S. iniae. Los resultados se analizaron mediante una simulación estocástica de la distribución beta usando el programa de incertidumbre @Risk, reportando una prevalencia media de 0.66% en tilapias enfermas. Conclusiones. Los peces analizados fueron positivos para bacterias del género Streptococcus, lo que confirma su presencia en la piscifactoría. Sin embargo, 19 aislados fueron beta-hemolíticos, y la presencia de S. iniae no fue positiva para la prevalencia límite de 2.7% en PCR en tiempo real

Detección molecular de Rickettsia typhi en perros de una comunidad rural de Yucatán, México

Introduction: Rickettsia typhi causes murine or endemic typhus, which is transmitted to humans primarily through flea bites contaminated with feces. Synanthropic and domestic animals also contribute to the infection cycle of R. typhi. Cases of murine typhus in humans were reported in the rural community of Bolmay, Yucatán, México, between 2007 and 2010. Objective: To identify the presence of R. typhi and estimate the frequency of infection in dogs from Bolmay, México, a locality with previous reports of murine typhus in humans. Materials and methods: Whole blood samples were taken from 128 dogs. Total DNA was extracted for use in the polymerase chain reaction (PCR) to amplify fragments of the 17 kDa and omp B genes and confirms the presence of Rickettsia spp. The reaction products were sequenced, and alignment analysis was performed using the BLAST tool. Results: The frequency of R. typhi infection in dogs was 5.5 % (7/128). The alignment identified 99% and 100% homology to the R. typhi 17 kDa and omp B genes, respectively. Conclusion: We confirmed the presence of R. typhi in dogs in the studied community but at a low frequency. However, there is potential risk of transmission to humans.Introducción. Rickettsia typhi es la bacteria causante del tifus múrido o endémico, el cual es transmitido al ser humano principalmente por medio de las heces infectadas de pulgas y en cuyo ciclo de infección se encuentran involucrados distintos animales sinantrópicos y domésticos. En la comunidad rural de Bolmay, Yucatán, México, se reportaron casos de tifus múrido en seres humanos durante el periodo 2007-2010.Objetivo. Identificar la presencia de R. typhi y estimar la frecuencia de infección en perros de Bolmay, México.Materiales y métodos. Se tomaron muestras de sangre completa de 128 perros, se les extrajo el ADN total y se analizaron mediante reacción en cadena de la polimerasa (PCR) para amplificar los fragmentos del gen de 17 kDa y omp B, y confirmar la presencia de Rickettsia spp. Los productos de las reacciones se enviaron a secuenciación y se les hizo un análisis de alineamiento con Basic Local Alignment Search Tool (BLAST).Resultados. Se encontró una frecuencia de infección de 5,5 % (7/128). El alineamiento demostró 99 % de homologación para el gen de 17 kDa y 100 % para el gen omp B en R. typhi.Conclusión. Se detectó la presencia de R. typhi pero una baja frecuencia de infección en perros de la comunidad de estudio; sin embargo, la especie podría representar un riesgo de transmisión para los seres humanos