Impact of Extracellular Vesicle Isolation Methods on Downstream Mirna Analysis in Semen: A Comparative Study.

Seminal plasma (SP) contains a unique concentration of miRNA, mostly contained in small extracellular vesicles (sEVs) such as exosomes, some of which could be clinically useful for diagnosis and/or prognosis of urogenital diseases such as prostate cancer (PCa). We optimized several exosome-EV isolation technologies for their use in semen, evaluating EV purifying effectiveness and impact on the downstream analysis of miRNAs against results from the standard ultracentrifugation (UC) method to implement the use of SP sEV_miRNAs as noninvasive biomarkers for PCa. Our results evidenced that commercial kits designed to isolate exosomes/EVs from blood or urine are mostly applicable to SP, but showed quantitative and qualitative variability between them. ExoGAG 3500× g and the miRCURY Cell/Urine/CSF 1500× g methods resulted as equivalent alternative procedures to UC for isolating exosomes/sEVs from semen for nanoparticle characteristics and quality of RNA contained in vesicles. Additionally, the expression profile of the altered semen sEV-miRNAs in PCa varies depending on the EV isolation method applied. This is possibly due to different extraction techniques yielding different proportions of sEV subtypes. This is evidence that the exosome-EV isolation method has a significant impact on the analysis of the miRNAs contained within, with important consequences for their use as clinical biomarkers. Therefore, miRNA analysis results for EVs cannot be directly extrapolated between different EV isolation methods until clear markers for delineation between microvesicles and exosomes are established. However, EV extraction methodology affects combined models (semen exosome miRNA signatures plus blood Prostate specific antigen (PSA) concentration for PCa diagnosis) less; specifically our previously described (miR-142-3p + miR-142-5p + miR-223-3p + PSA) model functions as molecular marker from EVs from any of the three isolation methods, potentially improving the efficiency of PSA PCa diagnosis.

[Off-label use of intravenous immunoglobulin g in a highly complex pediatric hospital in Argentina. A prospective observational study] / Uso "off label" de inmunoglobulina g endovenosa en un hospital pediátrico de alta complejidad de Argentina.

Introduction: The World Health Organization recommends prioritizing safe and effective drugs proven by clinical or epidemiological studies. However, in population groups with little research, a drug can be used for an indication or pharmaceutical form different from that approved by the regulatory agency (off-label), extrapolating data from studies in adults and exposing pediatric patients. to develop an Adverse Drug Reaction (ADR) due to safety considerations that have not been systematically studied. Intravenous immunoglobulin (IVIg), a high-cost drug, is used with scant evidence in some low-prevalence pathologies. This paper describes and analyzes the off-label use of IVIg at the J. P. Garrahan Pediatric Hospital. Methods: Observational, descriptive, prospective study on off-label indications of IVIg. The sampling technique was non-probabilistic and for convenience during 7 months. Results: 305 IVIg infusions were studied, corresponding to 111 patients. The indication classification showed that 22% (n=67) of the infusions were off-label. In neurology there was a higher percentage of off-label indications (46%) and within them 45% corresponded to the use in neurological disorders. 81% of the doses indicated off-label were in the range 0.8-1g/kg. The off-label infusions presented 61.5% (n=8) of the ADRs. Those from the Neurology service represented 87.5%; 75% being from the "Neurological disorders" group. Conclusion: In some cases, IVIg was indicated in an off-label manner, finding a statistically significant relationship with the appearance of ADR. This finding motivates the proposition of new hypotheses to carry out more studies.

Introducción: La Organización Mundial de la Salud recomienda priorizar fármacos seguros y eficaces comprobados mediante estudios clínicos o epidemiológicos. Sin embargo, en grupos poblacionales con escasa investigación, un fármaco puede utilizarse para una indicación o, forma farmacéutica diferente a la aprobada por la agencia reguladora ("off label"), extrapolando datos provenientes de estudios en adultos y, exponiendo a los pacientes pediátricos a desarrollar una Reacción Adversa Medicamentosa (RAM) por consideraciones de seguridad no estudiadas sistemáticamente. Inmunoglobulina G endovenosa (IgG EV), medicamento de alto costo, es utilizado con escasa evidencia en algunas patologías poco prevalentes. Este trabajo describe y analiza el uso "off label" de IgG EV en el Hospital de Pediatría J. P. Garrahan. Métodos: Estudio observacional, descriptivo, prospectivo sobre indicaciones "off label" de IgG EV. La técnica de muestreo fue no probabilística y por conveniencia durante 7 meses. Resultados: Se estudiaron 305 infusiones de IgG EV que correspondieron a 111 pacientes. La clasificación de la indicación mostró que 22% (n=67) de las infusiones fueron "off label". En neurología hubo mayor porcentaje de indicaciones "off label" (46%) y dentro de ellas el 45% correspondió al uso en desórdenes neurológicos. El 81% de dosis indicadas "off label" estuvieron en rango 0,8-1g/kg. Las infusiones indicadas "off label" presentaron el 61.5% (n=8) de las RAM. Las del servicio de Neurología, representaron el 87,5 %, siendo 75% del grupo "Desórdenes neurológicos". Conclusión: En algunos casos IgG EV fue indicada en forma "off label", encontrándose una relación estadísticamente significativa con la aparición de RAM. Este hallazgo motiva al planteo de nuevas hipótesis para realizar más estudios.

Extracellular Vesicles' Genetic Cargo as Noninvasive Biomarkers in Cancer: A Pilot Study Using ExoGAG Technology.

The two most developed biomarkers in liquid biopsy (LB)-circulating tumor cells and circulating tumor DNA-have been joined by the analysis of extracellular vesicles (EVs). EVs are lipid-bilayer enclosed structures released by all cell types containing a variety of molecules, including DNA, mRNA and miRNA. However, fast, efficient and a high degree of purity isolation technologies are necessary for their clinical routine implementation. In this work, the use of ExoGAG, a new easy-to-use EV isolation technology, was validated for the isolation of EVs from plasma and urine samples. After demonstrating its efficiency, an analysis of the genetic material contained in the EVs was carried out. Firstly, the sensitivity of the detection of point mutations in DNA from plasma EVs isolated by ExoGAG was analyzed. Then, a pilot study of mRNA expression using the nCounter NanoString platform in EV-mRNA from a healthy donor, a benign prostate hyperplasia patient and metastatic prostate cancer patient plasma and urine samples was performed, identifying the prostate cancer pathway as one of the main ones. This work provides evidence for the value of using ExoGAG for the isolation of EVs from plasma and urine samples, enabling downstream applications of the analysis of their genetic cargo.

Modeling ANXA2-overexpressing circulating tumor cells homing and high throughput screening for metastasis impairment in endometrial carcinomas.

Endometrial cancer (EC) is the most common neoplasm of the female reproductive tract in the developed world. Patients usually are diagnosed in early stage having a good prognosis. However, up to 20-25% of patients are diagnosed in advanced stages and have a higher risk of recurrence, making the prognosis worse. Previously studies identified ANXA2 as a predictor of recurrent disease in EC even in low risk patients. Furthermore, Circulating Tumor Cells (CTC) released from the primary tumor into the bloodstream, are plasticity entities responsible of the process of metastasis, becoming into an attractive clinical target. In this work we validated ANXA2 expression in CTC from high-risk EC patients. After that, we modelled in vitro and in vivo the tumor cell attachment of ANXA2-expressing CTC to the endothelium and the homing for the generation of micrometastasis. ANXA2 overexpression does not provide an advantage in the adhesion process of CTC, but it could be playing an important role in more advanced steps, conferring a greater homing capacity. We also performed a high-throughput screening (HTS) for compounds specifically targeting ANXA2, and selected Daunorubicin as candidate hit. Finally, we validated Daunorubicin in a 3D transendothelial migration system and also in a in vivo model of advanced EC, demonstrating the ability of Daunorubicin to inhibit the proliferation of ANXA2-overexpressing tumor cells.

Circulating Extracellular Vesicles in Gynecological Tumors: Realities and Challenges.

Although liquid biopsy can be considered a reality for the clinical management of some cancers, such as lung or colorectal cancer, it remains a promising field in gynecological tumors. In particular, circulating extracellular vesicles (cEVs) secreted by tumor cells represent a scarcely explored type of liquid biopsy in gynecological tumors. Importantly, these vesicles are responsible for key steps in tumor development and dissemination and are recognized as major players in cell-to-cell communication between the tumor and the microenvironment. However, limited work has been reported about the biologic effects and clinical value of EVs in gynecological tumors. Therefore, here we review the promising but already relatively limited data on the role of circulating EVs in promoting gynecological tumor spread and also their value as non-invasive biomarkers to improve the management of these type of tumors.

Utilization and reusability of hydroxyethyl cellulose alumina based aerogels for the removal of spilled oil.

Nowadays millions of oil tons are spilled into the environment causing important damage. Therefore, the development of new technologies and materials are needed to remediate this problem. In this study, hydroxyethyl cellulose alumina-based aerogels are synthesized by an environmentally friendly freeze-drying process to be used as sorbents for oil spills. It is demonstrated that the oil retention coefficient depends on the viscosity of the oil and the amount of hydroxyethyl cellulose contained in the aerogel, being 10% the optimal proportion. The aerogel synthetized with this content of hydroxyethyl cellulose displays the most favourable physicochemical and morphological properties to retain different oil spills, achieving 5.5 times its weight in comparison to its dry state. In addition, reusability experiments washing the aerogel with acetone or ethanol after the oil retention are carried out. Results show an improvement after a long washing of the sorbent with acetone, resulting in an oil weight gain of 38.7%.

Extracellular Vesicles-Based Biomarkers Represent a Promising Liquid Biopsy in Endometrial Cancer.

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EVs cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EVs isolation are tedious and low cost-efficient for routine clinical implementation. To explore the clinical value of circulating EVs analysis we attempted a proof-of-concept in endometrial cancer (EC) with ExoGAG, an easy to use and highly efficient new technology to enrich EVs. Technical performance was first evaluated using EVs secreted by Hec1A cells. Then, the clinical value of this strategy was questioned by analyzing the levels of two well-known tissue biomarkers in EC, L1 cell adhesion molecule (L1CAM) and Annexin A2 (ANXA2), in EVs purified from plasma in a cohort of 41 EC patients and 20 healthy controls. The results demonstrated the specific content of ANXA2 in the purified EVs fraction, with an accurate sensitivity and specificity for EC diagnosis. Importantly, high ANXA2 levels in circulating EVs were associated with high risk of recurrence and non-endometrioid histology suggesting a potential value as a prognostic biomarker in EC. These results also confirmed ExoGAG technology as a robust technique for the clinical implementation of circulating EVs analyses.

Immobilized laccase on polyimide aerogels for removal of carbamazepine.

Since it is known that conventional wastewater treatment plants cannot completely remove pharmaceutical compounds, such as carbamazepine, the need for their removal has intensified. The use of biocatalysts, such as enzyme is an environmentally friendly method for carbamazepine biodegradation. Nevertheless, enzyme immobilization is required to facilitate the recovery and reusability and avoid the loss of enzyme. In this work, laccase was immobilized on modified polyimide aerogels by means of covalent bonding. Results showed that the immobilized laccase on polyimide aerogels possesses significantly improved activity under acidic or basic pH range in comparison with the free enzyme. Furthermore, for all the temperature range the activity of the immobilized enzyme was higher compared to the free enzyme form. The storage stability improved by the immobilization on this support material. The reusability tests towards oxidation of 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS) showed that the immobilized laccase maintained 22% of the initial activity after 7 cycles. Immobilized laccase on polyimide aerogels for carbamazepine (CBZ) degradation exhibited 76% and 74% removal in spiked water and secondary effluent, respectively. Furthermore, after 7 cycles the CBZ removal efficiency remained higher (50% and 65% for spiked water and secondary effluent, respectively).

Dental morphological markers as a proxy for ethnicity in Robinson Crusoe Islanders / Marcadores morfológicos dentarios en la estimación de la etnicidad poblacional de la Isla Robinson Crusoe

Chilean Robinson Crusoe Island is a semi-isolated location with unusually high rates of both consanguinity and language disorder. The current population of 633 inhabitants is descended almost exclusively from the colonization at the end of the 19th century, as there were few preceding immigrations to the island. This study investigates the genetic composition and degree of miscegenation within the island population, using dental morphological markers. The universe of island children was studied (n= 128, 3 to 15 years of age) using clinical exams, dental cast, and identification of each individual within a previously-constructed extensive genealogy for the island. The frequencies for Carabelli's cusp (61.7%), shovel-shaped incisor (9.4%), and sixth cusp (2.3%), along with the absence of seventh cusp, are consistent with a primarily Caucasian population. The estimated degree of miscegenation suggests an Amerindian component of 4.3%, which is consistent with the extensive known genealogies of the founders. Characterizing the genetic profile of Robinson Crusoe Island, a location with a remarkably high prevalence of language disorder, facilitates the comparison of the genetic variants underlying this pathology with those identified in European populations.

La isla chilena Robinson Crusoe es un semiaislado geográfico de alta consanguinidad. Su población actual de 633 habitantes proviene de la última colonización ocurrida a finales del siglo XIX y pocas migraciones posteriores, en quienes recientemente se ha descrito una alta incidencia de trastorno de lenguaje. Este estudio estimó el componente genético y grado de miscegenación de la población isleña usando marcadores morfológicos dentarios. Se estudió al universo de niños isleños (n= 128, 3 a 15 años de edad) con exámenes clínicos, modelos dentales y ubicación de cada individuo en genealogías extensas confeccionadas previamente. La frecuencia de Tubérculo de Carabelli fue 61,7%, Diente en Pala 9,4%, tubérculo sexto 2,3% y ausencia del rasgo tubérculo séptimo, lo que concuerda con una población eminentemente caucásica. El grado de miscegenación estima que el componente amerindio de esta población es de 4,3%, que también se evidencia al analizar las genealogías extensas originadas por los colonizadores. La descripción del perfil genético de esta población, donde se han reportado altas prevalencias de trastorno de lenguaje, permitirá comparar con las variantes genéticas subyacentes a esta patología descritas para poblaciones europeas.