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IntroductionAnaphylaxis is among the most severe manifestations of allergic disorders, but its molecular basis remains largely unknown and reliable diagnostic markers are not currently available. MicroRNAs (miRNAs) regulate several pathophysiological processes and have been proposed as non-invasive biomarkers. Therefore, this study aims to evaluate their involvement in anaphylactic reaction and their value as biomarkers.MethodsAcute (anaphylaxis) and baseline (control) serum samples from 67 patients with anaphylaxis were studied. Among them, 35 were adults with drug-induced anaphylaxis, 13 adults with food-induced anaphylaxis and 19 children with food-induced anaphylaxis. The circulating serum miRNAs profile was characterized by next-generation sequencing (NGS). For this purpose, acute and baseline samples from 5 adults with drug-induced anaphylaxis were used. RNA was extracted, retrotranscribed, sequenced and the readings obtained were mapped to the human database miRBase_20. In addition, a system biology analysis (SBA) was performed with its target genes and revealed pathways related to anaphylactic mediators signaling. Moreover, functional and molecular endothelial permeability assays were conducted with miR-375-3p-transfected cells in response to cAMP.ResultsA total of 334 miRNAs were identified, of which 21 were significant differentially expressed between both phases. Extracellular vesicles (EVs) were characterized by Western blot, electron microscopy and NanoSight. A decrease of miR-375-3p levels was determined by qPCR in both serum and EVs of patients with anaphylaxis (****p

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

IntroductionDiabetic Retinopathy (DR) is a potentially blinding retinal disorder that develops through the pathogenesis of diabetes. The lack of disease predictors implies a poor prognosis with frequent irreversible retinal damage and vision loss. Extracellular Vesicles (EVs) present a novel opportunity for pre-symptomatic disease diagnosis and prognosis, both severely limited in DR. All biological fluids contain EVs, which are currently being studied as disease biomarkers. EV proteins derived from urine have emerged as potential noninvasive biomarkers.MethodsIn this study, we isolated EVs from DR retinal tissue explants and from DR patients’ urine, and characterized the vesicles, finding differences in particle number and size. Next, we performed proteomic analysis on human explanted DR retinal tissue conditioned media, DR retinal EVs and DR urinary EVs and compared to normal human retinal tissue, retinal EVs, and urinary EVs, respectivelyResultsOur system biology analysis of DR tissue and EV expression profiles revealed biological pathways related to cell-to-cell junctions, vesicle biology, and degranulation processes. Junction Plakoglobin (JUP), detected in DR tissue-derived EVs and DR urinary EVs, but not in controls, was revealed to be a central node in many identified pathogenic pathways. Proteomic results were validated by western blot. Urinary EVs obtained from healthy donors and diabetic patient without DR did not contain JUP.ConclusionThe absence of JUP in healthy urinary EVs provide the basis for development of a novel Diabetic Retinopathy biomarker, potentially facilitating diagnosis.

Abstract The mechanisms underlying retinal development have not been completely elucidated. Extracellular vesicles (EVs) are novel essential mediators of cell-to-cell communication with emerging roles in developmental processes. Nevertheless, the identification of EVs in human retinal tissue, characterization of their cargo, and analysis of their potential role in retina development has not been accomplished. Three-dimensional retinal tissue derived from human induced pluripotent stem cells (hiPSC) provide an ideal developmental system to achieve this goal. Here we report that hiPSC-derived retinal organoids release exosomes and microvesicles with small noncoding RNA cargo. EV miRNA cargo-predicted targetome correlates with Gene Ontology (GO) pathways involved in mechanisms of retinogenesis relevant to specific developmental stages corresponding to hallmarks of native human retina development. Furthermore, uptake of EVs by human retinal progenitor cells leads to changes in gene expression correlated with EV miRNA cargo predicted gene targets, and mechanisms involved in retinal development, ganglion cell and photoreceptor differentiation and function.

The implantation of oligodendrocyte precursor cells may be a useful therapeutic strategy for targeting remyelination. However, it is yet to be established how these cells behave after implantation and whether they retain the capacity to proliferate or differentiate into myelin-forming oligodendrocytes. One essential issue is the creation of administration protocols and determining which factors need to be well established. There is controversy around whether these cells may be implanted simultaneously with corticosteroid treatment, which is widely used in many clinical situations. This study assesses the influence of corticosteroids on the capacity for proliferation and differentiation and the survival of human oligodendroglioma cells. Our findings show that corticosteroids reduce the capacity of these cells to proliferate and to differentiate into oligodendrocytes and decrease cell survival. Thus, their effect does not favour remyelination; this is consistent with the results of studies with rodent cells. In conclusion, protocols for the administration of oligodendrocyte lineage cells with the aim of repopulating oligodendroglial niches or repairing demyelinated axons should not include corticosteroids, given the evidence that the effects of these drugs may undermine the objectives of cell transplantation.

Introduction: Cerebrospinal fluid (CSF) from amyotrophic lateral sclerosis (ALS) patients induces cytotoxic effects in in vitro cultured motor neurons. Materials and methods: We selected CSF with previously reported cytotoxic effects from 32 ALS patients. Twenty-eight adult male rats were intracerebroventricularly implanted with osmotic mini-pumps and divided into 3 groups: 9 rats injected with CSF from non-ALS patients, 15 rats injected with cytotoxic ALS-CSF, and 4 rats injected with a physiological saline solution. CSF was intracerebroventricularly and continuously infused for periods of 20 or 43 days after implantation. We conducted clinical assessments and electromyographic examinations, and histological analyses were conducted in rats euthanised 20, 45, and 82 days after surgery. Results: Immunohistochemical studies revealed tissue damage with similar characteristics to those found in the sporadic forms of ALS, such as overexpression of cystatin C, transferrin, and TDP-43 protein in the cytoplasm. The earliest changes observed seemed to play a protective role due to the overexpression of peripherin, AKTpan, AKTphospho, and metallothioneins; this expression had diminished by the time we analysed rats euthanised on day 82, when an increase in apoptosis was observed. The first cellular changes identified were activated microglia followed by astrogliosis and overexpression of GFAP and S100B proteins. Conclusion: Our data suggest that ALS could spread through CSF and that intracerebroventricular administration of cytotoxic ALS-CSF provokes changes similar to those found in sporadic forms of the disease. Resumen: Introducción: La exposición de líquido cefalorraquídeo (LCR) de pacientes con esclerosis lateral amiotrófica (ELA) induce efectos citotóxicos en cultivos celulares de neuronas motoras in vitro. Material y métodos: Se seleccionó LCR de 32 pacientes con ELA que previamente habían demostrado efectos citotóxicos. Se implantaron con minibombas osmóticas intracerebroventriculares (ICV) en 28 ratas macho adultas y se dividieron en 3 grupos: 9 ratas de LCR de pacientes no-ELA, 15 ratas de ELA-LCR citotóxico y 4 ratas de una solución salina fisiológica. El LCR se administró por vía ICV de forma continua durante periodos de 20 o 43 días. Se realizó la evaluación clínica, electromiográfica y análisis de tejidos después de sacrificio a los 20, 45 y 82 días tras la cirugía. Resultados: Los estudios inmunohistoquímicos muestran daño en los tejidos con características similares a las encontradas en formas esporádicas de ELA, tales como sobreexpresión de cistatina C, transferrina y la proteína en el TDP-43 citoplasmática. Los primeros cambios observados parecían jugar un papel protector por la sobreexpresión de periferina, panAKT, fosfoAKT y metalotioneínas; esta expresión habría disminuido al momento de analizar las ratas que se sacrificaron al día 82, en el que hay un aumento de apoptosis. Los primeros cambios celulares identificados fueron la constatación de activación de la microglía seguido por astrogliosis con sobreexpresión de GFAP y proteína S100B. Conclusiones: Nuestros datos parecen indicar que la ELA podría propagarse a través del LCR, y que la administración ICV de ELA-LCR citotóxico produce cambios similares a los encontrados en las formas esporádicas de la enfermedad. Keywords: Amyotrophic lateral sclerosis, ALS experimental model, Peripherin, TDP-43, Neurodegenerative diseases, Cytotoxicity, Palabras clave: Esclerosis lateral amiotrófica, Modelo experimental ELA, Periferina, TDP-43, Enfermedades neurodegenerativas, Citotoxicidad

Resumen: Introducción: La exposición de líquido cefalorraquídeo (LCR) de pacientes con esclerosis lateral amiotrófica (ELA) induce efectos citotóxicos en cultivos celulares de neuronas motoras in vitro. Material y métodos: Se seleccionó LCR de 32 pacientes con ELA que previamente habían demostrado efectos citotóxicos. Se implantaron con minibombas osmóticas intracerebroventriculares (ICV) en 28 ratas macho adultas y se dividieron en 3 grupos: 9 ratas de LCR de pacientes no-ELA, 15 ratas de ELA-LCR citotóxico y 4 ratas de una solución salina fisiológica. El LCR se administró por vía ICV de forma continua durante periodos de 20 o 43 días. Se realizó la evaluación clínica, electromiográfica y análisis de tejidos después de sacrificio a los 20, 45 y 82 días tras la cirugía. Resultados: Los estudios inmunohistoquímicos muestran daño en los tejidos con características similares a las encontradas en formas esporádicas de ELA, tales como sobre expresión de cistatina C, transferrina y la proteína en el TDP-43 citoplasmática. Los primeros cambios observados parecían jugar un papel protector por la sobreexpresión de periferina, panAKT, fosfoAKT y metalotioneínas; esta expresión habría disminuido al momento de analizar las ratas que se sacrificaron al día 82, en el que hay un aumento de apoptosis. Los primeros cambios celulares identificados fueron la constatación de activación de la microglía seguido por astrogliosis con sobreexpresión de GFAP y proteína S100B. Conclusiones: Nuestros datos parecen indicar que la ELA podría propagarse a través del LCR, y que la administración ICV de ELA-LCR citotóxico produce cambios similares a los encontrados en las formas esporádicas de la enfermedad. Abstract: Introduction: Cerebrospinal fluid (CSF) from amyotrophic lateral sclerosis (ALS) patients induces cytotoxic effects in in vitro cultured motor neurons. Material and methods: We selected CSF with previously reported cytotoxic effects from 32 ALS patients. Twenty-eight adult male rats were intracerebroventricularly implanted with osmotic mini-pumps and divided into 3 groups: 9 rats injected with CSF from non-ALS patients, 15 rats injected with cytotoxic ALS-CSF, and 4 rats injected with a physiological saline solution. CSF was intracerebroventricularly and continuously infused for periods of 20 or 43 days after implantation. We conducted clinical assessments and electromyographic examinations, and histological analyses were conducted in rats euthanised 20, 45, and 82 days after surgery. Results: Immunohistochemical studies revealed tissue damage with similar characteristics to those found in the sporadic forms of ALS, such as overexpression of cystatin C, transferrin, and TDP-43 protein in the cytoplasm. The earliest changes observed seemed to play a protective role due to the overexpression of peripherin, AKTpan, AKTphospho, and metallothioneins; this expression had diminished by the time we analysed rats euthanised on day 82, when an increase in apoptosis was observed. The first cellular changes identified were activated microglia followed by astrogliosis and overexpression of GFAP and S100B proteins. Conclusion: Our data suggest that ALS could spread through CSF and that intracerebroventricular administration of cytotoxic ALS-CSF provokes changes similar to those found in sporadic forms of the disease. Palabras clave: Esclerosis lateral amiotrófica, Modelo experimental ELA, Periferina, TDP-43, Enfermedades neurodegenerativas, Citotoxicidad, Keywords: Amyotrophic lateral sclerosis, ALS experimental model, Peripherin, TDP-43, Neurodegenerative diseases, Cytotoxicity

Zur Bestimmung des Abbaus von Kohlenhydraten bei der anaeroben Fermentation wurde die photometrische Anthron-Methode angepasst und für die Modellsubstrate Glucose (wasserlöslich), Mais-Stärke und mikrokristalline Cellulose (beide wasserunlöslich) in mesophilen BatchVersuchen überprüft. Für die Bestimmung des Gehalts an löslichen Kohlenhydraten wurde die Probe mit einem Spritzenaufsatzfilter (Material PTFE) auf < 0,45 µm filtriert, um alle Partikel inklusive der partikulären Kohlenhydrate abzutrennen. Die erhaltene Lösung reagierte dann während 8 Minuten mit der Anthronlösung in einem zuvor auf 100 °C temperierten Heizblock. Eine rasche Abkühlung im Eiswasser auf ca. 4 °C Probentemperatur (Zeitdauer fünf Minuten) erwies sich als sinnvoll, um die Farbreaktion zu stoppen und insbesondere bei größerem Probenumfang mit bedingt höherem Zeitaufwand wiederholbare Ergebnisse zu erzielen. Vor der Messung der Extinktion am Photometer wurde die Probe bei Raumtemperatur für ca. 30 Minuten temperiert. Eine Verdünnung der Probe war notwendig, einerseits zur Reduzierung des Farbhintergrunds, andererseits um den vorgegebenen Messbereich (λ = 625 nm) von 10 bis 100 mg Glucoseäquivalent/l einzustellen. Der Gesamtkohlenhydratanteil, der sich aus der Gesamtheit der gelösten sowie auch partikulären Fraktion zusammensetzt, wurde über eine zusätzliche, vor der Anthron-Reaktion durchgeführten Vorhydrolyse mit circa 50%igem Säureanteil an H2SO4 bei 100 °C und 8 Minuten Reaktionsdauer bestimmt. Dieser separate Bestimmungsschritt, nachfolgend indirekte Methode genannt, ist notwendig, um den Anteil unlöslicher bzw. polymerer Kohlenhydrate einer Probe zu bestimmen. Die Berechnung erfolgt über Differenzbildung aus dem Gesamt- zum löslichen Kohlenhydratanteil (= partikulärer Kohlenhydratanteil).

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture “what the community needs in a tool”. Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Resumen: Introducción: Los efectos neurotóxicos del líquido cefalorraquídeo (LCR) procedentes de pacientes con esclerosis lateral amiotrófica (ELA) han sido descritos por varios autores que han atribuido esta neurotoxicidad al efecto de glutamato del LCR-ELA. Material y métodos: Se han expuesto cultivos de neuronas embrionarias corticales de rata con un incubación de 24 h y el LCR procedente de pacientes con ELA, valorando las alteraciones celulares a través de microscopía óptica en comparación con aquellas que produce con 100 mM de glutamato y la inmunohistoquímica de caspasa-3, TNF y periferina a través de microscopia confocal. Resultados: En el cultivo expuesto a LCR-ELA se observan células con fragmentación del núcleo con escasa o nula modificación estructural de los organelos citoplasmáticos y mantenimiento de la membrana plasmática, lo que no ocurre con la exposición a glutamato. Se observa un aumento de caspasa-3 y de TNFα y un incremento de periferina que co-localiza con caspasa-3 pero no con TNFα, lo hace sugerir que puede tener un papel precoz en el desarrollo de la apoptosis. Conclusiones: La citotoxicidad por LCR-ELA no se relaciona con el glutamato, que provoca una afectación nuclear precoz sin alteración de la membrana citoplasmática produciendo una apoptosis citoplasmática que conlleva un incremento de caspasa-3 que co-localiza con sobreexpresión anómala de periferina. Abstract: Introduction: The neurotoxic effects of cerebrospinal fluid (CSF) from patients with amyotrophic lateral sclerosis (ALS) have been reported by various authors who have attributed this neurotoxicity to the glutamate in CSF-ALS. Material and methods: Cultures of rat embryonic cortical neurons were exposed to CSF from ALS patients during an incubation period of 24 hours. Optical microscopy was used to compare cellular changes to those elicited by exposure to 100 μm glutamate, and confocal microscopy was used to evaluate immunohistochemistry for caspase-3, TNFα, and peripherin. Results: In the culture exposed to CSF-ALS, we observed cells with nuclear fragmentation and scarce or null structural modifications to the cytoplasmic organelles or to plasma membrane maintenance. This did not occur in the culture exposed to glutamate. The culture exposed to CSF-ALS also demonstrated increases in caspase-3, TNFα, and in peripherin co-locating with caspase-3, but not with TNFα, suggesting that TNFα may play an early role in the process of apoptosis. Conclusions: CFS-ALS cytotoxicity is not related to glutamate. It initially affects the nucleus without altering the cytoplasmic membrane. It causes cytoplasmic apoptosis that involves an increase in caspase-3 co-located with peripherin, which is also overexpressed. Palabras clave: Esclerosis lateral amiotrófica, Líquido cefalorraquídeo, Neurotoxicidad, Periferina, Caspasa-3, TNFα, Glutamato, Keywords: Amyotrophic lateral sclerosis, Cerebrospinal fluid, Neurotoxicity, Peripherin, Caspase-3, TNFα, Glutamate

Introduction: The neurotoxic effects of cerebrospinal fluid (CSF) from patients with amyotrophic lateral sclerosis (ALS) have been reported by various authors who have attributed this neurotoxicity to the glutamate in CSF-ALS. Materials and methods: Cultures of rat embryonic cortical neurons were exposed to CSF from ALS patients during an incubation period of 24 hours. Optical microscopy was used to compare cellular changes to those elicited by exposure to 100 μm glutamate, and confocal microscopy was used to evaluate immunohistochemistry for caspase-3, TNFα, and peripherin. Results: In the culture exposed to CSF-ALS, we observed cells with nuclear fragmentation and scarce or null structural modifications to the cytoplasmic organelles or to plasma membrane maintenance. This did not occur in the culture exposed to glutamate. The culture exposed to CSF-ALS also demonstrated increases in caspase-3, TNFα, and in peripherin co-locating with caspase-3, but not with TNFα, suggesting that TNFα may play an early role in the process of apoptosis. Conclusions: CFS-ALS cytotoxicity is not related to glutamate. It initially affects the nucleus without altering the cytoplasmic membrane. It causes cytoplasmic apoptosis that involves an increase in caspase-3 co-located with peripherin, which is also overexpressed. Resumen: Introducción: Los efectos neurotóxicos del líquido cefalorraquídeo (LCR) procedentes de pacientes con esclerosis lateral amiotrófica (ELA) han sido descritos por varios autores que han atribuido esta neurotoxicidad al efecto de glutamato del LCR-ELA. Material y métodos: Se han expuesto cultivos de neuronas embrionarias corticales de rata con un incubación de 24 h y el LCR procedente de pacientes con ELA, valorando las alteraciones celulares a través de microscopía óptica en comparación con aquellas que produce con 100 mM de glutamato y la inmunohistoquímica de caspasa-3, TNF y periferina a través de microscopia confocal. Resultados: En el cultivo expuesto a LCR-ELA se observan células con fragmentación del núcleo con escasa o nula modificación estructural de los organelos citoplasmáticos y mantenimiento de la membrana plasmática, lo que no ocurre con la exposición a glutamato. Se observa un aumento de caspasa-3 y de TNFα y un incremento de periferina que co-localiza con caspasa-3 pero no con TNFα, lo hace sugerir que puede tener un papel precoz en el desarrollo de la apoptosis. Conclusiones: La citotoxicidad por LCR-ELA no se relaciona con el glutamato, que provoca una afectación nuclear precoz sin alteración de la membrana citoplasmática produciendo una apoptosis citoplasmática que conlleva un incremento de caspasa-3 que co-localiza con sobreexpresión anómala de periferina. Keywords: Amyotrophic lateral sclerosis, Cerebrospinal fluid, Neurotoxicity, Peripherin, Caspase-3, TNFα, Glutamate, Palabras clave: Esclerosis lateral amiotrófica, Líquido cefalorraquídeo, Neurotoxicidad, Periferina, Caspasa-3, TNFα, Glutamato