Exploring motor neuron degeneration in ALS - prevention by glycoursodeoxycholic acid and signaling to microglia

Dissertação para obtenção do Grau de Mestre em Genética Molecular e BiomedicinaAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects mainly motor neurons. Neuronal pathology involves glial cells, in particular microglia. However it is not known how these cells interact with motor neurons. This is particularly important because till now no therapy has shown efficacy in ALS treatment. Here, we aim (i) to evaluate the suitability of NSC-34, a hybrid cell line of neuroblastoma and motor neurons, as a model of ALS, (ii) to explore the reactivity of microglia to the neuronal released factors and (iii) to assess the efficacy of glycoursodeoxycholic acid (GUDCA), which already has shown beneficial effects in several neurodevelopmental and neurodegenerative diseases. For that, we used NSC-34 cells transfected with human superoxide dismutase 1 (hSOD1), either wild type or mutated in G93A and the microglial N9 cell line. We observed mitochondrial dysfunction, energy impairment, NO production and metalloproteinase-9 activation, with consequent apoptosis in NSC-34/hSOD1G93A cells after 4 days of differentiation, in comparison to NSC-34/hSOD1wt cells. In addition, we established GUDCA as an anti-apoptotic and anti-inflammatory agent, able to prevent all the above mentioned features. Finally, released neuronal factors induced N9 microglia apoptosis and decreased their phagocytic ability. Overall, our results emphasize NSC-34/hSOD1G93A cells as a good ALS model, highlight GUDCA as having beneficial effects and point to microglia neuroprotective failure as a determinant mechanism of ALS pathogenesis.Fundação para a Ciência e Tecnologia - PTDC/SAU-FAR/118787/2010, entitled “Are microglia a new potential target in amyotrophic lateral sclerosis (ALS)?” to DB and PEst- OE/SAU/UI4013/2011 to iMed.U

Role of glial cells as contributors to the onset and propagation of als disease

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2017Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease comprehending critical neuroinflammatory pathways, where microglia and astrocytes play a crucial role. ALS onset events are largely unknown and identification of disease steps during progression and dissemination, including the possible role of exosomes, are not clarified. Several models were used to improve data validity and deepen knowledge in ALS. We identified innovative targets to regulate microglia M1 polarization, including NLRP3-inflammasome, HMGB1 alarmin and MFG-E8/lactadherin, and demonstrated the sorting of microglial microRNA(miR) 155/miR-146a into exosomes. We showed that ALS NSC-34 MNs and their exosomes are enriched in miR-124, which are captured and drive early N9-microglia M1 polarization, with later development of M1/M2 subpopulations containing increased miR-124/miR-146a/miR-155. Moving from in vitro models to the spinal cord of the SOD1G93A ALS mouse model, we observed that depressed intercellular communication and increased miR-155 were early disease events preceding the inflammatory status of the symptomatic stage. Upregulated CX3CL1-CX3CR1, connexin-43/pannexin-1 and miR-124/miR-125b/miR-146a/miR-21 emerged as candidate targets for pathological neuroinflammation. Reduced MN number, together with aberrant/reactive astrocytes showing deficient glutamate transporters and GFAP, additionally characterized such state. Differently deregulated profiles of microglia isolated from the spinal cord of 7-day old SOD1G93A mice, after short- and long-term cultures, highlighted that cells present transient phenotypes accordingly to ALS environmental progression-stimuli and ultimately acquire a less responsive phenotype to stimulation. Astrocytes isolated from these mice promoted diverse inflammatory polarized subtypes in wild-type and ALS microglia, thus accounting to microglia heterogeneous populations, while strengthened deregulated microglia-astrocyte cross-talk as part of ALS neurodegenerative mechanisms. Our studies in ALS models reveal early promising biomarkers and novel targets to control excessive neuroinflammation and spread, including exosomal microRNAs. Due to multiple microglia phenotypes induced by MNs and their exosomes, and by reactive astrocytes, in the ALS disease, differentiated and combined therapeutic approaches may be recommended.Santa Casa da Misericórdia de Lisboa, programa de Investigação Científica em Esclerose Lateral Amiotrófica, projeto ELA-2015-002, The EU Joint Programme-Neurodegenerative Disease Research (JPND), projeto JPCOFUND/003/201

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data

NEOTROPICAL CARNIVORES: a data set on carnivore distribution in the Neotropics

Mammalian carnivores are considered a key group in maintaining ecological health and can indicate potential ecological integrity in landscapes where they occur. Carnivores also hold high conservation value and their habitat requirements can guide management and conservation plans. The order Carnivora has 84 species from 8 families in the Neotropical region: Canidae; Felidae; Mephitidae; Mustelidae; Otariidae; Phocidae; Procyonidae; and Ursidae. Herein, we include published and unpublished data on native terrestrial Neotropical carnivores (Canidae; Felidae; Mephitidae; Mustelidae; Procyonidae; and Ursidae). NEOTROPICAL CARNIVORES is a publicly available data set that includes 99,605 data entries from 35,511 unique georeferenced coordinates. Detection/non-detection and quantitative data were obtained from 1818 to 2018 by researchers, governmental agencies, non-governmental organizations, and private consultants. Data were collected using several methods including camera trapping, museum collections, roadkill, line transect, and opportunistic records. Literature (peer-reviewed and grey literature) from Portuguese, Spanish and English were incorporated in this compilation. Most of the data set consists of detection data entries (n = 79,343; 79.7%) but also includes non-detection data (n = 20,262; 20.3%). Of those, 43.3% also include count data (n = 43,151). The information available in NEOTROPICAL CARNIVORES will contribute to macroecological, ecological, and conservation questions in multiple spatio-temporal perspectives. As carnivores play key roles in trophic interactions, a better understanding of their distribution and habitat requirements are essential to establish conservation management plans and safeguard the future ecological health of Neotropical ecosystems. Our data paper, combined with other large-scale data sets, has great potential to clarify species distribution and related ecological processes within the Neotropics. There are no copyright restrictions and no restriction for using data from this data paper, as long as the data paper is cited as the source of the information used. We also request that users inform us of how they intend to use the data

NEOTROPICAL ALIEN MAMMALS: a data set of occurrence and abundance of alien mammals in the Neotropics

Biological invasion is one of the main threats to native biodiversity. For a species to become invasive, it must be voluntarily or involuntarily introduced by humans into a nonnative habitat. Mammals were among first taxa to be introduced worldwide for game, meat, and labor, yet the number of species introduced in the Neotropics remains unknown. In this data set, we make available occurrence and abundance data on mammal species that (1) transposed a geographical barrier and (2) were voluntarily or involuntarily introduced by humans into the Neotropics. Our data set is composed of 73,738 historical and current georeferenced records on alien mammal species of which around 96% correspond to occurrence data on 77 species belonging to eight orders and 26 families. Data cover 26 continental countries in the Neotropics, ranging from Mexico and its frontier regions (southern Florida and coastal-central Florida in the southeast United States) to Argentina, Paraguay, Chile, and Uruguay, and the 13 countries of Caribbean islands. Our data set also includes neotropical species (e.g., Callithrix sp., Myocastor coypus, Nasua nasua) considered alien in particular areas of Neotropics. The most numerous species in terms of records are from Bos sp. (n = 37,782), Sus scrofa (n = 6,730), and Canis familiaris (n = 10,084); 17 species were represented by only one record (e.g., Syncerus caffer, Cervus timorensis, Cervus unicolor, Canis latrans). Primates have the highest number of species in the data set (n = 20 species), partly because of uncertainties regarding taxonomic identification of the genera Callithrix, which includes the species Callithrix aurita, Callithrix flaviceps, Callithrix geoffroyi, Callithrix jacchus, Callithrix kuhlii, Callithrix penicillata, and their hybrids. This unique data set will be a valuable source of information on invasion risk assessments, biodiversity redistribution and conservation-related research. There are no copyright restrictions. Please cite this data paper when using the data in publications. We also request that researchers and teachers inform us on how they are using the data

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)