Development and characterization of a non-human primate model of disseminated synucleinopathy

IntroductionThe presence of a widespread cortical synucleinopathy is the main neuropathological hallmark underlying clinical entities such as Parkinson’s disease with dementia (PDD) and dementia with Lewy bodies (DLB). There currently is a pressing need for the development of non-human primate (NHPs) models of PDD and DLB to further overcome existing limitations in drug discovery.MethodsHere we took advantage of a retrogradely-spreading adeno-associated viral vector serotype 9 coding for the alpha-synuclein A53T mutated gene (AAV9-SynA53T) to induce a widespread synucleinopathy of cortical and subcortical territories innervating the putamen. Four weeks post-AAV deliveries animals were sacrificed and a comprehensive biodistribution study was conducted, comprising the quantification of neurons expressing alpha-synuclein, rostrocaudal distribution and their specific location.ResultsIntraputaminal deliveries of AAV9-SynA53T lead to a disseminated synucleinopathy throughout ipsi- and contralateral cerebral cortices, together with transduced neurons located in the ipsilateral caudal intralaminar nuclei and in the substantia nigra pars compacta (leading to thalamostriatal and nigrostriatal projections, respectively). Cortical afferent systems were found to be the main contributors to putaminal afferents (superior frontal and precentral gyri in particular).DiscussionObtained data extends current models of synucleinopathies in NHPs, providing a reproducible platform enabling the adequate implementation of end-stage preclinical screening of new drugs targeting alpha-synuclein

Detection of cannabinoid receptors CB1 and CB2 within basal ganglia output neurons in macaques: changes following experimental parkinsonism

Abstract Although type 1 cannabinoid receptors (CB1- Rs) are expressed abundantly throughout the brain, the presence of type 2 cannabinoid receptors (CB2Rs) in neurons is still somewhat controversial. Taking advantage of newly designed CB1R and CB2R mRNA riboprobes, we demonstrate by PCR and in situ hybridization that transcripts for both cannabinoid receptors are present within labeled pallidothalamic-projecting neurons of control and MPTP-treated macaques, whereas the expression is markedly reduced in dyskinetic animals. Moreover, an in situ proximity ligation assay was used to qualitatively assess the presence of CB1Rs and CB2Rs, as well as CB1R–CB2R heteromers within basal ganglia output neurons in all animal groups (control, parkinsonian and dyskinetic macaques). A marked reduction in the number of CB1Rs, CB2Rs and CB1R–CB2R heteromers was found in dyskinetic animals, mimicking the observed reduction in CB1R and CB2R mRNA expression levels. The fact that chronic levodopa treatment disrupted CB1R–CB2R heteromeric complexes should be taken into consideration when designing new drugs acting on cannabinoid receptor heteromers

Influencia de la limpieza del aire y de las superficies en instalaciones de envasado sobre la vida útil de productos agroalimentarios

[SPA] Algunos de los factores que más influyen sobre la calidad y la vida útil de los alimentos envasados no asépticamente son la carga microbiana inicial del producto antes de su procesado y la carga aportada durante el desarrollo de este procesado y del envasado, que tendrá como resultado la carga microbiana final del producto envasado. Esta carga microbiana dependerá en gran medida de la contaminación del aire de la sala de envasado y de las superficies de los equipos en contacto con el producto. Es por ello, que se ha llevado a cabo este estudio de caracterización y correlación de toda una serie de parámetros relativos al aire de la sala de envasado: temperatura, humedad relativa, velocidad del aire, concentración de partículas, recuento de unidades formadoras de colonias (UFC) en el aire, y la carga microbiana de las superficies de los equipos en contacto con el producto que se envasa. Esta carga microbiana del aire y de las superficies se ha relacionado con la evolución y vida útil de muestras de carne de cerdo y pavo cocido, que se han loncheado y envasado bajo dos condiciones de limpieza del aire y superficies: (i) en aire filtrado con flujo laminar (FL) (equivalente a sala blanca clase 100), y (ii) en condiciones de salas convencionales (SC) (en sala clase 100.000), y, posteriormente, se han almacenado a 4ºC. Las muestras se analizaron periódicamente hasta el final de su vida útil, determinando: la carga de enterobacterias, microflora total mesófila, microflora psicrotrofa, microflora láctica, Staphylococcus spp. Y Listeria monocytogenes; también, se determinaron los cambios organolépticos y de pH. Con la tecnología de loncheado y envasado en condiciones de flujo laminar se consiguió una mayor proporción de producto con recuentos inferiores a 10 UFC/g. En todos los casos, las muestras de carne de cerdo cocido presentaron valores inferiores a 104 UFC/g, y las de pavo cocido inferiores a 103 UFC/g. Esta significativa disminución de la carga microbiana inicial de los productos recién loncheados y envasados se tradujo en un claro aumento de su vida útil, en refrigeración a 4º C, que se amplió desde 18,6 días a 35,0 días, en el caso de la carne de cerdo cocida, y desde 32,3 a 49,2 días en el caso de la carne de pavo cocida. [ENG] The most important factors influencing the shelf life of cooked, sliced and packed meat products are the initial product quality and the factors of cutting and packaging process, mainly that’s concerning to the contamination control of air and equipment surfaces in contact with the product. This contamination will influences on the microbial load of the packaged product. Therefore the interest of the accomplishment of these operations protecting the The most important factors influencing the shelf life of cooked, sliced and packed meat products are the initial product quality and the factors of cutting and packaging process, mainly that’s concerning to the contamination control of air and equipment surfaces in contact with the product. This contamination will influences on the microbial load of the packaged product. Therefore the interest of the accomplishment of these operations protecting the product from the microbial contamination, using a cover of laminar flow with filtered air on the packaging line. In this study the shelf life of cooked ham and cooked turkey meat, sliced, packaged and stored in refrigeration (at 4º C) is analyzed, as function on the initial microbial load of the product, and the packaging technology: (i) under laminar flow (class 100 clean room) and (ii) in conventional clean room (class 100,000). The sliced and packaged product samples have been analyzed periodically until the end of their shelf life, determining the load of enterobacteriaceae, total mesophilic microflora, psychrotrophic microflora, lactic acid microflora, Staphylococcus spp. and L. monocytogenes in the samples. Also, they have been determined the changes in organoleptic properties, and pH. With the packaging technology using laminar flow a greater proportion of product just packaged with counts less than 10 UFC/g is obtained, and, in all the cases, sliced meat samples with counts less than of 104 UFC/g were observed in cooked ham, and less than 103 CFU/g were observed in cooked turkey meat. This significant diminution of microbial load in the product just packaged results in a clear increase of its shelf life at 4ºC, extending it from 18,6 days to 35 days in the case of sliced cooked ham, and from 32,3 to 49,2 days in the case of sliced cooked turkey meat

Efectividad de desinfectantes de superficies de los equipos en instalaciones de envasado de productos listos para su consumo

[SPA] Una de las principales fuentes de contaminación de los alimentos envasados listos para su consumo son las superficies de los equipos en contacto con los alimentos. En dichas superficies se desarrollan biofilms que con frecuencia son muy resistentes a los agentes desinfectantes utilizados en las plantas de fabricación. En este trabajo se han aislado diferentes microorganismos de biofilms en superficies de equipos, y se han estudiado frente a desinfectantes comerciales utilizados en diversas industrias alimentarias, en un equipo multiskan ascent y placas microtiter. Se estudiaron tres cepas bacterianas que pueden ser encontradas en las superficies de los equipos: Pseudomonas sp., Escherichia coli y Listeria monocytogenes. Los desinfectantes ensayados han sido quacide p40, dectocide yo2, dexacide b10, divosan activ, quacide mc7, limoseat, asep 150 y destocide. Respecto a las determinaciones de concentración minina inhibitoria (cmi) los desinfectantes mas efectivos resultaron ser quacide mc7, que a una concentración de 0.125 % v/v inhibió el crecimiento de todas las cepas diana y asep 150 que produjo el mismo efecto a una concentración de 0,06%. La definición de concentración mínima bactericida (cmb) se estableció como el valor de concentración del desinfectante que destruye una población de 104 cel./ml durante 20 min. Las cmb más bajas se obtuvieron con dexacide b10 con cmb = 0,125% para todas las cepas estudiadas y quacide mc7 con cmb = 0,0625% para l. Monocytogenes y e. Coli y de cmb =0,125% para Pseudomonas sp. Como conclusión se recomienda utilizar una alternancia de desinfectantes dependiendo del tipo de cepa predominante que contamine la superficie de los equipos. [ENG] The main contamination sources of packaged ready to eat products are equipment surfaces which keep in touch with them during the production process. Microbial biofilms are developed on these surfaces and frequently they are resistant to common disinfectants used in the food industry. Different spoilage microorganisms which usually are found on equipment surfaces biofilms have been tested in the present work. Their resistance against comercial disinfectants was studied in a Multiskan Ascent spectrophotometer and microtitter plates (96 wells). Three strains isolated from the equipment surfaces were tested: Pseudomonas sp., Escherichia coli y Listeria monocytogenes. Disinfectants were Quacide P40, Dectocide YO2, Dexacide B10, Divisan activ, Quacide MC7, Limoseat, Asep 150 y Destocide. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) were determined for each strain. The most effective disinfectants regarding the MIC values were Quacide MC7 and Asep 150 which inhibited the growing of the target strains at concentrations of 0,125 and 0,06 % v/v respectively. The MBC was defined as the minimal concentration at which the strain population of 104 cell/ml was killed during 20 minutes of interaction between the strain and each disinfectant. The lowest values of MBC were 0,125 % v/v with Dexacide B10 for all the target strains and 0,0625 % v/v with Quacide MC7 for L. monocytogenes and E.coli and 0,125 % v/v for Pseudomonas sp. As conclusion we recommend the use of a disinfection plan in relation to the predominant spoiling microflora and the potential risk that represents for the food product. This plan would include several disinfectants changing them each few days

Glucocerebrosidase expression patterns in the non-human primate brain

Glucocerebrosidase (GCase) is a lysosomal enzyme encoded by the GBA1 gene. Mutations in GBA1 gene lead to Gaucher’s disease, the most prevalent lysosomal storage disorder. GBA1 mutations reduce GCase activity, therefore promoting the aggregation of alphasynuclein, a common neuropathological finding underlying Parkinson’s disease (PD) and dementia with Lewy bodies. However, it is also worth noting that a direct link between GBA1 mutations and alpha-synuclein aggregation indicating cause and effect is still lacking, with limited experimental evidence to date. Bearing in mind that a number of strategies increasing GCase expression for the treatment of PD are currently under development, here we sought to analyze the baseline expression of GCase in the brain of Macaca fascicularis, which has often been considered as the gold-standard animal model of PD. Although as with other lysosomal enzymes, GCase is expected to be ubiquitously expressed, here a number of regional variations have been consistently found, together with several specific neurochemical phenotypes expressing very high levels of GCase. In this regard, the most enriched expression of GCase was constantly found in cholinergic neurons from the nucleus basalis of Meynert, dopaminergic cells in the substantia nigra pars compacta, serotoninergic neurons from the raphe nuclei, as well as in noradrenergic neurons located in the locus ceruleus. Moreover, it is also worth noting that moderate levels of expression were also found in a number of areas within the paleocortex and archicortex, such as the entorhinal cortex and the hippocampal formation, respectively

Calbindin content and differential vulnerability of midbrain efferent dopaminergic neurons in macaques

Calbindin (CB) is a calcium binding protein reported to protect dopaminergic neurons from degeneration. Although a direct link between CB content and differential vulnerability of dopaminergic neurons has long been accepted, factors other than CB have also been suggested, particularly those related to the dopamine transporter. Indeed, several studies have reported that CB levels are not causally related to the differential vulnerability of dopaminergic neurons against neurotoxins. Here we have used dual stains for tyrosine hydroxylase (TH) and CB in 3 control and 3 MPTP-treated monkeys to visualize dopaminergic neurons in the ventral tegmental area (VTA) and in the dorsal and ventral tiers of the substantia nigra pars compacta (SNcd and SNcv) co-expressing TH and CB. In control animals, the highest percentages of co-localization were found in VTA (58.2%), followed by neurons located in the SNcd (34.7%). As expected, SNcv neurons lacked CB expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA was similar to control monkeys (62.1%), whereas most of the few surviving neurons in the SNcd were CB-ir/TH-ir (88.6%). Next, we have elucidated the presence of CB within identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate nucleus and one injection of cholera toxin (CTB) into the postcommissural putamen, whereas two more monkeys were injected with CTB into the internal division of the globus pallidus (GPi). As expected, all the nigrocaudate- and nigroputamen-projecting neurons were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTPinduced degeneration, our data clearly demonstrated that these neurons are not giving rise to nigrostriatal projections and indeed CB-ir/TH-ir neurons only originate nigroextrastriatal projections

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Search for high-mass diphoton resonances in pp collisions at √s = 8 TeV with the ATLAS detector

This article describes a search for high-mass resonances decaying to a pair of photons using a sample of 20.3  fb−¹ of pp collisions at √s = 8 TeV recorded with the ATLAS detector at the Large Hadron Collider. The data are found to be in agreement with the Standard Model prediction, and limits are reported in the framework of the Randall-Sundrum model. This theory leads to the prediction of graviton states, the lightest of which could be observed at the Large Hadron Collider. A lower limit of 2.66 (1.41) TeV at 95% confidence level is set on the mass of the lightest graviton for couplings of k/M̄Pl=0.1(0.01)

Transverse momentum, rapidity, and centrality dependence of inclusive charged-particle production in √snn = 5.02 TeV p + Pb collisions measured by the ATLAS experiment

Measurements of the per-event charged-particle yield as a function of the charged-particle transverse momentum and rapidity are performed using p + Pb collision data collected by the ATLAS experiment at the LHC at a centre-of-mass energy of √snn = 5.02 TeV. Charged particles are reconstructed over pseudorapidity | η | < 2.3 and transverse momentum between 0.1 GeV and 22 GeV in a dataset corresponding to an integrated luminosity of 1 μb − 1. The results are presented in the form of charged-particle nuclear modification factors, where the p + Pb charged-particle multiplicities are compared between central and peripheral p + Pb collisions as well as to charged-particle cross sections measured in pp collisions. The p + Pb collision centrality is characterized by the total transverse energy measured in − 4.9 < η < − 3.1, which is in the direction of the outgoing lead beam. Three different estimations of the number of nucleons participating in the p + Pb collision are carried out using the Glauber model and two Glauber–Gribov colour-fluctuation extensions to the Glauber model. The values of the nuclear modification factors are found to vary significantly as a function of rapidity and transverse momentum. A broad peak is observed for all centralities and rapidities in the nuclear modification factors for charged-particle transverse momentum values around 3 GeV. The magnitude of the peak increases for more central collisions as well as rapidity ranges closer to the direction of the outgoing lead nucleus

Search for invisible particles produced in association with single-top-quarks in proton–proton collisions at √s = 8 TeV with the ATLAS detector

A search for the production of single-top-quarks in association with missing energy is performed in proton– proton collisions at a centre-of-mass energy of √s = 8 TeV with the ATLAS experiment at the large hadron collider using data collected in 2012, corresponding to an integrated luminosity of 20.3 fb−1. In this search, the W boson from the top quark is required to decay into an electron or a muon and a neutrino. No deviation from the standard model prediction is observed, and upper limits are set on the production cross-section for resonant and non-resonant production of an invisible exotic state in association with a right-handed top quark. In the case of resonant production, for a spin-0 resonance with a mass of 500 GeV, an effective coupling strength above 0.15 is excluded at 95 % confidence level for the top quark and an invisible spin-1/2 state with mass between 0 and 100 GeV. In the case of non-resonant production, an effective coupling strength above 0.2 is excluded at 95 % confidence level for the top quark and an invisible spin-1 state with mass between 0 and 657 GeV