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Funder: Community Jameel Imperial College Covid‐19 Excellence Fund, Funder: NIHR Imperial College Healthcare NHS Trust Biomedical Research Centre, Funder: North West London Pathology Research Grant Commission, SARS-CoV-2, the causative agent of COVID-19, typically manifests as a respiratory illness, although extrapulmonary involvement, such as in the gastrointestinal tract and nervous system, as well as frequent thrombotic events, are increasingly recognised. How this maps onto SARS-CoV-2 organ tropism at the histological level, however, remains unclear. Here, we perform a comprehensive validation of a monoclonal antibody against the SARS-CoV-2 nucleocapsid protein (NP) followed by systematic multisystem organ immunohistochemistry analysis of the viral cellular tropism in tissue from 36 patients, 16 postmortem cases and 16 biopsies with polymerase chain reaction (PCR)-confirmed SARS-CoV-2 status from the peaks of the pandemic in 2020 and four pre-COVID postmortem controls. SARS-CoV-2 anti-NP staining in the postmortem cases revealed broad multiorgan involvement of the respiratory, digestive, haematopoietic, genitourinary and nervous systems, with a typical pattern of staining characterised by punctate paranuclear and apical cytoplasmic labelling. The average time from symptom onset to time of death was shorter in positively versus negatively stained postmortem cases (mean = 10.3 days versus mean = 20.3 days, p = 0.0416, with no cases showing definitive staining if the interval exceeded 15 days). One striking finding was the widespread presence of SARS-CoV-2 NP in neurons of the myenteric plexus, a site of high ACE2 expression, the entry receptor for SARS-CoV-2, and one of the earliest affected cells in Parkinson's disease. In the bone marrow, we observed viral SARS-CoV-2 NP within megakaryocytes, key cells in platelet production and thrombus formation. In 15 tracheal biopsies performed in patients requiring ventilation, there was a near complete concordance between immunohistochemistry and PCR swab results. Going forward, our findings have relevance to correlating clinical symptoms with the organ tropism of SARS-CoV-2 in contemporary cases as well as providing insights into potential long-term complications of COVID-19. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Barrel cortex integrates contra- and ipsilateral whiskers’ inputs. While contralateral inputs depend on the thalamocortical innervation, ipsilateral ones are thought to rely on callosal axons. These are more abundant in the barrel cortex region bordering with S2 and containing the row A-whiskers representation, the row lying nearest to the facial midline. Here we ask what role this callosal axonal arrangement plays in ipsilateral tactile signalling. We found that novel object exploration with ipsilateral whiskers confines c-Fos expression within the highly callosal subregion. Targeting this area within vivopatch-clamp recordings revealed neurons with uniquely strong ipsilateral responses dependent on the corpus callosum, as assessed by tetrodotoxin silencing and by optogenetic activation of the contralateral hemisphere. Still, in this area, stimulation of contra- or ipsilateral row A-whiskers evoked an indistinguishable response in some neurons, mostly located in layers 5/6, indicating their involvement in the midline representation of the whiskers’ sensory space.

Giant cell arteritis (GCA) usually presents with headache, scalp tenderness and raised inflammatory markers. GCA presenting with a clinically evident cranial nerve palsy is rare and may result in a delayed or missed diagnosis if not suspected. We present the rare case of a woman in her 70s with histologically confirmed GCA presenting with a unilateral sixth nerve palsy, which responded to treatment with high-dose oral prednisolone.

We describe a human single-nuclei transcriptomic atlas for the substantia nigra (SN), generated by sequencing approximately 17,000 nuclei from matched cortical and SN samples. We show that the common genetic risk for Parkinson’s disease (PD) is associated with dopaminergic neuron (DaN)-specific gene expression, including mitochondrial functioning, protein folding and ubiquitination pathways. We identify a distinct cell type association between PD risk and oligodendrocyte-specific gene expression. Unlike Alzheimer’s disease (AD), we find no association between PD risk and microglia or astrocytes, suggesting that neuroinflammation plays a less causal role in PD than AD. Beyond PD, we find associations between SN DaNs and GABAergic neuron gene expression and multiple neuropsychiatric disorders. Conditional analysis reveals that distinct neuropsychiatric disorders associate with distinct sets of neuron-specific genes but converge onto shared loci within oligodendrocytes and oligodendrocyte precursors. This atlas guides our aetiological understanding by associating SN cell type expression profiles with specific disease risk., The substantia nigra is important in neurological disease, particularly movement disorders. Here the authors provide a single cell transcriptomic atlas for the human substantia nigra.

Rising detrimental global environmental statistics and measurable effects have caused the sustainable building concept to gain momentum. As a result, many sustainable building rating systems and as...

In vitro neuronal cultures embodied in a closed-loop control system have been used recently to study neuronal dynamics. This allows the development of neurons in a controlled environment with the purpose of exploring the computational capabilities of such biological neural networks. Due to the intrinsic properties of in vitro neuronal cultures and how the neuronal tissue grows in them, the ways in which signals are transmitted and generated within and throughout the culture can be difficult to characterize. The neural code is formed by patterns of spikes whose properties are in essence nonlinear and non-stationary. The usual approach for this characterization has been the use of the post-stimulus time histogram (PSTH). PSTH is calculated by counting the spikes detected in each neuronal culture electrode during some time windows after a stimulus in one of the electrodes. The objective is to find pairs of electrodes where stimulation in one of the pairs produces a response in the other but not in the rest of the electrodes in other pairs. The aim of this work is to explore possible ways of extracting relevant information from the global response to culture stimulus by studying the patterns of variation over time for the firing rate, estimated from inverse inter-spike intervals, in each electrode. Machine learning methods can then be applied to distinguish the electrode being stimulated from the whole culture response, in order to obtain a better characterization of the culture and its computational capabilities so it can be useful for robotic applications.

BackgroundMultimerization is a key process in prion-like disorders such as Alzheimer’s disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau–tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers.MethodsHere, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-proximity ligation assay (tau-PLA). We then analyzedMAPTKO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold.ResultsOur novel tau-proximity ligation assay (tau-PLA) directly visualized tau–tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal inMAPTKO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large lesions.ConclusionsTau-PLA is the first method to directly visualize tau multimers both in vitro and in situ with high specificity. We find that tau multimerization appears extensively from the earliest presymptomatic Braak stages as a previously unreported type of diffuse pathology. Importantly, in our study multimerization is the earliest detectable molecular event of AD tau pathology. Our findings open a new window to the study of early tau pathology, with potential implications in early diagnosis and the design of therapeutic strategies.

Synchrony of neural activity among cortical areas arises from functional coupling between those areas. Such a strong synchrony characterises the two mouse barrel fields (BFs) when the animal is deeply anaesthetised or asleep. In these conditions, neurons in the two hemispheres depolarise (up-state) and hyperpolarise to their resting potential (down-state) in a remarkably coordinated fashion. Callosal glutamatergic axons provide a means to functionally couple supra- and infragranular neurons of the two BFs. However, little is known about their relationship with the BF grid-like architecture: Are they able to influence the activity of barrel and/or septal neurons? Are there specific barrels more sensitive to the contralateral activity? To respond to these questions, we localised and counted the BF cells positive to c-Fos (c-Fos +) resulting from a contralateral whiskers deprivation when mice were free to explore a novel environment. In layer 4, we found a greater number of c-Fos + cells in septa compared to barrels, which mainly localised in the posterior and lateral aspects of the sensory-deprived BF. To learn more about such interhemispheric recruitment, we studied the propagation of slow-oscillatory activity in anaesthetised mice. We performed whole-cell patch-clamp in the ipsilateral BF while recording LFPs in the contralateral BF. In the BF lateral region, neurons showed faster oscillatory cycles, shorter up-state duration and faster down-to-up transitions compared to neurons recorded in BF regions with a sparser c-Fos signal, suggesting the reception of extra inputs in the former. We thus propose that the lateral BF is a critical sub-region for BFs activity-coupling.

Background: Although primarily a neurodegenerative process, there is increasing awareness of peripheral disease mechanisms in Parkinson's disease. To investigate disease processes in accessible patient cells, we studied peripheral blood mononuclear cells in recently diagnosed PD patients and rapid eye movement‐sleep behavior disorder patients who have a greatly increased risk of developing PD. We hypothesized that peripheral blood mononuclear cells may recapitulate cellular pathology found in the PD brain and investigated these cells for mitochondrial dysfunction and oxidative stress. Methods: Peripheral blood mononuclear cells were isolated and studied from PD patients, rapid eye movement‐sleep behavior disorder patients and age‐ and sex‐matched control individuals from the well‐characterized Oxford Discovery cohort. All participants underwent thorough clinical assessment. Results: Initial characterization showed that PD patients had elevated levels of CD14 + monocytes and monocytes expressing C‐C motif chemokine receptor 2. Mitochondrial dysfunction and oxidative stress were increased in PD patient peripheral blood mononuclear cells, with elevated levels of mitochondrial reactive oxygen species specifically in patient monocytes. This was combined with reduced levels of the antioxidant superoxide dismutase in blood cells from PD patients and, importantly, also in rapid eye movement‐sleep behavior disorder patients. This mitochondrial dysfunction was associated with a concomitant increase in glycolysis in both PD and rapid eye movement‐sleep behavior disorder patient blood cells independent of glucose uptake or monocyte activation. Conclusions: This work demonstrates functional bioenergetic deficits in PD and rapid eye movement‐sleep behavior disorder patient blood cells during the early stages of human disease. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Introduction: Langerhan cell histiocytosis (LCH) is a rare cause of eosinophilic granulomatous inflammation that can affect multiple organ systems, mainly diagnosed in children. Hypothalamic-pituitary axis involvement is well established and is the characteristic intracranial manifestation of LCH. Patients can present with several presentations including anterior pituitary axis failure; however, the most common presentation is Diabetes Insipidus (DI). Here, we present a rare case of isolated pituitary LHC in male adult. Clinical Case: 47 years old gentleman presented with 4 week history of insidious headaches associated with persistent nausea. He also noticed drooping of his left eyelid and presented to hospital with complete ptosis. His CT head did not show any abnormalities. However finding on his MRI brain were consistent with a central skull base inflammatory process centred on the pituitary, extending cranially to the chiasm and third ventricular floor as well as laterally to the cavernous sinuses. His anterior pituitary profile was as follow: LH - 0.7 U/L (1.2-8.6 U/L), FSH - 4.2 U/L (1.3-19.3 U/L), Testosterone

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation. Funding The European Commission FP7 NeurOmics project; CHDI Foundation; the Medical Research Council UK; the Brain Research Trust; and the Guarantors of Brain.

Alpha-synuclein is a protein implicated in Parkinson's disease and thought to be one of the main pathological drivers in the disease, although it remains unclear how this protein elicits its neurotoxic effects. Recent findings indicate that the assembly of toxic oligomeric species of alpha-synuclein may be one of the key processes for the pathology and spread of the disease. The absence of a sensitive in situ detection method has hindered the study of these oligomeric species and the role they play in the human brain until recently. In this review, we assess the evidence for the toxicity and prion-like activity of oligomeric forms of alpha-synuclein and discuss the advances in our understanding of the role of alpha-synuclein in Parkinson's disease that may be brought about by the specific and sensitive detection of distinct oligomeric species in post-mortem patient brain. Finally, we discuss current approaches being taken to therapeutically target alpha-synuclein oligomers and their implications.

La presente tesis versa sobre lo que hemos de denominar a lo largo de ella como concepción institucionalista del lenguaje, presente en distintas líneas del pragmatismo lingüístico contemporáneo. En una primera aproximación, ésta es sin dudas una investigación sobre el lenguaje, pero debido a que las perspectivas teóricas utilizadas entienden el lenguaje como institución y lo ponen en relación directa e inevitablemente con los procesos que se dan en las demás instituciones, en una inspección con mayor detalle es más apropiado aseverar que es una tesis que trata sobre lenguaje e instituciones, en la que –sí, indudablemente– la mirada está puesta especialmente en el lenguaje, pero –por ello mismo– está en tensión permanente hacia las esferas institucionales en sentido amplio. El foco central de interés, entonces, pasa por el lenguaje en tanto institución que tiene un rol decisivo en la conformación de las demás instituciones y en el carácter que puede llegar a tomar aquel en relación con las propiedades conferidas por las prácticas institucionales. Hacia allí está dirigida nuestra atención y nuestros esfuerzos. El punto básico de la perspectiva dentro de la que se mueve la tesis es que el lenguaje mismo es una institución debido a que está constituido por determinadas reglas, convenciones, prácticas, etc. que posibilitan y regulan su funcionamiento y que todo hablante debe manejar para poder desempeñarse dentro de su universo. Y, por otra parte, no hay instituciones extra-lingüísticas; lo cual no quiere decir que todas las instituciones existen en el lenguaje o que lo hagan gracias a él solamente, sino que todas ellas se conforman a través suyo, necesitan de la participación del lenguaje –y de varios elementos más según sea el caso de cada una de ellas– para constituirse como tales. No todo uso del lenguaje requiere obligatoriamente de una institución determinada, fuera de la institución misma del lenguaje por supuesto, pero toda institución necesita de la institución del lenguaje. Detener la mirada en que la necesaria participación del lenguaje en las instituciones establece determinados parámetros dentro de las prácticas institucionales conlleva asimismo, de modo indefectible, prestar atención a los rasgos y usos institucionales del lenguaje, no con el objetivo de centrar el análisis exclusivamente en ellos, sino, principalmente, para tratar de precisar lo que el lenguaje abre –implica o posibilita– en dichas prácticas en tanto instancia institucional él mismo., Facultad de Humanidades y Ciencias de la Educación

Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy are neurodegenerative disorders resulting in progressive motor/cognitive deficits among other symptoms. They are characterised by stereotypical brain cell loss accompanied by the formation of proteinaceous aggregations of the protein α-synuclein (α-syn), being, therefore, termed α-synucleinopathies. Although the presence of α-syn inclusions is a common hallmark of these disorders, the exact nature of the deposited protein is specific to each disease. Different neuroanatomical regions and cellular populations manifest a differential vulnerability to the appearance of protein deposits, cell dysfunction, and cell death, leading to phenotypic diversity. The present review describes the multiple factors that contribute to the selective vulnerability in α-synucleinopathies. We explore the intrinsic cellular properties in the affected regions, including the physiological and pathophysiological roles of endogenous α-syn, the metabolic and genetic build-up of the cells and their connectivity. These factors converge with the variability of the α-syn conformational strains and their spreading capacity to dictate the phenotypic diversity and regional vulnerability of each disease. Finally, we describe the exogenous and environmental factors that potentially contribute by igniting and modulating the differential pathology in α-synucleinopathies. In conclusion, we think that it is the confluence of this disruption of the cellular metabolic state and α-syn structural equilibrium through the anatomical connectivity which appears to initiate cascades of pathological processes triggered by genetic, environmental, or stochastic events that result in the “death by a thousand cuts” profile of α-synucleinopathies. Electronic supplementary material The online version of this article (10.1007/s00401-019-02010-2) contains supplementary material, which is available to authorized users.

Alpha-synuclein oligomers are thought to be toxic mediators of Parkinson's disease and other alpha-synucleinopathies, but their histological detection in situ in diseased brain has been a challenge in the field for some time. Here we describe a method, the alpha-synuclein proximity ligation assay (AS-PLA), to detect alpha-synuclein oligomers in paraffin-embedded brain sections. Using AS-PLA previously unobserved alpha-synuclein oligomeric pathology is revealed.

The management of different industrial by-products, such as recycled aggregates from construction and demolition waste and alumina by-products, as well as the reduction of landfill deposits by incorporating these products in a second life cycle, were the focus of this work. The aim of this study was to demonstrate the technical viability of using these waste and by-product as a material for road pavement base layers. For this purpose, a real-scale application was carried out, and the behavior of three types of materials, applied on a section of an experimental road under real vehicle traffic conditions, was studied and compared. Three materials were used in these sections applied in the road sub-bases. First, a control material composed of a type of artificial gravel was used to be compared with the rest of materials; the second material was composed of recycled aggregates, and the third was composed of a mix of recycled aggregates and alumina waste. The results concluded that the effectiveness of the sections built using recycled aggregates and alumina waste was very positive and similar those constructed using natural aggregates., Isolux-Corsan Construction

The application of mixed recycled aggregates (MRA) and recycled concrete aggregates (RCA) in road works has been studied intensively over the last two decades. One of the properties that has not been studied enough is the self-cementing capacity of recycled aggregates (RA), which favours improving the bearing capacity of compacted recycled aggregates. This research aims to analyse the variables that influence the self-cementing of recycled aggregates, both concrete and mixed. Elaborating recycled aggregate is carried out by crushing the material in a treatment plant which favours self-cementing. In this research part of the recycled material is crushed again in the laboratory in order to produce a better self-cementing process when the material is crushed and used in a short period of time. One of the causes for which the bearing capacity is improved is the appearance of old unhydrated cement particles, which can favour residual setting in the medium-term. In this work two series with different treatments, based on crushing and sieving part of the material, were studied to determine the influence of these changes on the bearing capacity: a series called All-in-one (AO) with a particle size of 0–20 mm, which includes coarse fraction (C) and fine fraction (F) and a second series, called Fine (F), which only include fine fraction with a particle size of 0–4 mm. In both series different samples of RA were processed by sieving, crushing and compaction in different ways. In addition, natural aggregate samples were studied to compare with recycled ones. The results show that the influence of the treatment in the material, mainly crushing and fresh compaction, as well as particle size, significantly improves the loading capacity of recycled aggregates.

Gastrointestinal (GI) alpha-synuclein (ASN) detection may represent a clinically useful biomarker of Parkinson’s disease (PD), but this has been challenged by conflicting results of recent studies employing different immunohistochemical (IHC) methods and reporting diverse morphological patterns with variable biological interpretation. To increase sensitivity and specificity, we applied three different techniques to detect different possible conformations of ASN in GI tissue derived from biopsies of the GI tract, which were obtained from a longitudinally followed, clinically well-characterized cohort of PD subjects and healthy controls (HC) (Oxford Discovery study). With IHC, we used antibodies reactive for total (T-ASN-Abs), phosphorylated (P-ASN-Abs) and oligomeric (O-ASN-Abs) ASN; with the ASN Proximity Ligation Assay (AS-PLA), we targeted oligomeric ASN species specifically; finally, with the Paraffin-Embedded Tissue Blot (PET-Blot) we aimed to detect fibrillary conformations of ASN specifically. Optimisation and validation of the PET-Blot and PLA techniques was carried out with studies on brain tissue from subjects with ASN pathology, and these experiments were used to gain insight into morphology and distribution of different conformational variants of ASN in the brain of subjects with Lewy pathology. We specified all the detected morphological staining patterns with each technique interpreting them as pathologic or non-specific. Correlation to clinical symptoms was assessed to investigate the potential predictive or diagnostic value of specific staining patterns as biomarkers. A total of 163 GI tissue blocks were collected from 51 PD patients (113 blocks) and 21 healthy controls (50 blocks). In 31 PD patients, GI biopsies had been taken before PD diagnosis (Prodromal PD group); while in 20 PD patients biopsies were obtained after PD diagnosis (Manifest PD group). The majority of these tissues blocks were from large intestine (62%), followed by small intestine (21%), stomach (10%) and oesophagus (7%). With IHC, four ASN staining patterns were detected in GI tissue (Neuritic, Ganglionic, Epithelial, and Cellular), while two distinct staining patterns were detected with AS-PLA (cellular and diffuse signal) and with AS-PET-Blot (ASN-localised and peri-crypt signal). The level of agreement between different techniques was generally low, and no single technique or staining pattern was able to reliably distinguish PD patients (Prodromal or Manifest) from HC. Overall, our study suggests that even specific detection of ASN conformational variants currently considered pathologic was not adequate for the prediction of PD. Future studies with these or other novel techniques focusing on the upper part of the GI tract could overcome current limitations in sensitivity and specificity.

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of ‘sporadic’ cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.

The transactive response DNA binding protein (TDP-43) is a major component of the characteristic neuronal cytoplasmic inclusions seen in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Furthermore, pathogenic mutations in the gene encoding TDP-43, TARDBP, are found in sporadic and familial ALS cases. To study the molecular mechanisms of cellular toxicity due to TDP-43 mutations we generated a novel in vitro cellular model using a fluorescently tagged human genomic TARDBP locus carrying one of two ALS-associated mutations, A382T or M337V, which were used to generate site-specific bacterial artificial chromosome (BAC) human stable cell lines and BAC transgenic mice. In cell lines and primary motor neurons in culture, TDP-M337V mislocalized to the cytoplasm more frequently than wild-type TDP (wt-TDP) and TDP-A382T, an effect potentiated by oxidative stress. Expression of mutant TDP-M337V correlated with increased apoptosis detected by cleaved caspase-3 staining. Cells expressing mislocalized TDP-M337V spontaneously developed cytoplasmic aggregates, while for TDP-A382T aggregates were only revealed after endoplasmic reticulum (ER) stress induced by the calcium-modifying drug thapsigargin. Lowering Ca(2+) concentration in the ER of wt-TDP cells partially recapitulated the effect of pathogenic mutations by increasing TDP-43 cytoplasmic mislocalization, suggesting Ca(2+) dysregulation as a potential mediator of pathology through alterations in Bcl-2 protein levels. Ca(2+) signaling from the ER was impaired in immortalized cells and primary neurons carrying TDP-43 mutations, with a 50% reduction in the levels of luminal ER Ca(2+) stores content and delayed Ca(2+) release compared with cells carrying wt-TDP. The deficits in Ca(2+) release in human cells correlated with the upregulation of Bcl-2 and siRNA-mediated knockdown of Bcl-2 restored the amplitude of Ca(2+) oscillations in TDP-M337V cells. These results suggest that TDP-43 pathogenic mutations elicit cytoplasmic mislocalization of TDP-43 and Bcl-2 mediated ER Ca(2+) signaling dysregulation.

Resumen Introduccion Las consultas de enfermeria en reumatologia (CER) son modelos organizativos asistenciales en el ambito de competencias de enfermeria. Hay diversos modelos de CER, pero no existe una definicion operacional. El objetivo del proyecto es elaborar estandares de calidad para definir y caracterizar una CER. Metodo Estudio Delphi a 2 rondas. El panel estuvo constituido por 67 expertos: reumatologos y enfermeras del Grupo de Trabajo de Enfermeria de la Sociedad Espanola de Reumatologia (SER). El cuestionario se elaboro tras revision bibliografica y experiencias de proyectos previos de la SER. El cuestionario consta de 7 apartados: consideraciones generales, estandares de estructura, de proceso, de tratamiento y seguimiento, educacion sanitaria, formacion e investigacion y calidad asistencial. Cada item se puntuo de 1 (menos importante) a 9 (mas importante) o mediante una cifra. El grado de acuerdo de los expertos se categorizo segun el coeficiente de variacion (CV) entre muy alto (CV ≤ 25%) y muy bajo (CV > 100%). Resultados El cuestionario de la segunda ronda (182 items) fue respondido por 46 panelistas (34 reumatologos y 12 enfermeras). Se obtuvo un grado de acuerdo muy importante en los estandares generales, de estructura, de proceso, de tratamiento y seguimiento, educacion sanitaria y calidad asistencial. Se encontro menor acuerdo en los estandares relacionados con el tiempo para formacion, el numero de proyectos de investigacion propios de enfermeria y de publicaciones recomendables. Conclusion Los estandares desarrollados en este estudio permitirian establecer minimos deseables de calidad de estructura, proceso, labor asistencial, investigadora y docente que se pueden utilizar para desarrollar y evaluar las CER.

Background Nursing clinics in rheumatology (NCR) are organizational models in the field of nursing care. There are various NCR models, but there is no consensus on its operational definition. Our objective is to develop quality standards to define and characterize a NCR. Method Two-round Delphi method. The panel consisted of 67 experts: rheumatologists and nurses of the nursing working group of the Spanish Society of Rheumatology (SSR). The Delphi questionnaire was developed after a literature and experience review from previous SSR projects. The questionnaire consists of 7 sections: general considerations, standards of structure, process, treatment and monitoring, health education, training and research and quality of care. Each item was scored from 1 (least important) to 9 (most important) or by assigning a number (e.g. waiting days). The degree of agreement among the experts was categorized according to the coefficient of variation (CoV) between very high (CoV≤25%) and very low (CoV>100%). Results The second round questionnaire (182 items) was answered by 46 panelists (34 rheumatologists and 12 nurses). A very important agreement was reached on the general standards of structure, process, treatment and monitoring, health education and quality of care. Less agreement was observed on standards related to training time, number of recommended nurses’ research projects and publications. Conclusion The standards developed in this study would be useful for establishing desirable quality standards of structure and process, and criteria for clinical work, research and teaching that can be used to develop and evaluate the NCRs.

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.