Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft

Hospital admission and emergency care attendance risk for SARS-CoV-2 delta (B.1.617.2) compared with alpha (B.1.1.7) variants of concern: a cohort study

Background: The SARS-CoV-2 delta (B.1.617.2) variant was first detected in England in March, 2021. It has since rapidly become the predominant lineage, owing to high transmissibility. It is suspected that the delta variant is associated with more severe disease than the previously dominant alpha (B.1.1.7) variant. We aimed to characterise the severity of the delta variant compared with the alpha variant by determining the relative risk of hospital attendance outcomes. Methods: This cohort study was done among all patients with COVID-19 in England between March 29 and May 23, 2021, who were identified as being infected with either the alpha or delta SARS-CoV-2 variant through whole-genome sequencing. Individual-level data on these patients were linked to routine health-care datasets on vaccination, emergency care attendance, hospital admission, and mortality (data from Public Health England's Second Generation Surveillance System and COVID-19-associated deaths dataset; the National Immunisation Management System; and NHS Digital Secondary Uses Services and Emergency Care Data Set). The risk for hospital admission and emergency care attendance were compared between patients with sequencing-confirmed delta and alpha variants for the whole cohort and by vaccination status subgroups. Stratified Cox regression was used to adjust for age, sex, ethnicity, deprivation, recent international travel, area of residence, calendar week, and vaccination status. Findings: Individual-level data on 43 338 COVID-19-positive patients (8682 with the delta variant, 34 656 with the alpha variant; median age 31 years [IQR 17–43]) were included in our analysis. 196 (2·3%) patients with the delta variant versus 764 (2·2%) patients with the alpha variant were admitted to hospital within 14 days after the specimen was taken (adjusted hazard ratio [HR] 2·26 [95% CI 1·32–3·89]). 498 (5·7%) patients with the delta variant versus 1448 (4·2%) patients with the alpha variant were admitted to hospital or attended emergency care within 14 days (adjusted HR 1·45 [1·08–1·95]). Most patients were unvaccinated (32 078 [74·0%] across both groups). The HRs for vaccinated patients with the delta variant versus the alpha variant (adjusted HR for hospital admission 1·94 [95% CI 0·47–8·05] and for hospital admission or emergency care attendance 1·58 [0·69–3·61]) were similar to the HRs for unvaccinated patients (2·32 [1·29–4·16] and 1·43 [1·04–1·97]; p=0·82 for both) but the precision for the vaccinated subgroup was low. Interpretation: This large national study found a higher hospital admission or emergency care attendance risk for patients with COVID-19 infected with the delta variant compared with the alpha variant. Results suggest that outbreaks of the delta variant in unvaccinated populations might lead to a greater burden on health-care services than the alpha variant. Funding: Medical Research Council; UK Research and Innovation; Department of Health and Social Care; and National Institute for Health Research

Changes in symptomatology, reinfection, and transmissibility associated with the SARS-CoV-2 variant B.1.1.7: an ecological study

Background The SARS-CoV-2 variant B.1.1.7 was first identified in December, 2020, in England. We aimed to investigate whether increases in the proportion of infections with this variant are associated with differences in symptoms or disease course, reinfection rates, or transmissibility. Methods We did an ecological study to examine the association between the regional proportion of infections with the SARS-CoV-2 B.1.1.7 variant and reported symptoms, disease course, rates of reinfection, and transmissibility. Data on types and duration of symptoms were obtained from longitudinal reports from users of the COVID Symptom Study app who reported a positive test for COVID-19 between Sept 28 and Dec 27, 2020 (during which the prevalence of B.1.1.7 increased most notably in parts of the UK). From this dataset, we also estimated the frequency of possible reinfection, defined as the presence of two reported positive tests separated by more than 90 days with a period of reporting no symptoms for more than 7 days before the second positive test. The proportion of SARS-CoV-2 infections with the B.1.1.7 variant across the UK was estimated with use of genomic data from the COVID-19 Genomics UK Consortium and data from Public Health England on spike-gene target failure (a non-specific indicator of the B.1.1.7 variant) in community cases in England. We used linear regression to examine the association between reported symptoms and proportion of B.1.1.7. We assessed the Spearman correlation between the proportion of B.1.1.7 cases and number of reinfections over time, and between the number of positive tests and reinfections. We estimated incidence for B.1.1.7 and previous variants, and compared the effective reproduction number, Rt, for the two incidence estimates. Findings From Sept 28 to Dec 27, 2020, positive COVID-19 tests were reported by 36 920 COVID Symptom Study app users whose region was known and who reported as healthy on app sign-up. We found no changes in reported symptoms or disease duration associated with B.1.1.7. For the same period, possible reinfections were identified in 249 (0·7% [95% CI 0·6–0·8]) of 36 509 app users who reported a positive swab test before Oct 1, 2020, but there was no evidence that the frequency of reinfections was higher for the B.1.1.7 variant than for pre-existing variants. Reinfection occurrences were more positively correlated with the overall regional rise in cases (Spearman correlation 0·56–0·69 for South East, London, and East of England) than with the regional increase in the proportion of infections with the B.1.1.7 variant (Spearman correlation 0·38–0·56 in the same regions), suggesting B.1.1.7 does not substantially alter the risk of reinfection. We found a multiplicative increase in the Rt of B.1.1.7 by a factor of 1·35 (95% CI 1·02–1·69) relative to pre-existing variants. However, Rt fell below 1 during regional and national lockdowns, even in regions with high proportions of infections with the B.1.1.7 variant. Interpretation The lack of change in symptoms identified in this study indicates that existing testing and surveillance infrastructure do not need to change specifically for the B.1.1.7 variant. In addition, given that there was no apparent increase in the reinfection rate, vaccines are likely to remain effective against the B.1.1.7 variant. Funding Zoe Global, Department of Health (UK), Wellcome Trust, Engineering and Physical Sciences Research Council (UK), National Institute for Health Research (UK), Medical Research Council (UK), Alzheimer's Society

Genomic assessment of quarantine measures to prevent SARS-CoV-2 importation and transmission

Mitigation of SARS-CoV-2 transmission from international travel is a priority. We evaluated the effectiveness of travellers being required to quarantine for 14-days on return to England in Summer 2020. We identified 4,207 travel-related SARS-CoV-2 cases and their contacts, and identified 827 associated SARS-CoV-2 genomes. Overall, quarantine was associated with a lower rate of contacts, and the impact of quarantine was greatest in the 16–20 age-group. 186 SARS-CoV-2 genomes were sufficiently unique to identify travel-related clusters. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to countries with no quarantine requirement. This difference was explained by fewer importation events per identified genome for these cases, as opposed to fewer onward contacts per case. Overall, our study demonstrates that a 14-day quarantine period reduces, but does not completely eliminate, the onward transmission of imported cases, mainly by dissuading travel to countries with a quarantine requirement

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

Genomic epidemiology of SARS-CoV-2 in a UK university identifies dynamics of transmission

AbstractUnderstanding SARS-CoV-2 transmission in higher education settings is important to limit spread between students, and into at-risk populations. In this study, we sequenced 482 SARS-CoV-2 isolates from the University of Cambridge from 5 October to 6 December 2020. We perform a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. We observe limited viral introductions into the university; the majority of student cases were linked to a single genetic cluster, likely following social gatherings at a venue outside the university. We identify considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and following a national lockdown. Transmission clusters were largely segregated within the university or the community. Our study highlights key determinants of SARS-CoV-2 transmission and effective interventions in a higher education setting that will inform public health policy during pandemics.</jats:p

Etude des propriétés enzymatiques et du rôle dans la biogénèse des ribosomes de la protéine humaine p120, marqueur de l'évolution tumorale, et de Nop2p son homologue chez Saccharomyces cerevisiae

The protein p120 is a proliferation marker that was originally identified in human tumor cells. The yeast homologue of p120, nucleolar protein Nop2p, was shown to be essential for viability of yeast. Alignment of Nop2p with human p120 points out the evolutionary conservation of a large domain that contains a SAM-binding motif and a protein domain having potential RNA: m5C-méthyltransférase activity. By in vitro methylation experiments and a sodium bisulfite approach, we show that Nop2p is a RNA: m5C-methyltransferase specific for 25S rRNA nucleotide 2870. In parallel, we succeeded to complement Nop2-deficient yeasts by human p120 and studied the importance of different sequence and structural domains of Nop2 and p120 for rRNA processing in vivo. In complementation assays, the C-terminal domain of Nop2 was essential for cell viability, whereas the absence of the N-terminal domain led to slow growth phenotype. Chimeric proteins formed by Nop2 and p120 fragments efficiently support growth if the N-terminal domain of Nop2 was present. In the absence of Nop2 or p120, the C1/C2 cleavages required to generate 5.8S and 25S rRNA were strongly reduced. The reduction of p120 content in human cells by specific siRNA leads to decreased 28S rRNA accumulation, supporting the idea on the implication of p120 in human pre-rRNA processingLa protéine p120, marqueur de prolifération, a été initialement identifiée dans les cellules humaines tumorales. Son homologue chez la levure, la protéine Nop2, est essentielle à la croissance et à la viabilité des cellules. La délétion du gène NOP2 bloque la maturation du pré-ARNr 27S en ARNr matures. Les protéines p120 et Nop2 possèdent des motifs de la famille des ARN : m5C-méthyltransférase. Dans un premier temps, j'ai démontré l'activité ARN : m5C-méthyltransférase de Nop2p par des expériences de méthylation in vitro en présence de S-adénosylméthionine tritiée. En parallèle, des expériences menées en collaboration avec une équipe allemande nous ont permis de localiser la méthylation catalysée par Nop2p à la position 2870 de l'ARNr 25S. Par la suite, j'ai étudié l'implication de Nop2p et de p120 dans la biogenèse de la sous-unité 60S du ribosome par plusieurs approches. Des expériences de complémentation dans une souche de levure [delta]NOP2 par la protéine humaine p120 et des protéines hybrides Nop2p-p120 ont permis de montrer que le domaine catalytique de p120, en présence du domaine N-terminal de Nop2p, permet une biogenèse normale des ribosomes chez S.cerevisiae. Parallèlement, des expériences d'ARN interférence dirigées contre p120 ont permis de montrer que, lorsque le taux de protéine p120 est diminué de 90% dans des cellules HEK293, la synthèse de l'ARNr mature 28S est ralentie. L'ensemble de ces données montrent que les protéines p120 et Nop2 appartiennent à la famille des protéines pré-ribosomiques essentielles au processus de maturation du pré-ARNr et semblent nécessaires à la structuration des pré-ARNr indispensable à leur maturatio

Characterization of the enzymatic activity and the role in ribosomes biogenesis of human nucleolar protein p120 and yeast homolog Nop2p

La protéine p120, marqueur de prolifération, a été initialement identifiée dans les cellules humaines tumorales. Son homologue chez la levure, la protéine Nop2, est essentielle à la croissance et à la viabilité des cellules. La délétion du gène NOP2 bloque la maturation du pré-ARNr 27S en ARNr matures. Les protéines p120 et Nop2 possèdent des motifs de la famille des ARN : m5C-méthyltransférase. Dans un premier temps, j'ai démontré l'activité ARN : m5C-méthyltransférase de Nop2p par des expériences de méthylation in vitro en présence de S-adénosylméthionine tritiée. En parallèle, des expériences menées en collaboration avec une équipe allemande nous ont permis de localiser la méthylation catalysée par Nop2p à la position 2870 de l'ARNr 25S. Par la suite, j'ai étudié l'implication de Nop2p et de p120 dans la biogenèse de la sous-unité 60S du ribosome par plusieurs approches. Des expériences de complémentation dans une souche de levure [delta]NOP2 par la protéine humaine p120 et des protéines hybrides Nop2p-p120 ont permis de montrer que le domaine catalytique de p120, en présence du domaine N-terminal de Nop2p, permet une biogenèse normale des ribosomes chez S.cerevisiae. Parallèlement, des expériences d'ARN interférence dirigées contre p120 ont permis de montrer que, lorsque le taux de protéine p120 est diminué de 90% dans des cellules HEK293, la synthèse de l'ARNr mature 28S est ralentie. L'ensemble de ces données montrent que les protéines p120 et Nop2 appartiennent à la famille des protéines pré-ribosomiques essentielles au processus de maturation du pré-ARNr et semblent nécessaires à la structuration des pré-ARNr indispensable à leur maturationThe protein p120 is a proliferation marker that was originally identified in human tumor cells. The yeast homologue of p120, nucleolar protein Nop2p, was shown to be essential for viability of yeast. Alignment of Nop2p with human p120 points out the evolutionary conservation of a large domain that contains a SAM-binding motif and a protein domain having potential RNA: m5C-méthyltransférase activity. By in vitro methylation experiments and a sodium bisulfite approach, we show that Nop2p is a RNA: m5C-methyltransferase specific for 25S rRNA nucleotide 2870. In parallel, we succeeded to complement Nop2-deficient yeasts by human p120 and studied the importance of different sequence and structural domains of Nop2 and p120 for rRNA processing in vivo. In complementation assays, the C-terminal domain of Nop2 was essential for cell viability, whereas the absence of the N-terminal domain led to slow growth phenotype. Chimeric proteins formed by Nop2 and p120 fragments efficiently support growth if the N-terminal domain of Nop2 was present. In the absence of Nop2 or p120, the C1/C2 cleavages required to generate 5.8S and 25S rRNA were strongly reduced. The reduction of p120 content in human cells by specific siRNA leads to decreased 28S rRNA accumulation, supporting the idea on the implication of p120 in human pre-rRNA processin

"Older adults, video games and the European francophone press: a mixed-methods discourse analysis"

International audienceOver the past few years, the French mainstream press has paid more and more attention to "silver gamers", adults over sixty who play video games. This article investigates the discursive and normative paradigms that underlie the unexpected enthusiasm of the French mainstream press for older adults who play video games. We use mixed methods on a corpus of French, Swiss and Belgian articles that mention both older people and video games. First, we produce topics, that is, sets of words related by their meanings and identified with a Bayesian statistical algorithm. Second, we cross the topic model results with a discursive analysis of selected articles. We preface the topic modeling's conclusions with a discussion of the representations of older people and video games in European French-language mainstream media. Our analysis explores how the press coverage of older people who play video games simultaneously erases moral panic about video games and reinforces the "successful ageing" discourse