CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Étude de la signature moléculaire des astrocytes réactifs induits par la voie JAK2-STAT3 à partir de différentes omiques : transcriptomique et protéomique

Les astrocytes sont des partenaires essentiels des neurones dans le cerveau. Ils présentent des changements morphologiques, moléculaires et fonctionnels complexes en conditions pathologiques qui restent à caractériser, tout comme leurs régulateurs. La voie de signalisation Janus kinase (JAK)2-signal transducer and activator of transcription (STAT)3 contrôle la réactivité des astrocytes, notamment dans les maladies neurodégénératives telles que la maladie de Huntington (MH), qui est caractérisée par une neurodégénérescence du striatum et l'agrégation de la Huntingtine mutante. Notre objectif est de définir les changements moléculaires régulés par JAK2-STAT3 dans les astrocytes striataux de souris et de mieux comprendre leur rôle dans la MH.Des vecteurs viraux codant pour une forme constitutivement active de JAK2 (JAK2ca) ont été développés pour activer cette voie dans les astrocytes de souris sauvages (WT) et dans un modèle murin de la MH. L'analyse histologique montre que JAK2ca induit des changements morphologiques et une surexpression des marqueurs classiques de réactivité. Une accumulation nucléaire de STAT3 est aussi observée dans le striatum de patients MH, ce qui confirme l'activation de cette voie dans les astrocytes MH.Nous avons d'abord réalisé une analyse sur puce à ADN sur des astrocytes striataux WT. JAK2ca induit des changements transcriptionnels importants dans les astrocytes, et induisant l'expression de gènes liés à l'inflammation, la signalisation des cytokines, la protéostase, le lysosome, le transport vésiculaire et le métabolisme énergétique. Pour évaluer si cette régulation transcriptionnelle se produit également dans le contexte de MH, nous avons effectué un séquençage ARN sur des astrocytes striataux de souris MH. Malgré un faible chevauchement des gènes induits par JAK2ca entre les astrocytes WT et MH, il y avait une convergence dans les fonctions associées, liées à la protéostase. Des expériences fonctionnelles complémentaires réalisées par les membres de l'équipe ont démontré que les astrocytes JAK2ca acquièrent des propriétés bénéfiques, aidant les neurones striataux à détoxifier la Huntingtine mutante grâce à leur activité protéolytique accrue et à la libération de chaperones (Abjean et al. 2023, Brain).Enfin, nous avons effectué une analyse protéomique des astrocytes JAK2ca striataux triés chez les souris WT par analyse de spectrométrie de masse. Après comparaison de six outils bioinformatiques, nous avons trouvé 87 protéines avec une abondance significativement différente, et majoritairement régulées à la baisse, entre les astrocytes GFP et JAK2ca. Ces protéines sont impliquées dans l'adhésion cellulaire, le cytosquelette et la phosphorylation oxydative. De nouveau, il y a un faible nombre de gènes/protéines communes régulées par JAK2ca, mais ces gènes/protéines sont impliqués dans des fonctions convergentes (système ubiquitine-protéasome, adhésion cellulaire et métabolisme). Plusieurs protéines induites par JAK2ca sont impliquées dans les jonctions cellulaires comme la protéine desmosomale DSP qui a été validée histologiquement.Ces analyses secondaires ont été réalisées dans DEVEA (Riquelme-Perez et al. 2023, F1000Research), un logiciel Shiny co-développé pour moi pour la visualisation, l'analyse d'expression différentielle et d'enrichissement des données omiques. Cet outil peut traiter des listes de gènes (ou de protéines) associés à des caractéristiques quantitatives, des matrices de comptages ou des objets DESeq complexes. Les analyses d'enrichissement s'appliquent à quatre organismes modèles différents.A partir de ces analyses transcriptomiques et protéomiques, nous avons découvert une signature cohérente dans les astrocytes JAK2ca du striatum qui pourrait sous-tendre les effets bénéfiques de l'activation de JAK2-STAT3 dans la MH. Ensemble, ces résultats contribuent à l'identification d'un état réactif astrocytaire spécifique qui pourrait être observé dans d'autres troubles cérébraux.Astrocytes are key partners to neurons in the brain. They undergo complex morphological, molecular and functional changes in disease. In particular, the specific molecular changes triggered in specific disease contexts and their upstream regulators are yet to be fully characterized. The Janus kinase (JAK)2-signal transducer and activator of transcription (STAT)3 signaling pathway is a central cascade controlling the reactive response of astrocytes, including in neurodegenerative disorders such as Huntington disease (HD), characterized by striatal neurodegeneration and aggregation of mutant Huntingtin. Here, we aimed to define JAK2-STAT3-dependent molecular changes in mouse striatal astrocytes and further understand their roles in HD.To activate the JAK2-STAT3 pathway, we designed viral vectors encoding a constitutively active form of JAK2 (JAK2ca) targeting astrocytes in WT mice and in a HD mouse model. Histological analysis showed that JAK2ca induced morphological changes and over-expression of standard astrocyte reactivity markers. Moreover, STAT3 nuclear accumulation was observed in the striatum of HD patient samples, supporting that this pathway is indeed activated in HD astrocytes.First, we performed microarray analysis on striatal WT astrocytes. We showed extensive transcriptional changes caused by JAK2ca in astrocytes, including an induction of genes linked to inflammation, cytokine signaling, immune reaction, proteostasis, lysosomal activity, vesicle-mediated transport and energy metabolism. To assess if such transcriptional regulation also occurred in a HD context, we performed RNA-sequencing on striatal astrocytes in HD mice. We confirmed that despite low gene overlap between genes induced by JAK2ca in WT versus HD astrocytes, there was a convergence on similar functions linked to proteostasis. Complementary functional experiments performed by team members, demonstrated that JAK2ca-astrocytes acquire beneficial properties, helping striatal neurons to handle toxic mutant Huntingtin through their enhanced proteolytic activity and release of chaperones (Abjean et al. 2023, Brain).Finally, we performed a proteomics analysis of sorted striatal JAK2ca-astrocytes in WT mice using label-free mass spectrometry analysis. After comparison of six analysis tools, we found 87 proteins with a significantly different abundance between GFP- and JAK2ca-astrocytes, with a majority of down-regulated proteins. These proteins were involved in cell adhesion, cytoskeleton and oxidative phosphorylation. There was low overlap between the mRNAs and proteins detected by microarray and mass spectrometry respectively in WT astrocytes, without large concordance on differential expressed genes/proteins. However, an overlap was found in the functions associated with the JAK2ca-regulated genes/proteins in WT astrocytes, linked to proteolytic activity through the ubiquitin-proteasome system, cell-cell adhesion and metabolic activity. Among the few proteins more abundant in WT-JAK2ca astrocytes, several of them were involved in cell junctions, including the desmosomal protein DSP, which was validated histologically.These omics analysis were mostly performed in a new Shiny user-friendly software called DEVEA (Riquelme-Perez et al. 2023, F1000Research), which I co-developed for differential expression analysis, visualization and enrichment analysis of omics data. This application tool can process simple gene (or protein) lists, gene lists with some statistical quantitative values, matrix of raw counts or complex DESeq objects. The enrichment analysis part applies to four model organisms (mouse, rat, human and A. thaliana).Using transcriptomics and proteomics analyses, we discovered a consistent signature in striatal JAK2ca-astrocytes that may underlie the beneficial effects of JAK2-STAT3 activation in HD. Together, these findings contribute to the identification of a specific astrocyte reactive state that may be observed in other brain disorders

DEVEA: an interactive shiny application for Differential Expression analysis, data Visualization and Enrichment Analysis of transcriptomics data

International audienceWe are at a time of considerable growth in the use and development of transcriptomics studies and subsequent in silico analysis. RNA sequencing is one of the most widely used approaches, now integrated in many studies. The processing of these data may typically require a noteworthy number of steps, statistical knowledge, and coding skills which is not accessible to all scientists. Despite the undeniable development of software applications over the years to address this concern, it is still possible to improve. Here we present DEVEA, an R shiny application tool developed to perform differential expression analysis, data visualization and enrichment pathway analysis mainly from transcriptomics data, but also from simpler gene lists with or without statistical values. Its intuitive and easy-to-manipulate interface facilitates gene expression exploration through numerous interactive figures and tables, statistical comparisons of expression profile levels between groups and further meta-analysis such as enrichment analysis, without bioinformatics expertise. DEVEA performs a thorough analysis from multiple and flexible input data representing distinct analysis stages. From them, it produces dynamic graphs and tables, to explore the expression levels and statistical differential expression analysis results. Moreover, it generates a comprehensive pathway analysis to extend biological insights. Finally, a complete and customizable HTML report can be extracted for further result exploration outside the application. DEVEA is accessible at https://shiny.imib.es/devea/ and the source code is available on our GitHub repository https://github.com/MiriamRiquelmeP/DEVEA

Reactive astrocytes promote proteostasis in Huntington's disease through the JAK2-STAT3 pathway

International audienc

Reactive astrocyte nomenclature, definitions, and future directions.

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions

Effect of general anaesthesia on functional outcome in patients with anterior circulation ischaemic stroke having endovascular thrombectomy versus standard care: a meta-analysis of individual patient data

Background: General anaesthesia (GA) during endovascular thrombectomy has been associated with worse patient outcomes in observational studies compared with patients treated without GA. We assessed functional outcome in ischaemic stroke patients with large vessel anterior circulation occlusion undergoing endovascular thrombectomy under GA, versus thrombectomy not under GA (with or without sedation) versus standard care (ie, no thrombectomy), stratified by the use of GA versus standard care. Methods: For this meta-analysis, patient-level data were pooled from all patients included in randomised trials in PuMed published between Jan 1, 2010, and May 31, 2017, that compared endovascular thrombectomy predominantly done with stent retrievers with standard care in anterior circulation ischaemic stroke patients (HERMES Collaboration). The primary outcome was functional outcome assessed by ordinal analysis of the modified Rankin scale (mRS) at 90 days in the GA and non-GA subgroups of patients treated with endovascular therapy versus those patients treated with standard care, adjusted for baseline prognostic variables. To account for between-trial variance we used mixed-effects modelling with a random effect for trials incorporated in all models. Bias was assessed using the Cochrane method. The meta-analysis was prospectively designed, but not registered. Findings: Seven trials were identified by our search; of 1764 patients included in these trials, 871 were allocated to endovascular thrombectomy and 893 were assigned standard care. After exclusion of 74 patients (72 did not undergo the procedure and two had missing data on anaesthetic strategy), 236 (30%) of 797 patients who had endovascular procedures were treated under GA. At baseline, patients receiving GA were younger and had a shorter delay between stroke onset and randomisation but they had similar pre-treatment clinical severity compared with patients who did not have GA. Endovascular thrombectomy improved functional outcome at 3 months both in patients who had GA (adjusted common odds ratio (cOR) 1·52, 95% CI 1·09–2·11, p=0·014) and in those who did not have GA (adjusted cOR 2·33, 95% CI 1·75–3·10, p<0·0001) versus standard care. However, outcomes were significantly better for patients who did not receive GA versus those who received GA (covariate-adjusted cOR 1·53, 95% CI 1·14–2·04, p=0·0044). The risk of bias and variability between studies was assessed to be low. Interpretation: Worse outcomes after endovascular thrombectomy were associated with GA, after adjustment for baseline prognostic variables. These data support avoidance of GA whenever possible. The procedure did, however, remain effective versus standard care in patients treated under GA, indicating that treatment should not be withheld in those who require anaesthesia for medical reasons

Analysis of Outcomes in Ischemic vs Nonischemic Cardiomyopathy in Patients With Atrial Fibrillation A Report From the GARFIELD-AF Registry

IMPORTANCE Congestive heart failure (CHF) is commonly associated with nonvalvular atrial fibrillation (AF), and their combination may affect treatment strategies and outcomes