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Biomarkers; Stroke; Stroke‐associated infection Biomarcadors; Ictus; Infecció associada a un ictus Biomarcadores; Ictus; Infección asociada a un ictus Background Acute decompensated heart failure (ADHF) and respiratory tract infections (RTIs) are potentially life‐threatening complications in patients experiencing stroke during hospitalization. We aimed to test whether blood biomarker panels might predict these complications early after admission. Methods and Results Nine hundred thirty‐eight patients experiencing ischemic stroke were prospectively recruited in the Stroke‐Chip study. Post‐stroke complications during hospitalization were retrospectively evaluated. Blood samples were drawn within 6 hours after stroke onset, and 14 biomarkers were analyzed by immunoassays. Biomarker values were normalized using log‐transformation and Z score. PanelomiX algorithm was used to select panels with the best accuracy for predicting ADHF and RTI. Logistic regression models were constructed with the clinical variables and the biomarker panels. The additional predictive value of the panels compared with the clinical model alone was evaluated by receiver operating characteristic curves. An internal validation through a 10‐fold cross‐validation with 3 repeats was performed. ADHF and RTI occurred in 19 (2%) and 86 (9.1%) cases, respectively. Three‐biomarker panels were developed as predictors: vascular adhesion protein‐1 >5.67, NT‐proBNP (N‐terminal pro‐B‐type natriuretic peptide) >4.98 and d‐dimer >5.38 (sensitivity, 89.5%; specificity, 71.7%) for ADHF; and interleukin‐6 >3.97, von Willebrand factor >3.67, and d‐dimer >4.58 (sensitivity, 82.6%; specificity, 59.8%) for RTI. Both panels independently predicted stroke complications (panel for ADHF: odds ratio [OR] [95% CI], 10.1 [3–52.2]; panel for RTI: OR, 3.73 [1.95–7.14]) after adjustment by clinical confounders. The addition of the panel to clinical predictors significantly improved areas under the curve of the receiver operating characteristic curves in both cases. Conclusions Blood biomarkers could be useful for the early prediction of ADHF and RTI. Future studies should assess the usefulness of these panels in front of patients experiencing stroke with respiratory symptoms such as dyspnea. This project received funding from Instituto de Salud Carlos III (ISCIII) [DTS14/00004, PI17/02130], co‐financed by the European Regional Development Fund (FEDER), and from Fundació La Marató de TV3 [201706] and the European Union's Horizon 2020 research and innovation program [754517]. Neurovascular Research Laboratory takes part into the Spanish stroke research network INVICTUS+ (RD16/0019/0021). The funders had no role in the study design and conduction.

II. Es van analitzar mostres de microdiàlisi cerebral in vivo de pacients d'ictus isquèmic pel descobriment de biomarcadors associats a la malaltia cerebrovascular. Es van obtenir microdialitzats (MDs) de la zona infartada, de la zona del periinfart i de la zona contralateral no afectada. Els MDs es van comparar, qualitativament i quantitativament, mitjançant tècniques de proteómica basades en marcatge isobàric i espectrometria de masses. Complementàriament, alguns candidats es van estudiar mitjançant immunotransferència en mostres de teixit de necròpsies cerebrals de pacients morts a causa d'un ictus isquèmic. Finalment, hem començat a realitzar ELISAs per la determinació d'aquests candidats en mostres de sang. I. Se analizaron muestras de microdiálisis cerebral in vivo de pacientes de ictus isquémico para el descubrimiento de biomarcadores asociados a la enfermedad cerebrovascular. Se obtuvieron microdializados (MDs) de la zona infartada, de la zona del periinfarto y de la zona contralateral no afectada. Los MDs fueron comparados, cualitativa y cuantitativamente, mediante técnicas de proteómica basadas en marcaje isobárico y espectrometría de masas. Complementariamente, algunos candidatos se estudiaron mediante inmunotransferencia en muestras de tejido de necropsias cerebrales de pacientes fallecidos a causa de un ictus isquémico. Finalmente, hemos comenzado a realizar ELISAs para la determinación de estos candidatos en muestras de sangre.

Ischemic stroke, the third leading cause of death in the Western world, affects mainly the elderly and is strongly associated with comorbid conditions such as atherosclerosis or diabetes, which are pathologically characterized by increased inflammation and are known to influence the outcome of stroke. Stroke incidence peaks during influenza seasons, and patients suffering from infections such as pneumonia prior to stroke exhibit a worse stroke outcome. Earlier studies have shown that comorbidities aggravate the outcome of stroke, yet the mediators of this phenomenon remain obscure. Here, we show that acute peripheral inflammation aggravates stroke‐induced neuronal damage and motor deficits specifically in aged mice. This is associated with increased levels of plasma proinflammatory cytokines, rather than with an increase of inflammatory mediators in the affected brain parenchyma. Nascent transcriptomics data with mature microRNA sequencing were used to identify the neuron‐specific miRNome, in order to decipher dysregulated miRNAs in the brains of aged animals with stroke and co‐existing inflammation. We pinpoint a previously uninvestigated miRNA in the brain, miR‐127, that is highly neuronal, to be associated with increased cell death in the aged, LPS‐injected ischemic mice. Target prediction tools indicate that miR‐127 interacts with several basally expressed neuronal genes, and of these we verify miR‐127 binding to Psmd3. Finally, we report reduced expression of miR‐127 in human stroke brains. Our results underline the impact of peripheral inflammation on the outcome of stroke in aged subjects and pinpoint molecular targets for restoring endogenous neuronal capacity to combat ischemic stroke., Peripheral inflammation preceeding ischemic stroke impairs neuronal survival specifically in the elderly. This increased vulnerability is associated with downregulation of the expression of miR‐127, in both animals and humans. Our results reveal novel molecular targets for treating ischemic stroke, and underline the impact of peripheral inflammation on the outcome of stroke in aged subjects.

Trabajo presentado en el VIII Congreso SEProt and 1st Joint online meeting of Spanish, French and Portuguese Proteomics associations, celebrado online el 12 de noviembre de 2020, The capability ofmonitoring largemolecules as possible biomarkers inwastewaterwill be an important contribution to the newfieldof sewage epidemiology.Here, we explore theuseofpolymer probes togetherwithuntargetedproteomics for large scale protein analysis in sewage and treated water. Polymeric probes were immersed in the influent, anoxic reactor and effluent waters of a SpanishWWTP during 11 days. Proteins sorbed were extracted and identified by mass spectrometry. A total of 690 proteins from bacteria, plants and animals, including human, were identified showing different proteome profiles in the different sites. Bacterial proteins (510) pointed at 175 genera distributed in 22 bacterial classes. Themost abundantwere EF-Tu, GroEL and ATP synthase whichwere contributed by a high number of species. Human was the species contributing the greatest number of identified proteins (57), some in high abundance like keratins. Human proteins dominated in the influent water and were efficiently removed at the effluent. Several of the proteins identified (S100A8, uromodulin, defensins) are known disease biomarkers. This study provides the first insight into the proteome profiles present in real wastewater.

[Rationale] Ischemic stroke is among the leading causes of adult disability. Part of the variability in functional outcome after stroke has been attributed to genetic factors but no locus has been consistently associated with stroke outcome., [Objective] Our aim was to identify genetic loci influencing the recovery process using accurate phenotyping to produce the largest GWAS (genome-wide association study) in ischemic stroke recovery to date., [Methods and Results] A 12-cohort, 2-phase (discovery-replication and joint) meta-analysis of GWAS included anterior-territory and previously independent ischemic stroke cases. Functional outcome was recorded using 3-month modified Rankin Scale. Analyses were adjusted for confounders such as discharge National Institutes of Health Stroke Scale. A gene-based burden test was performed. The discovery phase (n=1225) was followed by open (n=2482) and stringent joint-analyses (n=1791). Those cohorts with modified Rankin Scale recorded at time points other than 3-month or incomplete data on previous functional status were excluded in the stringent analyses. Novel variants in PATJ (Pals1-associated tight junction) gene were associated with worse functional outcome at 3-month after stroke. The top variant was rs76221407 (G allele, β=0.40, P=1.70×10−9)., [Conclusions] Our results identify a set of common variants in PATJ gene associated with 3-month functional outcome at genome-wide significance level. Future studies should examine the role of PATJ in stroke recovery and consider stringent phenotyping to enrich the information captured to unveil additional stroke outcome loci., Genetic contribution to functional outcome and disability after stroke (GODS) project, Fundació Marató-TV3 Grant 2011 (76/C/2011), Recercaixa’13; Generación Project, Instituto de Salud Carlos III; GENISIS (Genetics of Early Neurological InStability After Ischemic Stroke) project, National Institutes of Health (NIH); National Institute of Neurological Disorders and Stroke Stroke Genetics Network (SiGN) Project, NIH.

[Rationale] Ischemic stroke is among the leading causes of adult disability. Part of the variability in functional outcome after stroke has been attributed to genetic factors but no locus has been consistently associated with stroke outcome. [Objective] Our aim was to identify genetic loci influencing the recovery process using accurate phenotyping to produce the largest GWAS (genome-wide association study) in ischemic stroke recovery to date. [Methods and Results] A 12-cohort, 2-phase (discovery-replication and joint) meta-analysis of GWAS included anterior-territory and previously independent ischemic stroke cases. Functional outcome was recorded using 3-month modified Rankin Scale. Analyses were adjusted for confounders such as discharge National Institutes of Health Stroke Scale. A gene-based burden test was performed. The discovery phase (n=1225) was followed by open (n=2482) and stringent joint-analyses (n=1791). Those cohorts with modified Rankin Scale recorded at time points other than 3-month or incomplete data on previous functional status were excluded in the stringent analyses. Novel variants in PATJ (Pals1-associated tight junction) gene were associated with worse functional outcome at 3-month after stroke. The top variant was rs76221407 (G allele, β=0.40, P=1.70×10−9). [Conclusions] Our results identify a set of common variants in PATJ gene associated with 3-month functional outcome at genome-wide significance level. Future studies should examine the role of PATJ in stroke recovery and consider stringent phenotyping to enrich the information captured to unveil additional stroke outcome loci. Genetic contribution to functional outcome and disability after stroke (GODS) project, Fundació Marató-TV3 Grant 2011 (76/C/2011), Recercaixa’13; Generación Project, Instituto de Salud Carlos III; GENISIS (Genetics of Early Neurological InStability After Ischemic Stroke) project, National Institutes of Health (NIH); National Institute of Neurological Disorders and Stroke Stroke Genetics Network (SiGN) Project, NIH.

The limited accessibility to the brain has turned the cerebrospinal fluid (CSF) into a valuable source that may contribute to the complete understanding of the stroke pathophysiology. Here we have described the CSF proteome in the hyper-acute phase of cerebral ischemia by performing an aptamer-based proteomic assay (SOMAscan) in CSF samples collected before and 30 min after male Wistar rats had undergone a 90 min Middle Cerebral Artery Occlusion (MCAO) or sham-surgery. Proteomic results indicated that cerebral ischemia acutely increased the CSF levels of 716 proteins, mostly overrepresented in leukocyte chemotaxis and neuronal death processes. Seven promising candidates were further evaluated in rat plasma and brain (CKB, CaMK2A, CaMK2B, CaMK2D, PDXP, AREG, CMPK). The 3 CaMK2 family-members and CMPK early decreased in the infarcted brain area and, together with AREG, co-localized with neurons. Conversely, CKB levels remained consistent after the insult and specifically matched with astrocytes. Further exploration of these candidates in human plasma revealed the potential of CKB and CMPK to diagnose stroke, while CaMK2B and CMPK resulted feasible biomarkers of functional stroke outcome. Our findings provided insights into the CSF proteome following cerebral ischemia and identified new outstanding proteins that might be further considered as potential biomarkers of stroke.

Altres ajuts: The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The Neurovascular Research Laboratory acknowledges funding for this project through a grant from Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III (cofinanced by the European Regional Development Fund, FEDER). The Neurovascular Research Laboratory also takes part in the Spanish stroke research network INVICTUS+ (RD16/0019). L Ramiro is supported by a predoctoral fellowship grant from the Instituto de Salud Carlos III, and A Simats is supported by a predoctoral fellowship grant from AGAUR. Stroke is the fifth leading cause of death and the most frequent cause of disability worldwide. Currently, stroke diagnosis is based on neuroimaging; therefore, the lack of a rapid tool to diagnose stroke is still a major concern. In addition, therapeutic approaches to combat ischemic stroke are still scarce, since the only approved therapies are directed toward restoring blood flow to the affected brain area. However, due to the reduced time window during which these therapies are effective, few patients benefit from them; therefore, alternative treatments are urgently needed to reduce stroke brain damage in order to improve patients' outcome. The inflammatory response triggered after the ischemic event plays an important role in the progression of stroke; consequently, the study of inflammatory molecules in the acute phase of stroke has attracted increasing interest in recent decades. Here, we provide an overview of the inflammatory processes occurring during ischemic stroke, as well as the potential for these inflammatory molecules to become stroke biomarkers and the possibility that these candidates will become interesting neuroprotective therapeutic targets to be blocked or stimulated in order to modulate inflammation after stroke.