The impact of astrocytic NF-κB on healthy and Alzheimer's disease brains.

Astrocytes play a role in healthy cognitive function and Alzheimer's disease (AD). The transcriptional factor nuclear factor-κB (NF-κB) drives astrocyte diversity, but the mechanisms are not fully understood. By combining studies in human brains and animal models and selectively manipulating NF-κB function in astrocytes, we deepened the understanding of the role of astrocytic NF-κB in brain health and AD. In silico analysis of bulk and cell-specific transcriptomic data revealed the association of NF-κB and astrocytes in AD. Confocal studies validated the higher level of p50 NF-κB and phosphorylated-p65 NF-κB in glial fibrillary acidic protein (GFAP)+-astrocytes in AD versus non-AD subjects. In the healthy mouse brain, chronic activation of astrocytic NF-κB disturbed the proteomic milieu, causing a loss of mitochondrial-associated proteins and the rise of inflammatory-related proteins. Sustained NF-κB signaling also led to microglial reactivity, production of pro-inflammatory mediators, and buildup of senescence-related protein p16INK4A in neurons. However, in an AD mouse model, NF-κB inhibition accelerated ß-amyloid and tau accumulation. Molecular biology studies revealed that astrocytic NF-κB activation drives the increase in GFAP and inflammatory proteins and aquaporin-4, a glymphatic system protein that assists in mitigating AD. Our investigation uncovered fundamental mechanisms by which NF-κB enables astrocytes' neuroprotective and neurotoxic responses in the brain.

Monomeric α-synuclein activates the plasma membrane calcium pump.

Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.

Assessment of models for calculating the hydrodynamic radius of intrinsically disordered proteins.

Diffusion measurements by pulsed-field gradient NMR and fluorescence correlation spectroscopy can be used to probe the hydrodynamic radius of proteins, which contains information about the overall dimension of a protein in solution. The comparison of this value with structural models of intrinsically disordered proteins is nonetheless impaired by the uncertainty of the accuracy of the methods for computing the hydrodynamic radius from atomic coordinates. To tackle this issue, we here build conformational ensembles of 11 intrinsically disordered proteins that we ensure are in agreement with measurements of compaction by small-angle x-ray scattering. We then use these ensembles to identify the forward model that more closely fits the radii derived from pulsed-field gradient NMR diffusion experiments. Of the models we examined, we find that the Kirkwood-Riseman equation provides the best description of the hydrodynamic radius probed by pulsed-field gradient NMR experiments. While some minor discrepancies remain, our results enable better use of measurements of the hydrodynamic radius in integrative modeling and for force field benchmarking and parameterization.

A differential therapeutic consideration for use of corticosteroids according to established COVID-19 clinical phenotypes in critically ill patients.

OBJECTIVE: To determine if the use of corticosteroids was associated with Intensive Care Unit (ICU) mortality among whole population and pre-specified clinical phenotypes. DESIGN: A secondary analysis derived from multicenter, observational study. SETTING: Critical Care Units. PATIENTS: Adult critically ill patients with confirmed COVID-19 disease admitted to 63 ICUs in Spain. INTERVENTIONS: Corticosteroids vs. no corticosteroids. MAIN VARIABLES OF INTEREST: Three phenotypes were derived by non-supervised clustering analysis from whole population and classified as (A: severe, B: critical and C: life-threatening). We performed a multivariate analysis after propensity optimal full matching (PS) for whole population and weighted Cox regression (HR) and Fine-Gray analysis (sHR) to assess the impact of corticosteroids on ICU mortality according to the whole population and distinctive patient clinical phenotypes. RESULTS: A total of 2017 patients were analyzed, 1171 (58%) with corticosteroids. After PS, corticosteroids were shown not to be associated with ICU mortality (OR: 1.0; 95% CI: 0.98-1.15). Corticosteroids were administered in 298/537 (55.5%) patients of "A" phenotype and their use was not associated with ICU mortality (HR=0.85 [0.55-1.33]). A total of 338/623 (54.2%) patients in "B" phenotype received corticosteroids. No effect of corticosteroids on ICU mortality was observed when HR was performed (0.72 [0.49-1.05]). Finally, 535/857 (62.4%) patients in "C" phenotype received corticosteroids. In this phenotype HR (0.75 [0.58-0.98]) and sHR (0.79 [0.63-0.98]) suggest a protective effect of corticosteroids on ICU mortality. CONCLUSION: Our finding warns against the widespread use of corticosteroids in all critically ill patients with COVID-19 at moderate dose. Only patients with the highest inflammatory levels could benefit from steroid treatment.

Deletion of MyD88 in astrocytes prevents ß-amyloid-induced neuropathology in mice.

As the understanding of immune responses in Alzheimer's disease (AD) is in its early phases, there remains an urgency to identify the cellular and molecular processes driving chronic inflammation. In AD, a subpopulation of astrocytes acquires a neurotoxic phenotype which prompts them to lose typical physiological features. While the underlying molecular mechanisms are still unknown, evidence suggests that myeloid differentiation primary response 88 (MyD88) adaptor protein may play a role in coordinating these cells' immune responses in AD. Herein, we combined studies in human postmortem samples with a conditional genetic knockout mouse model to investigate the link between MyD88 and astrocytes in AD. In silico analyses of bulk and cell-specific transcriptomic data from human postmortem brains demonstrated an upregulation of MyD88 expression in astrocytes in AD versus non-AD individuals. Proteomic studies revealed an increase in glial fibrillary acidic protein in multiple brain regions of AD subjects. These studies also showed that although overall MyD88 steady-state levels were unaffected by AD, this protein was enriched in astrocytes near amyloid plaques and neurofibrillary tangles. Functional studies in mice indicated that the deletion of astrocytic MyD88 protected animals from the acute synaptic toxicity and cognitive impairment caused by the intracerebroventricular administration of ß-amyloid (Aß). Lastly, unbiased proteomic analysis revealed that loss of astrocytic MyD88 resulted in altered astrocyte reactivity, lower levels of immune-related proteins, and higher expression of synaptic-related proteins in response to Aß. Our studies provide evidence of the pivotal role played by MyD88 in the regulation of astrocytes response to AD.

A differential therapeutic consideration for use of corticosteroids according to established COVID-19 clinical phenotypes in critically ill patients.

Objective: To determine if the use of corticosteroids was associated with Intensive Care Unit (ICU) mortality among whole population and pre-specified clinical phenotypes. Design: A secondary analysis derived from multicenter, observational study. Setting: Critical Care Units. Patients: Adult critically ill patients with confirmed COVID-19 disease admitted to 63 ICUs in Spain. Interventions: Corticosteroids vs. no corticosteroids. Main variables of interest: Three phenotypes were derived by non-supervised clustering analysis from whole population and classified as (A: severe, B: critical and C: life-threatening). We performed a multivariate analysis after propensity optimal full matching (PS) for whole population and weighted Cox regression (HR) and Fine-Gray analysis (sHR) to assess the impact of corticosteroids on ICU mortality according to the whole population and distinctive patient clinical phenotypes. Results: A total of 2017 patients were analyzed, 1171 (58%) with corticosteroids. After PS, corticosteroids were shown not to be associated with ICU mortality (OR: 1.0; 95% CI: 0.98-1.15). Corticosteroids were administered in 298/537 (55.5%) patients of "A" phenotype and their use was not associated with ICU mortality (HR = 0.85 [0.55-1.33]). A total of 338/623 (54.2%) patients in "B" phenotype received corticosteroids. No effect of corticosteroids on ICU mortality was observed when HR was performed (0.72 [0.49-1.05]). Finally, 535/857 (62.4%) patients in "C" phenotype received corticosteroids. In this phenotype HR (0.75 [0.58-0.98]) and sHR (0.79 [0.63-0.98]) suggest a protective effect of corticosteroids on ICU mortality. Conclusion: Our finding warns against the widespread use of corticosteroids in all critically ill patients with COVID-19 at moderate dose. Only patients with the highest inflammatory levels could benefit from steroid treatment.

Objetivo: Evaluar si el uso de corticoesteroides (CC) se asocia con la mortalidad en la unidad de cuidados intensivos (UCI) en la población global y dentro de los fenotipos clínicos predeterminados. Diseño: Análisis secundario de estudio multicéntrico observacional. Ámbito: UCI. Pacientes: Pacientes adultos con COVID-19 confirmado ingresados en 63 UCI de España. Intervención: Corticoides vs. no corticoides. Variables de interés principales: A partir del análisis no supervisado de grupos, 3 fenotipos clínicos fueron derivados y clasificados como: A grave, B crítico y C potencialmente mortal. Se efectuó un análisis multivariado después de un propensity optimal full matching (PS) y una regresión ponderada de Cox (HR) y análisis de Fine-Gray (sHR) para evaluar el impacto del tratamiento con CC sobre la mortalidad en la población general y en cada fenotipo clínico. Resultados: Un total de 2.017 pacientes fueron analizados, 1.171 (58%) con CC. Después del PS, el uso de CC no se relacionó significativamente con la mortalidad en UCI (OR: 1,0; IC 95%: 0,98-1,15). Los CC fueron administrados en 298/537 (55,5%) pacientes del fenotipo A y no se observó asociación significativa con la mortalidad (HR = 0,85; 0,55-1,33). Un total de 338/623 (54,2%) pacientes del fenotipo B recibieron CC sin efecto significativo sobre la mortalidad (HR = 0,72; 0,49-1,05). Por último, 535/857 (62,4%) pacientes del fenotipo C recibieron CC. En este fenotipo, se evidenció un efecto protector de los CC sobre la mortalidad HR (0,75; 0,58-0,98). Conclusión: Nuestros hallazgos alertan sobre el uso indiscriminado de CC a dosis moderadas en todos los pacientes críticos con COVID-19. Solamente pacientes con elevado estado de inflamación podrían beneficiarse con el tratamiento con CC.

How phosphorylation impacts intrinsically disordered proteins and their function.

Phosphorylation is the most common post-translational modification (PTM) in eukaryotes, occurring particularly frequently in intrinsically disordered proteins (IDPs). These proteins are highly flexible and dynamic by nature. Thus, it is intriguing that the addition of a single phosphoryl group to a disordered chain can impact its function so dramatically. Furthermore, as many IDPs carry multiple phosphorylation sites, the number of possible states increases, enabling larger complexities and novel mechanisms. Although a chemically simple and well-understood process, the impact of phosphorylation on the conformational ensemble and molecular function of IDPs, not to mention biological output, is highly complex and diverse. Since the discovery of the first phosphorylation site in proteins 75 years ago, we have come to a much better understanding of how this PTM works, but with the diversity of IDPs and their capacity for carrying multiple phosphoryl groups, the complexity grows. In this Essay, we highlight some of the basic effects of IDP phosphorylation, allowing it to serve as starting point when embarking on studies into this topic. We further describe how recent complex cases of multisite phosphorylation of IDPs have been instrumental in widening our view on the effect of protein phosphorylation. Finally, we put forward perspectives on the phosphorylation of IDPs, both in relation to disease and in context of other PTMs; areas where deep insight remains to be uncovered.

Deciphering the Alphabet of Disorder-Glu and Asp Act Differently on Local but Not Global Properties.

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences between glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing database of experimentally verified IDPs (DisProt) and show that glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while the Glu/Asp variants support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity, where glutamates can support increased helicity, important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in, e.g., transactivation domains and ion-binding.

Prior influenza vaccine is not a risk factor for bacterial coinfection in patients admitted to the ICU due to severe influenza.

OBJECTIVE: To determine whether the prior usage of the flu vaccine is a risk factor for bacterial co-infection in patients with severe influenza. DESIGN: This was a retrospective observational cohort study of subjects admitted to the ICU. A propensity score matching, and logistic regression adjusted for potential confounders were carried out to evaluate the association between prior influenza vaccination and bacterial co-infection. SETTINGS: 184 ICUs in Spain due to severe influenza. PATIENTS: Patients included in the Spanish prospective flu registry. INTERVENTIONS: Flu vaccine prior to the hospital admission. RESULTS: A total of 4175 subjects were included in the study. 489 (11.7%) received the flu vaccine prior to develop influenza infection. Prior vaccinated patients were older 71 [61-78], and predominantly male 65.4%, with at least one comorbid condition 88.5%. Prior vaccination was not associated with bacterial co-infection in the logistic regression model (OR: 1.017; 95%CI 0.803-1.288; p=0.885). After matching, the average treatment effect of prior influenza vaccine on bacterial co-infection was not statistically significant when assessed by propensity score matching (p=0.87), nearest neighbor matching (p=0.59) and inverse probability weighting (p=0.99). CONCLUSIONS: No association was identified between prior influenza vaccine and bacterial coinfection in patients admitted to the ICU due to severe influenza. Post influenza vaccination studies are necessary to continue evaluating the possible benefits.

Clindamycin but not Intravenous Immunoglobulins reduces mortality in a retrospective cohort of critically ill patients with bacteremic Group A Streptococcal infections.

OBJECTIVE: Mortality of patients requiring Intensive Care Unit (ICU) admission for an invasive group A streptococcal (GAS) infection continues being high. In critically ill patients with bacteremic GAS infection we aimed at determining risk factors for mortality. METHODS: Retrospective multicentre study carried out in nine ICU in Southern Spain. All adult patients admitted to the participant ICUs from January 2014 to June 2019 with one positive blood culture for S. pyogenes were included in this study. Patient characteristics, infection-related variables, therapeutic interventions, failure of organs, and outcomes were registered. Risk factors independently associated with ICU and in-hospital mortalities were determined by multivariate regression analyses. RESULTS: Fifty-seven patients were included: median age was 63 (45-73) years, median SOFA score at admission was 11 (7-13). The most frequent source was skin and soft tissue infection (n=32) followed by unknown origin of bacteremia (n=12). In the multivariate analysis, age (OR 1.079; 95% CI 1.016-1.145), SOFA score (OR 2.129; 95% CI 1.339-3.383) were the risk factors for ICU mortality and the use of clindamycin was identified as a protective factor (OR 0.049; 95% CI 0.003-0.737). Age and SOFA were the independent factors associated with hospital mortality however the use of clindamycin showed a strong trend but without reaching statistical significance (OR 0.085; 95% CI 0.007-1.095). CONCLUSIONS: In this cohort of critically ill patients the use of intravenous immunoglobulin was not identified as a protective factor for ICU or hospital mortality treatment with clindamycin significantly reduced mortality after controlling for confounders.

Predictive factors of six-week mortality in critically ill patients with SARS-CoV-2: A multicenter prospective study.

OBJECTIVE: The objective of the study is to identify the risk factors associated with mortality at six weeks, especially by analyzing the role of antivirals and munomodulators. DESIGN: Prospective descriptive multicenter cohort study. SETTING: 26 Intensive care units (ICU) from Andalusian region in Spain. PATIENTS OR PARTICIPANTS: Consecutive critically ill patients with confirmed SARS-CoV-2 infection were included from March 8 to May 30. INTERVENTIONS: None. VARIABLES: Variables analyzed were demographic, severity scores and clinical condition. Support therapy, drug and mortality were analyzed. An univariate followed by multivariate Cox regression with propensity score analysis was applied. RESULTS: 495 patients were enrolled, but 73 of them were excluded for incomplete data. Thus, 422 patients were included in the final analysis. Median age was 63 years and 305 (72.3%) were men. ICU mortality: 144/422 34%; 14 days mortality: 81/422 (19.2%); 28 days mortality: 121/422 (28.7%); 6-week mortality 152/422 36.5%. By multivariable Cox proportional analysis, factors independently associated with 42-day mortality were age, APACHE II score, SOFA score at ICU admission >6, Lactate dehydrogenase at ICU admission >470U/L, Use of vasopressors, extrarenal depuration, %lymphocytes 72h post-ICU admission <6.5%, and thrombocytopenia whereas the use of lopinavir/ritonavir was a protective factor. CONCLUSION: Age, APACHE II, SOFA>value of 6 points, along with vasopressor requirements or renal replacement therapy have been identified as predictor factors of mortality at six weeks. Administration of corticosteroids showed no benefits in mortality, as did treatment with tocilizumab. Lopinavir/ritonavir administration is identified as a protective factor.

ADENI-UCI study: Analysis of non-income decisions in ICU as a measure of limitation of life support treatments.

OBJECTIVE: To analyze the variables associated with ICU refusal decisions as a life support treatment limitation measure. DESIGN: Prospective, multicentrico. SCOPE: 62 ICU from Spain between February 2018 and March 2019. PATIENTS: Over 18 years of age who were denied entry into ICU as a life support treatment limitation measure. INTERVENTIONS: None. MAIN INTEREST VARIABLES: Patient comorities, functional situation as measured by the KNAUS and Karnosfky scale; predicted scales of Lee and Charlson; severity of the sick person measured by the APACHE II and SOFA scales, which justifies the decision-making, a person to whom the information is transmitted; date of discharge or in-hospital death, destination for hospital discharge. RESULTS: A total of 2312 non-income decisions were recorded as an LTSV measure of which 2284 were analyzed. The main reason for consultation was respiratory failure (1080 [47.29%]). The poor estimated quality of life of the sick (1417 [62.04%]), the presence of a severe chronic disease (1367 [59.85%]) and the prior functional limitation of patients (1270 [55.60%]) were the main reasons for denying admission. The in-hospital mortality rate was 60.33%. The futility of treatment was found as a risk factor associated with mortality (OR: 3.23; IC95%: 2.62-3.99). CONCLUSIONS: Decisions to limit ICU entry as an LTSV measure are based on the same reasons as decisions made within the ICU. The futility valued by the intensivist is adequately related to the final result of death.

Recommendations for the management of critically ill patients with COVID-19 in Intensive Care Units.

The COVID-19 pandemic has led to the admission of a high number of patients to the ICU, generally due to severe respiratory failure. Since the appearance of the first cases of SARS-CoV-2 infection, at the end of 2019, in China, a huge number of treatment recommendations for this entity have been published, not always supported by sufficient scientific evidence or with methodological rigor necessary. Thanks to the efforts of different groups of researchers, we currently have the results of clinical trials, and other types of studies, of higher quality. We consider it necessary to create a document that includes recommendations that collect this evidence regarding the diagnosis and treatment of COVID-19, but also aspects that other guidelines have not considered and that we consider essential in the management of critical patients with COVID-19. For this, a drafting committee has been created, made up of members of the SEMICYUC Working Groups more directly related to different specific aspects of the management of these patients.

[Recommendations for the management of critically ill patients with COVID-19 in Intensive Care Units]. / Recomendaciones para el manejo de los pacientes críticos con COVID-19 en las Unidades de Cuidados Intensivos.

The COVID-19 pandemic has led to the admission of a high number of patients to the ICU, generally due to severe respiratory failure. Since the appearance of the first cases of SARS-CoV-2 infection, at the end of 2019, in China, a huge number of treatment recommendations for this entity have been published, not always supported by sufficient scientific evidence or with methodological rigor necessary. Thanks to the efforts of different groups of researchers, we currently have the results of clinical trials, and other types of studies, of higher quality. We consider it necessary to create a document that includes recommendations that collect this evidence regarding the diagnosis and treatment of COVID-19, but also aspects that other guidelines have not considered and that we consider essential in the management of critical patients with COVID-19. For this, a drafting committee has been created, made up of members of the SEMICYUC Working Groups more directly related to different specific aspects of the management of these patients.

Recommendations for antibiotic selection for severe nosocomial infections.

Severe infection and its evolution to sepsis are becoming more prevalent every day and are among the leading causes of critical illness and mortality. Proper management is crucial to improve prognosis. This document addresses three essential points that have a significant impact on this objective: a) early recognition of patients with sepsis criteria, b) identification of those patients who suffer from an infection and have a high risk of progressing to sepsis, and c) adequate selection and optimization of the initial antimicrobial treatment.

Management of infectious complications associated with coronavirus infection in severe patients admitted to ICU.

Infections have become one of the main complications of patients with severe SARS-CoV-2 pneumonia admitted in ICU. Poor immune status, frequent development of organic failure requiring invasive supportive treatments, and prolonged ICU length of stay in saturated structural areas of patients are risk factors for infection development. The Working Group on Infectious Diseases and Sepsis GTEIS of the Spanish Society of Intensive Medicine and Coronary Units SEMICYUC emphasizes the importance of infection prevention measures related to health care, the detection and early treatment of major infections in the patient with SARS-CoV-2 infections. Bacterial co-infection, respiratory infections related to mechanical ventilation, catheter-related bacteremia, device-associated urinary tract infection and opportunistic infections are review in the document.

Insight into Calcium-Binding Motifs of Intrinsically Disordered Proteins.

Motifs within proteins help us categorize their functions. Intrinsically disordered proteins (IDPs) are rich in short linear motifs, conferring them many different roles. IDPs are also frequently highly charged and, therefore, likely to interact with ions. Canonical calcium-binding motifs, such as the EF-hand, often rely on the formation of stabilizing flanking helices, which are a key characteristic of folded proteins, but are absent in IDPs. In this study, we probe the existence of a calcium-binding motif relevant to IDPs. Upon screening several carefully selected IDPs using NMR spectroscopy supplemented with affinity quantification by colorimetric assays, we found calcium-binding motifs in IDPs which could be categorized into at least two groups-an Excalibur-like motif, sequentially similar to the EF-hand loop, and a condensed-charge motif carrying repetitive negative charges. The motifs show an affinity for calcium typically in the ~100 µM range relevant to regulatory functions and, while calcium binding to the condensed-charge motif had little effect on the overall compaction of the IDP chain, calcium binding to Excalibur-like motifs resulted in changes in compaction. Thus, calcium binding to IDPs may serve various structural and functional roles that have previously been underreported.