Obesity, Inflammation, and Clinical Outcomes in COVID-19: A Multicenter Prospective Cohort Study.

CONTEXT: Obesity is a risk factor for coronavirus disease 2019 (COVID-19)-related outcomes; however, the mechanism remains unclear. OBJECTIVE: The objective of this analysis was to determine whether inflammation mediates the association between obesity and COVID-19 outcomes. DESIGN: The International Study of Inflammation in Covid-19 (ISIC): A Prospective Multi-Center Observational Study Examining the Role of Biomarkers of Inflammation in Predicting Covid-19 Related Outcomes in Hospitalized Patients. SETTING: Ten hospitals in the United States and Europe. PARTICIPANTS: Adults hospitalized specifically for COVID-19 between February 1, 2020, through October 19, 2022. MAIN OUTCOME MEASURES: Inflammatory biomarkers, including soluble urokinase plasminogen activator receptor (suPAR), were measured at admission. Associations were examined between body-mass index (BMI, kg/m2) and a composite of death, need for mechanical ventilation, and renal replacement therapy, stratified by pre- and post-Omicron variants. The contribution of inflammation to the relationship between obesity and outcomes was assessed. RESULTS: Among 4644 participants (mean age 59.3, 45.6% male, 21.8% BMI≥35), those with BMI>40 (n=485) had 55% higher odds of the composite outcome (95% CI[1.21 to 1.98]) compared to non-obese individuals (BMI<30, n=2358) in multivariable analysis. In multiple mediation analysis, only suPAR remained a significant mediator between BMI and composite outcome. Associations were amplified for participants younger than 65 years and with pre-Omicron variants. CONCLUSION: Obesity is associated with worse outcomes in COVID-19, notably in younger participants and in the pre-Omicron era. Inflammation, as measured by suPAR, is a significant mediator of the association between obesity and COVID-19 outcomes.

Deep-time phylogenetic inference by paleoproteomic analysis of dental enamel.

In temperate and subtropical regions, ancient proteins are reported to survive up to about 2 million years, far beyond the known limits of ancient DNA preservation in the same areas. Accordingly, their amino acid sequences currently represent the only source of genetic information available to pursue phylogenetic inference involving species that went extinct too long ago to be amenable for ancient DNA analysis. Here we present a complete workflow, including sample preparation, mass spectrometric data acquisition and computational analysis, to recover and interpret million-year-old dental enamel protein sequences. During sample preparation, the proteolytic digestion step, usually an integral part of conventional bottom-up proteomics, is omitted to increase the recovery of the randomly degraded peptides spontaneously generated by extensive diagenetic hydrolysis of ancient proteins over geological time. Similarly, we describe other solutions we have adopted to (1) authenticate the endogenous origin of the protein traces we identify, (2) detect and validate amino acid variation in the ancient protein sequences and (3) attempt phylogenetic inference. Sample preparation and data acquisition can be completed in 3-4 working days, while subsequent data analysis usually takes 2-5 days. The workflow described requires basic expertise in ancient biomolecules analysis, mass spectrometry-based proteomics and molecular phylogeny. Finally, we describe the limits of this approach and its potential for the reconstruction of evolutionary relationships in paleontology and paleoanthropology.

Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling.

Receptor tyrosine kinases (RTKs) initiate cellular signaling pathways, which are regulated through a delicate balance of phosphorylation and dephosphorylation events. While many studies of RTKs have focused on downstream-activated kinases catalyzing the site-specific phosphorylation, few studies have focused on the phosphatases carrying out the dephosphorylation. In this study, we analyzed six protein phosphatase networks using chemical inhibitors in context of epidermal growth factor receptor (EGFR) signaling by mass spectrometry-based phosphoproteomics. Specifically, we focused on protein phosphatase 2C (PP2C), involved in attenuating p38-dependent signaling pathways in various cellular responses, and confirmed its effect in regulating p38 activity in EGFR signaling. Furthermore, utilizing a p38 inhibitor, we classified phosphosites whose phosphorylation status depends on PP2C inhibition into p38-dependent and p38-independent sites. This study provides a large-scale dataset of phosphatase-regulation of EGF-responsive phosphorylation sites, which serves as a useful resource to deepen our understanding of EGFR signaling.

Development and validation of prognostic machine learning models for short- and long-term mortality among acutely admitted patients based on blood tests.

Several scores predicting mortality at the emergency department have been developed. However, all with shortcomings either simple and applicable in a clinical setting, with poor performance, or advanced, with high performance, but clinically difficult to implement. This study aimed to explore if machine learning algorithms could predict all-cause short- and long-term mortality based on the routine blood test collected at admission. METHODS: We analyzed data from a retrospective cohort study, including patients > 18 years admitted to the Emergency Department (ED) of Copenhagen University Hospital Hvidovre, Denmark between November 2013 and March 2017. The primary outcomes were 3-, 10-, 30-, and 365-day mortality after admission. PyCaret, an automated machine learning library, was used to evaluate the predictive performance of fifteen machine learning algorithms using the area under the receiver operating characteristic curve (AUC). RESULTS: Data from 48,841 admissions were analyzed, of these 34,190 (70%) were randomly divided into training data, and 14,651 (30%) were in test data. Eight machine learning algorithms achieved very good to excellent results of AUC on test data in a of range 0.85-0.93. In prediction of short-term mortality, lactate dehydrogenase (LDH), leukocyte counts and differentials, Blood urea nitrogen (BUN) and mean corpuscular hemoglobin concentration (MCHC) were the best predictors, whereas prediction of long-term mortality was favored by age, LDH, soluble urokinase plasminogen activator receptor (suPAR), albumin, and blood urea nitrogen (BUN). CONCLUSION: The findings suggest that measures of biomarkers taken from one blood sample during admission to the ED can identify patients at high risk of short-and long-term mortality following emergency admissions.

One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes.

Mass spectrometry (MS)-based proteomics workflows typically involve complex, multi-step processes, presenting challenges with sample losses, reproducibility, requiring substantial time and financial investments, and specialized skills. Here we introduce One-Tip, a proteomics methodology that seamlessly integrates efficient, one-pot sample preparation with precise, narrow-window data-independent acquisition (nDIA) analysis. One-Tip substantially simplifies sample processing, enabling the reproducible identification of >9000 proteins from ~1000 HeLa cells. The versatility of One-Tip is highlighted by nDIA identification of ~6000 proteins in single cells from early mouse embryos. Additionally, the study incorporates the Uno Single Cell Dispenser™, demonstrating the capability of One-Tip in single-cell proteomics with >3000 proteins identified per HeLa cell. We also extend One-Tip workflow to analysis of extracellular vesicles (EVs) extracted from blood plasma, demonstrating its high sensitivity by identifying >3000 proteins from 16 ng EV preparation. One-Tip expands capabilities of proteomics, offering greater depth and throughput across a range of sample types.

Systematic Optimization of Automated Phosphopeptide Enrichment for High-Sensitivity Phosphoproteomics.

Improving coverage, robustness, and sensitivity is crucial for routine phosphoproteomics analysis by single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) from minimal peptide inputs. Here, we systematically optimized key experimental parameters for automated on-bead phosphoproteomics sample preparation with a focus on low-input samples. Assessing the number of identified phosphopeptides, enrichment efficiency, site localization scores, and relative enrichment of multiply-phosphorylated peptides pinpointed critical variables influencing the resulting phosphoproteome. Optimizing glycolic acid concentration in the loading buffer, percentage of ammonium hydroxide in the elution buffer, peptide-to-beads ratio, binding time, sample, and loading buffer volumes allowed us to confidently identify >16,000 phosphopeptides in half-an-hour LC-MS/MS on an Orbitrap Exploris 480 using 30 µg of peptides as starting material. Furthermore, we evaluated how sequential enrichment can boost phosphoproteome coverage and showed that pooling fractions into a single LC-MS/MS analysis increased the depth. We also present an alternative phosphopeptide enrichment strategy based on stepwise addition of beads thereby boosting phosphoproteome coverage by 20%. Finally, we applied our optimized strategy to evaluate phosphoproteome depth with the Orbitrap Astral MS using a cell dilution series and were able to identify >32,000 phosphopeptides from 0.5 million HeLa cells in half-an-hour LC-MS/MS using narrow-window data-independent acquisition (nDIA).

Capacidad del biomarcador suPAR para estratificar el pronóstico en pacientes atendidos en servicios de urgencias hospitalarios / Value of the soluble urokinase-type plasminogen activator receptor as a predictor of prognosis in patients attended in hospital emergency departments

Objetivos. Determinar la capacidad del receptor soluble del activador del plasminógeno tipo uroquinasa (suPAR) para la estratificación pronóstica en pacientes atendidos en servicios de urgencias hospitalarios (SUH). Los objetivos secundarios son: 1)medir la capacidad de los puntos de decisión habituales, 2)identificar una población de bajo riesgo de mortalidad que puede darse de alta de forma segura desde el SUH, y 3)medir la correlación entre suPAR y otros biomarcadores. Métodos. Estudio observacional de cohortes prospectivo de pacientes atendidos en SUH. Se registraron variables sociodemográficas, de comorbilidad, datos del episodio agudo, biomarcadores de uso común en urgencias y suPAR. Las variables de resultado fueron la necesidad de ingreso en el episodio índice, reconsulta al SUH y mortalidad a los 90 días. Resultados. Se incluyeron 990 pacientes, la edad fue de 68 (53-81) años, 50,8% eran hombres, la mediana de suPAR fue de 3,8 (2,8-6,0) ng/ml, 112 pacientes (11,31%) requirieron ingreso. En el seguimiento a 90 días hubo 276 reconsultas (27,9%) y 47 pacientes (4,74%) fallecieron. Los pacientes con suPAR<4 ng/ml (52,5%) tenían menor mortalidad (1%), menor reconsulta (24,4%) y menor necesidad de ingreso hospitalario (20,6%), que pacientes con suPAR>6 ng/ml (mortalidad 13,5%, reconsulta 39,6% e ingreso 56,3%). Un suPAR>6 ng/ml mostró una hazard ratio (IC 95%) ajustada de 4,61 (1,68-12,67) para predecir mortalidad a 90 días y de 1,59 (1,13-2,10) para la reconsulta, y una odds ratio de 1,62 (0,99-2,62) para la necesidad de ingreso hospitalario. Conclusiones. Un valor de suPAR < 4 ng/ml identifica pacientes con riesgo bajo de mortalidad a 90 días, de reconsulta y de necesidad de ingreso, mientras que los pacientes con suPAR>6 ng/ml tienen mayor mortalidad, reconsulta y necesidad de ingreso. (AU)

Objectives. To determine the value of the soluble urokinase-type plasminogen activator receptor (suPAR) for predicting outcomes in emergency department (ED) patients. Secondary objectives were 1)to measure the predictive value of the usual decision points, 2)to identify patients at low risk for mortality who could be safely discharged from the ED, and 3)to measure the correlation between suPAR and other biomarkers. Methods. Prospective observational cohort study of patients attended in the EDs of participating hospitals. We recorded sociodemographic variables, comorbidity, variables related to the acute episode, prognostic markers commonly used in EDs, and suPAR concentration. Outcome variables were the need for hospital admission during the index episode, ED revisits within 90 days, and 90-day mortality. Results. A total of 990 patients with a median (interquartile range) age of 68 (53-81 years) were studied; 50.8% were men. The median suPAR concentration was 3.8 (2.8-6.0) ng/mL, and 112 patients (11.31%) required admission. At 90 days there were 276 revisits (27.9% of the cohort), and 47 patients (4.74%) had died. Mortality was lower (1%) in patients with suPAR concentrations less than 4 ng/mL (52.5%), and fewer of these patients revisited (24.4%) or required hospitalization (20.6%) than patients with suPAR concentrations higher than 6 ng/mL (mortality, 13.5%; revisits, 39.6%; admissions, 56.3%). A suPAR concentration over 6 ng/mL was associated with 90-day mortality and revisits (adjusted hazard ratios and 95% CIs of 4.61 [1.68-12.67] and 1.59 [1.13-2.10]), respectively. The high suPAR concentration was also associated with hospital admission (odds ratio, 1.62 [0.99-2.62]). Conclusions. A suPAR concentration of less than 4 ng/mL identifies patients at low risk of 90-day mortality and revisits or need for hospitalization, whereas a suPAR concentration higher than 6 ng/mL is associated with higher risk for these outcomes. (AU)

Capacidad del biomarcador suPAR para estratificar el pronóstico en pacientes atendidos en servicios de urgencias hospitalarios / Value of the soluble urokinase-type plasminogen activator receptor as a predictor of prognosis in patients attended in hospital emergency departments

Objetivos. Determinar la capacidad del receptor soluble del activador del plasminógeno tipo uroquinasa (suPAR) para la estratificación pronóstica en pacientes atendidos en servicios de urgencias hospitalarios (SUH). Los objetivos secundarios son: 1)medir la capacidad de los puntos de decisión habituales, 2)identificar una población de bajo riesgo de mortalidad que puede darse de alta de forma segura desde el SUH, y 3)medir la correlación entre suPAR y otros biomarcadores. Métodos. Estudio observacional de cohortes prospectivo de pacientes atendidos en SUH. Se registraron variables sociodemográficas, de comorbilidad, datos del episodio agudo, biomarcadores de uso común en urgencias y suPAR. Las variables de resultado fueron la necesidad de ingreso en el episodio índice, reconsulta al SUH y mortalidad a los 90 días. Resultados. Se incluyeron 990 pacientes, la edad fue de 68 (53-81) años, 50,8% eran hombres, la mediana de suPAR fue de 3,8 (2,8-6,0) ng/ml, 112 pacientes (11,31%) requirieron ingreso. En el seguimiento a 90 días hubo 276 reconsultas (27,9%) y 47 pacientes (4,74%) fallecieron. Los pacientes con suPAR<4 ng/ml (52,5%) tenían menor mortalidad (1%), menor reconsulta (24,4%) y menor necesidad de ingreso hospitalario (20,6%), que pacientes con suPAR>6 ng/ml (mortalidad 13,5%, reconsulta 39,6% e ingreso 56,3%). Un suPAR>6 ng/ml mostró una hazard ratio (IC 95%) ajustada de 4,61 (1,68-12,67) para predecir mortalidad a 90 días y de 1,59 (1,13-2,10) para la reconsulta, y una odds ratio de 1,62 (0,99-2,62) para la necesidad de ingreso hospitalario. Conclusiones. Un valor de suPAR < 4 ng/ml identifica pacientes con riesgo bajo de mortalidad a 90 días, de reconsulta y de necesidad de ingreso, mientras que los pacientes con suPAR>6 ng/ml tienen mayor mortalidad, reconsulta y necesidad de ingreso. (AU)

Objectives. To determine the value of the soluble urokinase-type plasminogen activator receptor (suPAR) for predicting outcomes in emergency department (ED) patients. Secondary objectives were 1)to measure the predictive value of the usual decision points, 2)to identify patients at low risk for mortality who could be safely discharged from the ED, and 3)to measure the correlation between suPAR and other biomarkers. Methods. Prospective observational cohort study of patients attended in the EDs of participating hospitals. We recorded sociodemographic variables, comorbidity, variables related to the acute episode, prognostic markers commonly used in EDs, and suPAR concentration. Outcome variables were the need for hospital admission during the index episode, ED revisits within 90 days, and 90-day mortality. Results. A total of 990 patients with a median (interquartile range) age of 68 (53-81 years) were studied; 50.8% were men. The median suPAR concentration was 3.8 (2.8-6.0) ng/mL, and 112 patients (11.31%) required admission. At 90 days there were 276 revisits (27.9% of the cohort), and 47 patients (4.74%) had died. Mortality was lower (1%) in patients with suPAR concentrations less than 4 ng/mL (52.5%), and fewer of these patients revisited (24.4%) or required hospitalization (20.6%) than patients with suPAR concentrations higher than 6 ng/mL (mortality, 13.5%; revisits, 39.6%; admissions, 56.3%). A suPAR concentration over 6 ng/mL was associated with 90-day mortality and revisits (adjusted hazard ratios and 95% CIs of 4.61 [1.68-12.67] and 1.59 [1.13-2.10]), respectively. The high suPAR concentration was also associated with hospital admission (odds ratio, 1.62 [0.99-2.62]). Conclusions. A suPAR concentration of less than 4 ng/mL identifies patients at low risk of 90-day mortality and revisits or need for hospitalization, whereas a suPAR concentration higher than 6 ng/mL is associated with higher risk for these outcomes. (AU)

Atypical cofilin signaling drives dendritic cell migration through the extracellular matrix via nuclear deformation.

To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to T cells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-µm diameter limit for 3D migration through gaps in the extracellular matrix. We show that the nuclear shape change is partially attained through reduced cell adhesion, whereas improved 3D migration is achieved through reprogramming of the actin cytoskeleton. Specifically, our data point to a model whereby the phosphorylation of cofilin-1 at serine 41 drives the assembly of a cofilin-actomyosin ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe signaling events through which dendritic cells deform their nucleus and enhance their migratory capacity.

Digging deeper into ancient skeletal proteomes through consecutive digestion with multiple proteases.

An increasing number of studies utilise the recovery of ancient skeletal proteomes for phylogenetic and evolutionary analysis. Although these studies manage to extract and analyse ancient peptides, the recovered proteomes are generally small in size and with low protein sequence coverage. We expand on previous observations which have shown that the parallel digestion and analysis of Pleistocene skeletal proteomes increases overall proteome size and protein sequence coverage. Furthermore, we demonstrate that the consecutive digestion of a skeletal proteome using two proteases, particularly the combination of Glu-C or chymotrypsin followed by trypsin digestion, enables the recovery of alternative proteome components not reachable through trypsin digestion alone. The proteomes preserved in Pleistocene skeletal specimens are larger than previously anticipated, but unlocking this protein sequence information requires adaptation of extraction and protein digestion protocols. The sequential utilisation of several proteases is, in this regard, a promising avenue for the study of highly degraded but unique hominin proteomes for phylogenetic purposes. SIGNIFICANCE: Palaeoproteomic analysis of archaeological materials, such as hominin skeletal elements, show great promise in studying past organisms and evolutionary relationships. However, as most proteomic methods are inherently destructive, it is essential to aim to recover as much information as possible from every sample. Currently, digestion with trypsin is the standard approach in most palaeoproteomic studies. We find that parallel or consecutive digestion with multiple proteases can improve proteome size and coverage for both Holocene and Pleistocene bone specimens. This allows for recovery of more proteomic data from a sample and maximises the chance of recovering phylogenetically relevant information.

Value of the soluble urokinase-type plasminogen activator receptor as a predictor of prognosis in patients attended in hospital emergency departments. / Capacidad del biomarcador suPAR para estratificar el pronóstico en pacientes atendidos en servicios de urgencias hospitalarios.

OBJECTIVES: To determine the value of the soluble urokinase-type plasminogen activator receptor (suPAR) for predicting outcomes in emergency department (ED) patients. Secondary objectives were 1) to measure the predictive value of the usual decision points, 2) to identify patients at low risk for mortality who could be safely discharged from the ED, and 3) to measure the correlation between suPAR and other biomarkers. MATERIAL AND METHODS: Prospective observational cohort study of patients attended in the EDs of participating hospitals. We recorded sociodemographic variables, comorbidity, variables related to the acute episode, prognostic markers commonly used in EDs, and suPAR concentration. Outcome variables were the need for hospital admission during the index episode, ED revisits within 90 days, and 90-day mortality. RESULTS: A total of 990 patients with a median (interquartile range) age of 68 (53-81 years) were studied; 50.8% were men. The median suPAR concentration was 3.8 (2.8-6.0) ng/mL, and 112 patients (11.31%) required admission. At 90 days there were 276 revisits (27.9% of the cohort), and 47 patients (4.74%) had died. Mortality was lower (1%) in patients with suPAR concentrations less than 4 ng/mL (52.5%), and fewer of these patients revisited (24.4%) or required hospitalization (20.6%) than patients with suPAR concentrations higher than 6 ng/mL (mortality, 13.5%; revisits, 39.6%; admissions, 56.3%). A suPAR concentration over 6 ng/mL was associated with 90-day mortality and revisits (adjusted hazard ratios and 95% CIs of 4.61 [1.68-12.67] and 1.59 [1.13-2.10]), respectively. The high suPAR concentration was also associated with hospital admission (odds ratio, 1.62 [0.99-2.62]). CONCLUSION: A suPAR concentration of less than 4 ng/mL identifies patients at low risk of 90-day mortality and revisits or need for hospitalization, whereas a suPAR concentration higher than 6 ng/mL is associated with higher risk for these outcomes.

OBJETIVO: Determinar la capacidad del receptor soluble del activador del plasminógeno tipo uroquinasa (suPAR) para la estratificación pronóstica en pacientes atendidos en servicios de urgencias hospitalarios (SUH). Los objetivos secundarios son: 1) medir la capacidad de los `puntos de decisión habituales, 2) identificar una población de bajo riesgo de mortalidad que puede darse de alta de forma segura desde el SUH, y 3) medir la correlación entre suPAR y otros biomarcadores. METODO: Estudio observacional de cohortes prospectivo de pacientes atendidos en SUH. Se registraron variables sociodemográficas, de comorbilidad, datos del episodio agudo, biomarcadores de uso común en urgencias y suPAR. Las variables de resultado fueron la necesidad de ingreso en el episodio índice, reconsulta al SUH y mortalidad a los 90 días. RESULTADOS: Se incluyeron 990 pacientes, la edad fue de 68 (53-81) años, 50,8% eran hombres, la mediana de suPAR fue de 3,8 (2,8-6,0) ng/ml, 112 pacientes (11,31%) requirieron ingreso. En el seguimiento a 90 días hubo 276 reconsultas (27,9%) y 47 pacientes (4,74%) fallecieron. Los pacientes con suPAR 4 ng/ml (52,5%) tenían menor mortalidad (1%), menor reconsulta (24,4%) y menor necesidad de ingreso hospitalario (20,6%), que pacientes con suPAR 6 ng/ml (mortalidad 13,5%, reconsulta 39,6% e ingreso 56,3%). Un suPAR 6 ng/ml mostró una hazard ratio (IC 95%) ajustada de 4,61 (1,68-12,67) para predecir mortalidad a 90 días y de 1,59 (1,13-2,10) para la reconsulta, y una odds ratio de 1,62 (0,99-2,62) para la necesidad de ingreso hospitalario. CONCLUSIONES: Un valor de suPAR 4 ng/ml identifica pacientes con riesgo bajo de mortalidad a 90 días, de reconsulta y de necesidad de ingreso, mientras que los pacientes con suPAR 6 ng/ml tienen mayor mortalidad, reconsulta y necesidad de ingreso.

qSOFA combined with suPAR for early risk detection and guidance of antibiotic treatment in the emergency department: a randomized controlled trial.

BACKGROUND: Sepsis guidelines suggest immediate start of resuscitation for patients with quick Sequential Organ Failure Assessment (qSOFA) 2 or 3. However, the interpretation of qSOFA 1 remains controversial. We investigated whether measurements of soluble urokinase plasminogen activator receptor (suPAR) may improve risk detection when qSOFA is 1. METHODS: The study had two parts. At the first part, the combination of suPAR with qSOFA was analyzed in a prospective cohort for early risk detection. At the second part, the double-blind, randomized controlled trial (RCT) SUPERIOR evaluated the efficacy of the suPAR-guided medical intervention. SUPERIOR took place between November 2018 and December 2020. Multivariate stepwise Cox regression was used for the prospective cohort, while univariate and multivariate logistic regression was used for the RCT. Consecutive admissions at the emergency department (ED) with suspected infection, qSOFA 1 and suPAR ≥ 12 ng/mL were allocated to single infusion of placebo or meropenem. The primary endpoint was early deterioration, defined as at least one-point increase of admission Sequential Organ Failure Assessment (SOFA) score the first 24 h. RESULTS: Most of the mortality risk was for patients with qSOFA 2 and 3. Taking the hazard ratio (HR) for death of patients with qSOFA = 1 and suPAR < 12 ng/mL as reference, the HR of qSOFA = 1 and suPAR ≥ 12 ng/mL for 28-day mortality was 2.98 (95% CI 2.11-3.96). The prospective RCT was prematurely ended due to pandemia-related ED re-allocations, with 91 patients enrolled: 47 in the placebo and 44 in the meropenem arm. The primary endpoint was met in 40.4% (n = 19) and 15.9% (n = 7), respectively (difference 24.5% [5.9-40.8]; odds ratio 0.14 [0.04-0.50]). One post hoc analysis showed significant median changes of SOFA score after 72 and 96 h equal to 0 and - 1, respectively. CONCLUSIONS: Combining qSOFA 1 with the biomarker suPAR improves its prognostic performance for unfavorable outcome and can help decision for earlier treatment. Trial registration EU Clinical Trials Register (EudraCT, 2018-001008-13) and Clinical-Trials.gov (NCT03717350). Registered 24 October 2018.

Intubation Decision Based on Illness Severity and Mortality in COVID-19: An International Study.

OBJECTIVES: To evaluate the impact of intubation timing, guided by severity criteria, on mortality in critically ill COVID-19 patients, amidst existing uncertainties regarding optimal intubation practices. DESIGN: Prospective, multicenter, observational study conducted from February 1, 2020, to November 1, 2022. SETTING: Ten academic institutions in the United States and Europe. PATIENTS: Adults (≥ 18 yr old) confirmed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and hospitalized specifically for COVID-19, requiring intubation postadmission. Exclusion criteria included patients hospitalized for non-COVID-19 reasons despite a positive SARS-CoV-2 test. INTERVENTIONS: Early invasive mechanical ventilation (EIMV) was defined as intubation in patients with less severe organ dysfunction (Sequential Organ Failure Assessment [SOFA] < 7 or Pao2/Fio2 ratio > 250), whereas late invasive mechanical ventilation (LIMV) was defined as intubation in patients with SOFA greater than or equal to 7 and Pao2/Fio2 ratio less than or equal to 250. MEASUREMENTS AND MAIN RESULTS: The primary outcome was mortality within 30 days of hospital admission. Among 4464 patients, 854 (19.1%) required mechanical ventilation (mean age 60 yr, 61.7% male, 19.3% Black). Of those, 621 (72.7%) were categorized in the EIMV group and 233 (27.3%) in the LIMV group. Death within 30 days after admission occurred in 278 patients (42.2%) in the EIMV and 88 patients (46.6%) in the LIMV group (p = 0.28). An inverse probability-of-treatment weighting analysis revealed a statistically significant association with mortality, with patients in the EIMV group being 32% less likely to die either within 30 days of admission (adjusted hazard ratio [HR] 0.68; 95% CI, 0.52-0.90; p = 0.008) or within 30 days after intubation irrespective of its timing from admission (adjusted HR 0.70; 95% CI, 0.51-0.90; p = 0.006). CONCLUSIONS: In severe COVID-19 cases, an early intubation strategy, guided by specific severity criteria, is associated with a reduced risk of death. These findings underscore the importance of timely intervention based on objective severity assessments.

Mass spectrometry-based proteomics of cerebrospinal fluid in pediatric central nervous system malignancies: a systematic review with meta-analysis of individual patient data.

BACKGROUND: The cerebrospinal fluid (CSF) proteome could offer important insights into central nervous system (CNS) malignancies. To advance proteomic research in pediatric CNS cancer, the current study aims to (1) evaluate past mass spectrometry-based workflows and (2) synthesize previous CSF proteomic data, focusing on both qualitative summaries and quantitative re-analysis. MAIN: In our analysis of 11 studies investigating the CSF proteome in pediatric patients with acute lymphoblastic leukemia (ALL) or primary brain tumors, we observed significant methodological variability. This variability negatively affects comparative analysis of the included studies, as per GRADE criteria for quality of evidence. The qualitative summaries covered 161 patients and 134 non-tumor controls, while the application of validation cohort varied among the studies. The quantitative re-analysis comprised 15 B-ALL vs 6 "healthy" controls and 15 medulloblastoma patients vs 22 non-tumor controls. Certain CSF proteins were identified as potential indicators of specific malignancies or stages of neurotoxicity during chemotherapy, yet definitive conclusions were impeded by inconsistent data. There were no proteins with statistically significant differences when comparing cases versus controls that were corroborated across studies where quantitative reanalysis was feasible. From a gene ontology enrichment, we observed that age disparities between unmatched case and controls may mislead to protein correlations more indicative of age-related CNS developmental stages rather than neuro-oncological disease. Despite efforts to batch correct (HarmonizR) and impute missing values, merging of dataset proved unfeasible and thereby limited meaningful data integration across different studies. CONCLUSION: Infrequent publications on rare pediatric cancer entities, which often involve small sample sizes, are inherently prone to result in heterogeneous studies-particularly when conducted within a rapidly evolving field like proteomics. As a result, obtaining clear evidence, such as CSF proteome biomarkers for CNS dissemination or early-stage neurotoxicity, is currently impractical. Our general recommendations comprise the need for standardized methodologies, collaborative efforts, and improved data sharing in pediatric CNS malignancy research. We specifically emphasize the possible importance of considering natural age-related variations in CSF due to different CNS development stages when matching cases and controls in future studies.

Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition.

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here we present the narrow-window data-independent acquisition (nDIA) strategy consisting of high-resolution MS1 scans with parallel tandem MS (MS/MS) scans of ~200 Hz using 2-Th isolation windows, dissolving the differences between data-dependent and -independent methods. This is achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer which provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. The nDIA strategy enables profiling of >100 full yeast proteomes per day, or 48 human proteomes per day at the depth of ~10,000 human protein groups in half-an-hour or ~7,000 proteins in 5 min, representing 3× higher coverage compared with current state-of-the-art MS. Multi-shot acquisition of offline fractionated samples provides comprehensive coverage of human proteomes in ~3 h. High quantitative precision and accuracy are demonstrated in a three-species proteome mixture, quantifying 14,000+ protein groups in a single half-an-hour run.

Homo sapiens reached the higher latitudes of Europe by 45,000 years ago.

The Middle to Upper Palaeolithic transition in Europe is associated with the regional disappearance of Neanderthals and the spread of Homo sapiens. Late Neanderthals persisted in western Europe several millennia after the occurrence of H. sapiens in eastern Europe1. Local hybridization between the two groups occurred2, but not on all occasions3. Archaeological evidence also indicates the presence of several technocomplexes during this transition, complicating our understanding and the association of behavioural adaptations with specific hominin groups4. One such technocomplex for which the makers are unknown is the Lincombian-Ranisian-Jerzmanowician (LRJ), which has been described in northwestern and central Europe5-8. Here we present the morphological and proteomic taxonomic identification, mitochondrial DNA analysis and direct radiocarbon dating of human remains directly associated with an LRJ assemblage at the site Ilsenhöhle in Ranis (Germany). These human remains are among the earliest directly dated Upper Palaeolithic H. sapiens remains in Eurasia. We show that early H. sapiens associated with the LRJ were present in central and northwestern Europe long before the extinction of late Neanderthals in southwestern Europe. Our results strengthen the notion of a patchwork of distinct human populations and technocomplexes present in Europe during this transitional period.

ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates.

Protein arginine methylations are important post-translational modifications (PTMs) in eukaryotes, regulating many biological processes. However, traditional collision-based mass spectrometry methods inevitably cause neutral losses of methylarginines, preventing the deep mining of biologically important sites. Herein we developed an optimized mass spectrometry workflow based on electron-transfer dissociation (ETD) with supplemental activation for proteomic profiling of arginine methylation in human cells. Using symmetric dimethylarginine (sDMA) as an example, we show that the ETD-based optimized workflow significantly improved the identification and site localization of sDMA. Quantitative proteomics identified 138 novel sDMA sites as potential PRMT5 substrates in HeLa cells. Further biochemical studies on SERBP1, a newly identified PRMT5 substrate, confirmed the coexistence of sDMA and asymmetric dimethylarginine in the central RGG/RG motif, and loss of either methylation caused increased the recruitment of SERBP1 to stress granules under oxidative stress. Overall, our optimized workflow not only enabled the identification and localization of extensive, nonoverlapping sDMA sites in human cells but also revealed novel PRMT5 substrates whose sDMA may play potentially important biological functions.

Extending the Coverage of Lys-C/Trypsin-Based Bottom-up Proteomics by Cysteine S-Aminoethylation.

To improve the coverage in bottom-up proteomics, S-aminoethylation of cysteine residues (AE-Cys) was carried out with 2-bromoethylamine, followed by cleavage with lysyl endopeptidase (Lys-C) or Lys-C/trypsin. A model study with bovine serum albumin showed that the C-terminal side of AE-Cys was successfully cleaved by Lys-C. The frequency of side reactions at amino acids other than Cys was less than that in the case of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts in the data-dependent acquisition mode after AE-Cys modification afforded a greater number of identified protein groups, especially membrane proteins. In addition, label-free quantification of proteins in mouse nonsmall cell lung cancer (NSCLC) tissue in the data-independent acquisition mode after AE-Cys modification showed improved NSCLC pathway coverage and greater reproducibility. Furthermore, the AE-Cys method could identify an epidermal growth factor receptor peptide containing the T790 M mutation site, a well-established lung-cancer-related mutation site that has evaded conventional bottom-up methods. Finally, AE-Cys was found to fully mimic Lys in terms of collision-induced dissociation fragmentation, ion mobility separation, and cleavage by Lys-C/trypsin, except for sulfoxide formation during sample preparation.

SuPAR, biomarkers of inflammation, and severe outcomes in patients hospitalized for COVID-19: The International Study of Inflammation in COVID-19.

Severe coronavirus disease 2019 (COVID-19) is a hyperinflammatory syndrome. The biomarkers of inflammation best suited to triage patients with COVID-19 are unknown. We conducted a prospective multicenter observational study of adult patients hospitalized specifically for COVID-19 from February 1, 2020 to October 19, 2022. Biomarkers measured included soluble urokinase plasminogen activator receptor (suPAR), C-reactive protein, interleukin-6, procalcitonin, ferritin, and D-dimer. In-hospital outcomes examined include death and the need for mechanical ventilation. Patients admitted in the United States (US, n = 1962) were used to compute area under the curves (AUCs) and identify biomarker cutoffs. The combined European cohorts (n = 1137) were used to validate the biomarker cutoffs. In the US cohort, 356 patients met the composite outcome of death (n = 197) or need for mechanical ventilation (n = 290). SuPAR was the most important predictor of the composite outcome and had the highest AUC (0.712) followed by CRP (0.642), ferritin (0.619), IL-6 (0.614), D-dimer (0.606), and lastly procalcitonin (0.596). Inclusion of other biomarkers did not improve discrimination. A suPAR cutoff of 4.0 ng/mL demonstrated a sensitivity of 95.4% (95% CI: 92.4%-98.0%) and negative predictive value (NPV) of 92.5% (95% CI: 87.5%-96.9%) for the composite outcome. Patients with suPAR < 4.0 ng/mL comprised 10.6% of the cohort and had a 0.8% probability of the composite outcome. Applying this cutoff to the validation cohort yielded a sensitivity of 93.8% (90.4%-96.7%) and NPV of 95.5% (93.1%-97.8%) for the composite outcome. Among commonly measured biomarkers, suPAR offered stronger discriminatory ability and may be useful in triaging low-risk patients with COVID-19.