A reduced proteomic signature in critically ill Covid-19 patients determined with plasma antibody micro-array and machine learning.

BACKGROUND: COVID-19 is a complex, multi-system disease with varying severity and symptoms. Identifying changes in critically ill COVID-19 patients' proteomes enables a better understanding of markers associated with susceptibility, symptoms, and treatment. We performed plasma antibody microarray and machine learning analyses to identify novel proteins of COVID-19. METHODS: A case-control study comparing the concentration of 2000 plasma proteins in age- and sex-matched COVID-19 inpatients, non-COVID-19 sepsis controls, and healthy control subjects. Machine learning was used to identify a unique proteome signature in COVID-19 patients. Protein expression was correlated with clinically relevant variables and analyzed for temporal changes over hospitalization days 1, 3, 7, and 10. Expert-curated protein expression information was analyzed with Natural language processing (NLP) to determine organ- and cell-specific expression. RESULTS: Machine learning identified a 28-protein model that accurately differentiated COVID-19 patients from ICU non-COVID-19 patients (accuracy = 0.89, AUC = 1.00, F1 = 0.89) and healthy controls (accuracy = 0.89, AUC = 1.00, F1 = 0.88). An optimal nine-protein model (PF4V1, NUCB1, CrkL, SerpinD1, Fen1, GATA-4, ProSAAS, PARK7, and NET1) maintained high classification ability. Specific proteins correlated with hemoglobin, coagulation factors, hypertension, and high-flow nasal cannula intervention (P < 0.01). Time-course analysis of the 28 leading proteins demonstrated no significant temporal changes within the COVID-19 cohort. NLP analysis identified multi-system expression of the key proteins, with the digestive and nervous systems being the leading systems. CONCLUSIONS: The plasma proteome of critically ill COVID-19 patients was distinguishable from that of non-COVID-19 sepsis controls and healthy control subjects. The leading 28 proteins and their subset of 9 proteins yielded accurate classification models and are expressed in multiple organ systems. The identified COVID-19 proteomic signature helps elucidate COVID-19 pathophysiology and may guide future COVID-19 treatment development.

The plasma proteome differentiates the multisystem inflammatory syndrome in children (MIS-C) from children with SARS-CoV-2 negative sepsis.

BACKGROUND: The Multi-System Inflammatory Syndrome in Children (MIS-C) can develop several weeks after SARS-CoV-2 infection and requires a distinct treatment protocol. Distinguishing MIS-C from SARS-CoV-2 negative sepsis (SCNS) patients is important to quickly institute the correct therapies. We performed targeted proteomics and machine learning analysis to identify novel plasma proteins of MIS-C for early disease recognition. METHODS: A case-control study comparing the expression of 2,870 unique blood proteins in MIS-C versus SCNS patients, measured using proximity extension assays. The 2,870 proteins were reduced in number with either feature selection alone or with a prior COMBAT-Seq batch effect adjustment. The leading proteins were correlated with demographic and clinical variables. Organ system and cell type expression patterns were analyzed with Natural Language Processing (NLP). RESULTS: The cohorts were well-balanced for age and sex. Of the 2,870 unique blood proteins, 58 proteins were identified with feature selection (FDR-adjusted P < 0.005, P < 0.0001; accuracy = 0.96, AUC = 1.00, F1 = 0.95), and 15 proteins were identified with a COMBAT-Seq batch effect adjusted feature selection (FDR-adjusted P < 0.05, P < 0.0001; accuracy = 0.92, AUC = 1.00, F1 = 0.89). All of the latter 15 proteins were present in the former 58-protein model. Several proteins were correlated with illness severity scores, length of stay, and interventions (LTA4H, PTN, PPBP, and EGF; P < 0.001). NLP analysis highlighted the multi-system nature of MIS-C, with the 58-protein set expressed in all organ systems; the highest levels of expression were found in the digestive system. The cell types most involved included leukocytes not yet determined, lymphocytes, macrophages, and platelets. CONCLUSIONS: The plasma proteome of MIS-C patients was distinct from that of SCNS. The key proteins demonstrated expression in all organ systems and most cell types. The unique proteomic signature identified in MIS-C patients could aid future diagnostic and therapeutic advancements, as well as predict hospital length of stays, interventions, and mortality risks.

Characterization of cardiac fibroblast-extracellular matrix crosstalk across developmental ages provides insight into age-related changes in cardiac repair.

Heart failure afflicts an estimated 6.5 million people in the United States, driven largely by incidents of coronary heart disease (CHD). CHD leads to heart failure due to the inability of adult myocardial tissue to regenerate after myocardial infarction (MI). Instead, immune cells and resident cardiac fibroblasts (CFs), the cells responsible for the maintenance of the cardiac extracellular matrix (cECM), drive an inflammatory wound healing response, which leads to fibrotic scar tissue. However, fibrosis is reduced in fetal and early (<1-week-old) neonatal mammals, which exhibit a transient capability for regenerative tissue remodeling. Recent work by our laboratory and others suggests this is in part due to compositional differences in the cECM and functional differences in CFs with respect to developmental age. Specifically, fetal cECM and CFs appear to mitigate functional loss in MI models and engineered cardiac tissues, compared to adult CFs and cECM. We conducted 2D studies of CFs on solubilized fetal and adult cECM to investigate whether these age-specific functional differences are synergistic with respect to their impact on CF phenotype and, therefore, cardiac wound healing. We found that the CF migration rate and stiffness vary with respect to cell and cECM developmental age and that CF transition to a fibrotic phenotype can be partially attenuated in the fetal cECM. However, this effect was not observed when cells were treated with cytokine TGF-ß1, suggesting that inflammatory signaling factors are the dominant driver of the fibroblast phenotype. This information may be valuable for targeted therapies aimed at modifying the CF wound healing response and is broadly applicable to age-related studies of cardiac remodeling.

Pediatric sepsis inflammatory blood biomarkers that correlate with clinical variables and severity of illness scores.

BACKGROUND: Sepsis is a dysregulated systemic inflammatory response triggered by infection, resulting in organ dysfunction. A major challenge in clinical pediatrics is to identify sepsis early and then quickly intervene to reduce morbidity and mortality. As blood biomarkers hold promise as early sepsis diagnostic tools, we aimed to measure a large number of blood inflammatory biomarkers from pediatric sepsis patients to determine their predictive ability, as well as their correlations with clinical variables and illness severity scores. METHODS: Pediatric patients that met sepsis criteria were enrolled, and clinical data and blood samples were collected. Fifty-eight inflammatory plasma biomarker concentrations were determined using immunoassays. The data were analyzed with both conventional statistics and machine learning. RESULTS: Twenty sepsis patients were enrolled (median age 13 years), with infectious pathogens identified in 75%. Vasopressors were administered to 85% of patients, while 55% received invasive ventilation and 20% were ventilated non-invasively. A total of 24 inflammatory biomarkers were significantly different between sepsis patients and age/sex-matched healthy controls. Nine biomarkers (IL-6, IL-8, MCP-1, M-CSF, IL-1RA, hyaluronan, HSP70, MMP3, and MMP10) yielded AUC parameters > 0.9 (95% CIs: 0.837-1.000; p < 0.001). Boruta feature reduction yielded 6 critical biomarkers with their relative importance: IL-8 (12.2%), MCP-1 (11.6%), HSP70 (11.6%), hyaluronan (11.5%), M-CSF (11.5%), and IL-6 (11.5%); combinations of 2 biomarkers yielded AUC values of 1.00 (95% CI: 1.00-1.00; p < 0.001). Specific biomarkers strongly correlated with illness severity scoring, as well as other clinical variables. IL-3 specifically distinguished bacterial versus viral infection (p < 0.005). CONCLUSIONS: Specific inflammatory biomarkers were identified as markers of pediatric sepsis and strongly correlated to both clinical variables and sepsis severity.

A novel multiplex biomarker panel for profiling human acute and chronic kidney disease.

Acute and chronic kidney disease continues to confer significant morbidity and mortality in the clinical setting. Despite high prevalence of these conditions, few validated biomarkers exist to predict kidney dysfunction. In this study, we utilized a novel kidney multiplex panel to measure 21 proteins in plasma and urine to characterize the spectrum of biomarker profiles in kidney disease. Blood and urine samples were obtained from age-/sex-matched healthy control subjects (HC), critically-ill COVID-19 patients with acute kidney injury (AKI), and patients with chronic or end-stage kidney disease (CKD/ESKD). Biomarkers were measured with a kidney multiplex panel, and results analyzed with conventional statistics and machine learning. Correlations were examined between biomarkers and patient clinical and laboratory variables. Median AKI subject age was 65.5 (IQR 58.5-73.0) and median CKD/ESKD age was 65.0 (IQR 50.0-71.5). Of the CKD/ESKD patients, 76.1% were on hemodialysis, 14.3% of patients had kidney transplant, and 9.5% had CKD without kidney replacement therapy. In plasma, 19 proteins were significantly different in titer between the HC versus AKI versus CKD/ESKD groups, while NAG and RBP4 were unchanged. TIMP-1 (PPV 1.0, NPV 1.0), best distinguished AKI from HC, and TFF3 (PPV 0.99, NPV 0.89) best distinguished CKD/ESKD from HC. In urine, 18 proteins were significantly different between groups except Calbindin, Osteopontin and TIMP-1. Osteoactivin (PPV 0.95, NPV 0.95) best distinguished AKI from HC, and ß2-microglobulin (PPV 0.96, NPV 0.78) best distinguished CKD/ESKD from HC. A variety of correlations were noted between patient variables and either plasma or urine biomarkers. Using a novel kidney multiplex biomarker panel, together with conventional statistics and machine learning, we identified unique biomarker profiles in the plasma and urine of patients with AKI and CKD/ESKD. We demonstrated correlations between biomarker profiles and patient clinical variables. Our exploratory study provides biomarker data for future hypothesis driven research on kidney disease.

One Billion hiPSC-Cardiomyocytes: Upscaling Engineered Cardiac Tissues to Create High Cell Density Therapies for Clinical Translation in Heart Regeneration.

Despite the overwhelming use of cellularized therapeutics in cardiac regenerative engineering, approaches to biomanufacture engineered cardiac tissues (ECTs) at clinical scale remain limited. This study aims to evaluate the impact of critical biomanufacturing decisions-namely cell dose, hydrogel composition, and size-on ECT formation and function-through the lens of clinical translation. ECTs were fabricated by mixing human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) and human cardiac fibroblasts into a collagen hydrogel to engineer meso-(3 × 9 mm), macro- (8 × 12 mm), and mega-ECTs (65 × 75 mm). Meso-ECTs exhibited a hiPSC-CM dose-dependent response in structure and mechanics, with high-density ECTs displaying reduced elastic modulus, collagen organization, prestrain development, and active stress generation. Scaling up, cell-dense macro-ECTs were able to follow point stimulation pacing without arrhythmogenesis. Finally, we successfully fabricated a mega-ECT at clinical scale containing 1 billion hiPSC-CMs for implantation in a swine model of chronic myocardial ischemia to demonstrate the technical feasibility of biomanufacturing, surgical implantation, and engraftment. Through this iterative process, we define the impact of manufacturing variables on ECT formation and function as well as identify challenges that must still be overcome to successfully accelerate ECT clinical translation.

Action potential metrics and automated data analysis pipeline for cardiotoxicity testing using optically mapped hiPSC-derived 3D cardiac microtissues.

Recent advances in human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues provide a unique opportunity for cardiotoxic assessment of pharmaceutical and environmental compounds. Here, we developed a series of automated data processing algorithms to assess changes in action potential (AP) properties for cardiotoxicity testing in 3D engineered cardiac microtissues generated from hiPSC-derived cardiomyocytes (hiPSC-CMs). Purified hiPSC-CMs were mixed with 5-25% human cardiac fibroblasts (hCFs) under scaffold-free conditions and allowed to self-assemble into 3D spherical microtissues in 35-microwell agarose gels. Optical mapping was performed to quantify electrophysiological changes. To increase throughput, AP traces from 4x4 cardiac microtissues were simultaneously acquired with a voltage sensitive dye and a CMOS camera. Individual microtissues showing APs were identified using automated thresholding after Fourier transforming traces. An asymmetric least squares method was used to correct non-uniform background and baseline drift, and the fluorescence was normalized (ΔF/F0). Bilateral filtering was applied to preserve the sharpness of the AP upstroke. AP shape changes under selective ion channel block were characterized using AP metrics including stimulation delay, rise time of AP upstroke, APD30, APD50, APD80, APDmxr (maximum rate change of repolarization), and AP triangulation (APDtri = APDmxr-APD50). We also characterized changes in AP metrics under various ion channel block conditions with multi-class logistic regression and feature extraction using principal component analysis of human AP computer simulations. Simulation results were validated experimentally with selective pharmacological ion channel blockers. In conclusion, this simple and robust automated data analysis pipeline for evaluating key AP metrics provides an excellent in vitro cardiotoxicity testing platform for a wide range of environmental and pharmaceutical compounds.

Organ and cell-specific biomarkers of Long-COVID identified with targeted proteomics and machine learning.

BACKGROUND: Survivors of acute COVID-19 often suffer prolonged, diffuse symptoms post-infection, referred to as "Long-COVID". A lack of Long-COVID biomarkers and pathophysiological mechanisms limits effective diagnosis, treatment and disease surveillance. We performed targeted proteomics and machine learning analyses to identify novel blood biomarkers of Long-COVID. METHODS: A case-control study comparing the expression of 2925 unique blood proteins in Long-COVID outpatients versus COVID-19 inpatients and healthy control subjects. Targeted proteomics was accomplished with proximity extension assays, and machine learning was used to identify the most important proteins for identifying Long-COVID patients. Organ system and cell type expression patterns were identified with Natural Language Processing (NLP) of the UniProt Knowledgebase. RESULTS: Machine learning analysis identified 119 relevant proteins for differentiating Long-COVID outpatients (Bonferonni corrected P < 0.01). Protein combinations were narrowed down to two optimal models, with nine and five proteins each, and with both having excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, F1 = 1.00). NLP expression analysis highlighted the diffuse organ system involvement in Long-COVID, as well as the involved cell types, including leukocytes and platelets, as key components associated with Long-COVID. CONCLUSIONS: Proteomic analysis of plasma from Long-COVID patients identified 119 highly relevant proteins and two optimal models with nine and five proteins, respectively. The identified proteins reflected widespread organ and cell type expression. Optimal protein models, as well as individual proteins, hold the potential for accurate diagnosis of Long-COVID and targeted therapeutics.

Cross-immunity against SARS-COV-2 variants of concern in naturally infected critically ill COVID-19 patients.

Critically ill patients infected with SARS-CoV-2 display adaptive immunity, but it is unknown if they develop cross-reactivity to variants of concern (VOCs). We profiled cross-immunity against SARS-CoV-2 VOCs in naturally infected, non-vaccinated, critically ill COVID-19 patients. Wave-1 patients (wild-type infection) were similar in demographics to Wave-3 patients (wild-type/alpha infection), but Wave-3 patients had higher illness severity. Wave-1 patients developed increasing neutralizing antibodies to all variants, as did patients during Wave-3. Wave-3 patients, when compared to Wave-1, developed more robust antibody responses, particularly for wild-type, alpha, beta and delta variants. Within Wave-3, neutralizing antibodies were significantly less to beta and gamma VOCs, as compared to wild-type, alpha and delta. Patients previously diagnosed with cancer or chronic obstructive pulmonary disease had significantly fewer neutralizing antibodies. Naturally infected ICU patients developed adaptive responses to all VOCs, with greater responses in those patients more likely to be infected with the alpha variant, versus wild-type.

Beyond pharmaceuticals: Fit-for-purpose new approach methodologies for environmental cardiotoxicity testing.

Environmental factors play a substantial role in determining cardiovascular health, but data informing the risks presented by environmental toxicants is insufficient. In vitro new approach methodologies (NAMs) offer a promising approach with which to address the limitations of traditional in vivo and in vitro assays for assessing cardiotoxicity. Driven largely by the needs of pharmaceutical toxicity testing, considerable progress in developing NAMs for cardiotoxicity analysis has already been made. As the scientific and regulatory interest in NAMs for environmental chemicals continues to grow, a thorough understanding of the unique features of environmental cardiotoxicants and their associated cardiotoxicities is needed. Here, we review the key characteristics of as well as important regulatory and biological considerations for fit-for-purpose NAMs for environmental cardiotoxicity. By emphasizing the challenges and opportunities presented by NAMs for environmental cardiotoxicity we hope to accelerate their development, acceptance, and application.

Novel plasma protein biomarkers from critically ill sepsis patients.

BACKGROUND: Despite the high morbidity and mortality associated with sepsis, the relationship between the plasma proteome and clinical outcome is poorly understood. In this study, we used targeted plasma proteomics to identify novel biomarkers of sepsis in critically ill patients. METHODS: Blood was obtained from 15 critically ill patients with suspected/confirmed sepsis (Sepsis-3.0 criteria) on intensive care unit (ICU) Day-1 and Day-3, as well as age- and sex-matched 15 healthy control subjects. A total of 1161 plasma proteins were measured with proximal extension assays. Promising sepsis biomarkers were narrowed with machine learning and then correlated with relevant clinical and laboratory variables. RESULTS: The median age for critically ill sepsis patients was 56 (IQR 51-61) years. The median MODS and SOFA values were 7 (IQR 5.0-8.0) and 7 (IQR 5.0-9.0) on ICU Day-1, and 4 (IQR 3.5-7.0) and 6 (IQR 3.5-7.0) on ICU Day-3, respectively. Targeted proteomics, together with feature selection, identified the leading proteins that distinguished sepsis patients from healthy control subjects with ≥ 90% classification accuracy; 25 proteins on ICU Day-1 and 26 proteins on ICU Day-3 (6 proteins overlapped both ICU days; PRTN3, UPAR, GDF8, NTRK3, WFDC2 and CXCL13). Only 7 of the leading proteins changed significantly between ICU Day-1 and Day-3 (IL10, CCL23, TGFα1, ST2, VSIG4, CNTN5, and ITGAV; P < 0.01). Significant correlations were observed between a variety of patient clinical/laboratory variables and the expression of 15 proteins on ICU Day-1 and 14 proteins on ICU Day-3 (P < 0.05). CONCLUSIONS: Targeted proteomics with feature selection identified proteins altered in critically ill sepsis patients relative to healthy control subjects. Correlations between protein expression and clinical/laboratory variables were identified, each providing pathophysiological insight. Our exploratory data provide a rationale for further hypothesis-driven sepsis research.

Elevated vascular transformation blood biomarkers in Long-COVID indicate angiogenesis as a key pathophysiological mechanism.

BACKGROUND: Long-COVID is characterized by prolonged, diffuse symptoms months after acute COVID-19. Accurate diagnosis and targeted therapies for Long-COVID are lacking. We investigated vascular transformation biomarkers in Long-COVID patients. METHODS: A case-control study utilizing Long-COVID patients, one to six months (median 98.5 days) post-infection, with multiplex immunoassay measurement of sixteen blood biomarkers of vascular transformation, including ANG-1, P-SEL, MMP-1, VE-Cad, Syn-1, Endoglin, PECAM-1, VEGF-A, ICAM-1, VLA-4, E-SEL, thrombomodulin, VEGF-R2, VEGF-R3, VCAM-1 and VEGF-D. RESULTS: Fourteen vasculature transformation blood biomarkers were significantly elevated in Long-COVID outpatients, versus acutely ill COVID-19 inpatients and healthy controls subjects (P < 0.05). A unique two biomarker profile consisting of ANG-1/P-SEL was developed with machine learning, providing a classification accuracy for Long-COVID status of 96%. Individually, ANG-1 and P-SEL had excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, P < 0.0001; validated in a secondary cohort). Specific to Long-COVID, ANG-1 levels were associated with female sex and a lack of disease interventions at follow-up (P < 0.05). CONCLUSIONS: Long-COVID patients suffer prolonged, diffuse symptoms and poorer health. Vascular transformation blood biomarkers were significantly elevated in Long-COVID, with angiogenesis markers (ANG-1/P-SEL) providing classification accuracy of 96%. Vascular transformation blood biomarkers hold potential for diagnostics, and modulators of angiogenesis may have therapeutic efficacy.

A Distinct Metabolite Signature in Military Personnel Exposed to Repetitive Low-Level Blasts.

Military Breachers and Range Staff (MBRS) are subjected to repeated sub-concussive blasts, and they often report symptoms that are consistent with a mild traumatic brain injury (mTBI). Biomarkers of blast injury would potentially aid blast injury diagnosis, surveillance and avoidance. Our objective was to identify plasma metabolite biomarkers in military personnel that were exposed to repeated low-level or sub-concussive blast overpressure. A total of 37 military members were enrolled (18 MBRS and 19 controls), with MBRS having participated in 8-20 breaching courses per year, with a maximum exposure of 6 blasts per day. The two cohorts were similar except that the number of blast exposures were significantly higher in the MBRS, and the MBRS cohort suffered significantly more post-concussive symptoms and poorer health on assessment. Metabolomics profiling demonstrated significant differences between groups with 74% MBRS classification accuracy (CA). Feature reduction identified 6 metabolites that resulted in a MBRS CA of 98%, and included acetic acid (23.7%), formate (22.6%), creatine (14.8%), acetone (14.2%), methanol (12,7%), and glutamic acid (12.0%). All 6 metabolites were examined with individual receiver operating characteristic (ROC) curve analyses and demonstrated areas-under-the-curve (AUCs) of 0.82-0.91 (P ≤ 0.001) for MBRS status. Several parsimonious combinations of three metabolites increased accuracy of ROC curve analyses to AUCs of 1.00 (P < 0.001), while a combination of volatile organic compounds (VOCs; acetic acid, acetone and methanol) yielded an AUC of 0.98 (P < 0.001). Candidate biomarkers for chronic blast exposure were identified, and if validated in a larger cohort, may aid surveillance and care of military personnel. Future point-of-care screening could be developed that measures VOCs from breath, with definitive diagnoses confirmed with plasma metabolomics profiling.

The Use of Cremation Data for Timely Mortality Surveillance During the COVID-19 Pandemic in Ontario, Canada: Validation Study.

BACKGROUND: Early estimates of excess mortality are crucial for understanding the impact of COVID-19. However, there is a lag of several months in the reporting of vital statistics mortality data for many jurisdictions, including across Canada. In Ontario, a Canadian province, certification by a coroner is required before cremation can occur, creating real-time mortality data that encompasses the majority of deaths within the province. OBJECTIVE: This study aimed to validate the use of cremation data as a timely surveillance tool for all-cause mortality during a public health emergency in a jurisdiction with delays in vital statistics data. Specifically, this study aimed to validate this surveillance tool by determining the stability, timeliness, and robustness of its real-time estimation of all-cause mortality. METHODS: Cremation records from January 2020 until April 2021 were compared to the historical records from 2017 to 2019, grouped according to week, age, sex, and whether COVID-19 was the cause of death. Cremation data were compared to Ontario's provisional vital statistics mortality data released by Statistics Canada. The 2020 and 2021 records were then compared to previous years (2017-2019) to determine whether there was excess mortality within various age groups and whether deaths attributed to COVID-19 accounted for the entirety of the excess mortality. RESULTS: Between 2017 and 2019, cremations were performed for 67.4% (95% CI 67.3%-67.5%) of deaths. The proportion of cremated deaths remained stable throughout 2020, even within age and sex categories. Cremation records are 99% complete within 3 weeks of the date of death, which precedes the compilation of vital statistics data by several months. Consequently, during the first wave (from April to June 2020), cremation records detected a 16.9% increase (95% CI 14.6%-19.3%) in all-cause mortality, a finding that was confirmed several months later with cremation data. CONCLUSIONS: The percentage of Ontarians cremated and the completion of cremation data several months before vital statistics did not change meaningfully during the COVID-19 pandemic period, establishing that the pandemic did not significantly alter cremation practices. Cremation data can be used to accurately estimate all-cause mortality in near real-time, particularly when real-time mortality estimates are needed to inform policy decisions for public health measures. The accuracy of this excess mortality estimation was confirmed by comparing it with official vital statistics data. These findings demonstrate the utility of cremation data as a complementary data source for timely mortality information during public health emergencies.

Pediatric severe traumatic brain injury mortality prediction determined with machine learning-based modeling.

INTRODUCTION: Severe traumatic brain injury (sTBI) is a leading cause of mortality in children. As clinical prognostication is important in guiding optimal care and decision making, our goal was to create a highly discriminative sTBI outcome prediction model for mortality. METHODS: Machine learning and advanced analytics were applied to the patient admission variables obtained from a comprehensive pediatric sTBI database. Demographic and clinical data, head CT imaging abnormalities and blood biochemical data from 196 children and adolescents admitted to a tertiary pediatric intensive care unit (PICU) with sTBI were integrated using feature ranking by way of a forest of randomized decision trees, and a model was generated from a reduced number of admission variables with maximal ability to discriminate outcome. RESULTS: In total, 36 admission variables were analyzed using feature ranking with variable weighting to determine their predictive importance for mortality following sTBI. Reduction analysis utilizing Borata feature selection resulted in a parsimonious six-variable model with a mortality classification accuracy of 82%. The final admission variables that predicted mortality were: partial thromboplastin time (22%); motor Glasgow Coma Scale (21%); serum glucose (16%); fixed pupil(s) (16%); platelet count (13%) and creatinine (12%). Using only these six admission variables, a t-distributed stochastic nearest neighbor embedding algorithm plot demonstrated visual separation of sTBI patients that lived or died, with high mortality predictive ability of this model on the validation dataset (AUC = 0.90) which was confirmed with a conventional area-under-the-curve statistical approach on the total dataset (AUC = 0.91; P < 0.001). CONCLUSIONS: Machine learning-based modeling identified the most clinically important prognostic factors resulting in a pragmatic, high performing prognostic tool for pediatric sTBI with excellent discriminative ability to predict mortality risk with 82% classification accuracy (AUC = 0.90). After external multicenter validation, our prognostic model might help to guide treatment decisions, aggressiveness of therapy and prepare family members and caregivers for timely end-of-life discussions and decision making. LEVEL OF EVIDENCE: III; Prognostic.

Human Atrial Cardiac Microtissues for Chamber-Specific Arrhythmic Risk Assessment.

INTRODUCTION: Although atrial fibrillation is the most prevalent disorder of electrical conduction, the mechanisms behind atrial arrhythmias remain elusive. To address this challenge, we developed a robust in vitro model of 3D atrial microtissue from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and evaluated chamber-specific chemical responses experimentally and computationally. METHODS: We differentiated atrial and ventricular cardiomyocytes (aCMs/vCMs) from GCaMP6f-expressing hiPSCs and assessed spontaneous AP activity using fluorescence imaging. Self-assembling 3D microtissues were formed with lactate purified CMs and 5% human cardiac fibroblasts and electrically stimulated for one week before high resolution action potential (AP) optical mapping. AP responses to the atrial-specific potassium repolarizing current I Kur-blocker 4-Aminopyridine (4-AP) and funny current I f-blocker Ivabradine were characterized within their therapeutic window. Finally, we expanded upon a published hiPSC-CM computational model by incorporating the atrial-specific I Kur current, modifying ion channel conductances to match the AP waveforms of our microtissues, and employing the updated model to reinforce our experimental findings. RESULTS: High purity CMs (> 75% cTnT+) demonstrated subtype specification by MLC2v expression. Spontaneous beating rates significantly decreased following 3D microtissue formation, with atrial microtissues characterized by their faster spontaneous beating rate, slower AP rise time, and shorter AP duration (APD) compared to ventricular microtissues. We measured atrial-specific responses, including dose-dependent APD prolongation with 4-AP treatment and dose-dependent reduction in spontaneous activity post-Ivabradine treatment. CONCLUSION: The presented in vitro platform for screening atrial-specific responses is both robust and sensitive, with high throughput, enabling studies focused at elucidating the mechanisms underlying atrial arrhythmias. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00703-x.

Novel severe traumatic brain injury blood outcome biomarkers identified with proximity extension assay.

OBJECTIVES: Severe traumatic brain injury (sTBI) patients suffer high mortality. Accurate prognostic biomarkers have not been identified. In this exploratory study, we performed targeted proteomics on plasma obtained from sTBI patients to identify potential outcome biomarkers. METHODS: Blood sample was collected from patients admitted to the ICU suffering a sTBI, using standardized clinical and computerized tomography (CT) imaging criteria. Age- and sex-matched healthy control subjects and sTBI patients were enrolled. Targeted proteomics was performed on plasma with proximity extension assays (1,161 proteins). RESULTS: Cohorts were well-balanced for age and sex. The majority of sTBI patients were injured in motor vehicle collisions and the most frequent head CT finding was subarachnoid hemorrhage. Mortality rate for sTBI patients was 40%. Feature selection identified the top performing 15 proteins for identifying sTBI patients from healthy control subjects with a classification accuracy of 100%. The sTBI proteome was dominated by markers of vascular pathology, immunity/inflammation, cell survival and macrophage/microglia activation. Receiver operating characteristic (ROC) curve analyses demonstrated areas-under-the-curves (AUC) for identifying sTBI that ranged from 0.870-1.000 (p≤0.005). When mortality was used as outcome, ROC curve analyses identified the top 3 proteins as Willebrand factor (vWF), Wnt inhibitory factor-1 (WIF-1), and colony stimulating factor-1 (CSF-1). Combining vWF with either WIF-1 or CSF-1 resulted in excellent mortality prediction with AUC of 1.000 for both combinations (p=0.011). CONCLUSIONS: Targeted proteomics with feature classification and selection distinguished sTBI patients from matched healthy control subjects. Two protein combinations were identified that accurately predicted sTBI patient mortality. Our exploratory findings require confirmation in larger sTBI patient populations.

Is Human Walking a Network Medicine Problem? An Analysis Using Symbolic Regression Models with Genetic Programming.

BACKGROUND AND OBJECTIVE: Human walking is typically assessed using a sensor placed on the lower back or the hip. Such analyses often ignore that the arms, legs, and body trunk movements all have significant roles during walking; in other words, these body nodes with accelerometers form a body sensor network (BSN). BSN refers to a network of wearable sensors or devices on the human body that collects physiological signals. Our study proposes that human locomotion could be considered as a network of well-connected nodes. METHODS: While hypothesizing that accelerometer data can model this BSN, we collected accelerometer signals from six body areas from ten healthy participants performing a cognitive task. Machine learning based on genetic programming was used to produce a collection of non-linear symbolic models of human locomotion. RESULTS: With implications in precision medicine, our primary finding was that our BSN models fit the data from the lower back's accelerometer and describe subject-specific data the best compared to all other models. Across subjects, models were less effective due to the diversity of human sizes. CONCLUSIONS: A BSN relationship between all six body nodes has been shown to describe the subject-specific data, which indicates that the network-medicine relationship between these nodes is essential in adequately describing human walking. Our gait analyses can be used for several clinical applications such as medical diagnostics as well as creating a baseline for healthy walking with and without a cognitive load.

Case Report: Inflammation and Endothelial Injury Profiling of COVID-19 Pediatric Multisystem Inflammatory Syndrome (MIS-C).

Introduction: COVID-19 is associated with a novel multi-system inflammatory syndrome that shares some characteristics with Kawasaki's Disease. The syndrome manifestation is delayed relative to COVID-19 onset, with a spectrum of clinical severity. Clinical signs may include persistent fever, gastrointestinal symptoms, cardiac inflammation and/or shock. Case Presentation: We measured 59 inflammatory and endothelial injury plasma analytes in an adolescent girl that presented with malaise, fever, cough, strawberry tongue and jaundice. Her COVID-19 status was positive with detection of 2 SARS-CoV-2 viral genes using polymerase chain reaction. She was treated with intravenous immunoglobulin prior to blood draw, but our plasma measurements suggested a unique analyte expression pattern associated with inflammation, endothelial injury and microvascular glycocalyx degradation. Conclusions: COVID-19 is associated with a multi-system inflammatory syndrome and a unique inflammatory and endothelial injury signature. Summary: Analyte markers of inflammation and endothelial cell injury might serve as putative biomarkers and/or be investigated further as potential therapeutic targets.

Repetitive mild traumatic brain injury in mice triggers a slowly developing cascade of long-term and persistent behavioral deficits and pathological changes.

We have previously reported long-term changes in the brains of non-concussed varsity rugby players using magnetic resonance spectroscopy (MRS), diffusion tensor imaging (DTI) and functional magnetic imaging (fMRI). Others have reported cognitive deficits in contact sport athletes that have not met the diagnostic criteria for concussion. These results suggest that repetitive mild traumatic brain injuries (rmTBIs) that are not severe enough to meet the diagnostic threshold for concussion, produce long-term consequences. We sought to characterize the neuroimaging, cognitive, pathological and metabolomic changes in a mouse model of rmTBI. Using a closed-skull model of mTBI that when scaled to human leads to rotational and linear accelerations far below what has been reported for sports concussion athletes, we found that 5 daily mTBIs triggered two temporally distinct types of pathological changes. First, during the first days and weeks after injury, the rmTBI produced diffuse axonal injury, a transient inflammatory response and changes in diffusion tensor imaging (DTI) that resolved with time. Second, the rmTBI led to pathological changes that were evident months after the injury including: changes in magnetic resonance spectroscopy (MRS), altered levels of synaptic proteins, behavioural deficits in attention and spatial memory, accumulations of pathologically phosphorylated tau, altered blood metabolomic profiles and white matter ultrastructural abnormalities. These results indicate that exceedingly mild rmTBI, in mice, triggers processes with pathological consequences observable months after the initial injury.