Ubiquitin C-terminal Hydrolase L1 Research Articles

Minor head injury in anticoagulated patients: performance of biomarkers S100B, NSE, GFAP, UCH-L1 and Alinity TBI in the detection of intracranial injury. A prospective observational study.

Data in literature indicate that in patients suffering a minor head injury (MHI), biomarkers serum levels could be effective to predict the absence of intracranial injury (ICI) on head CT scan. Use of these biomarkers in case of patients taking oral anticoagulants who experience MHI is very limited. We investigated biomarkers as predictors of ICI in anticoagulated patients managed in an ED. We conducted a single-cohort, prospective, observational study in an ED. Our structured clinical pathway included a first head CT scan, 24 h observation and a second CT scan. The outcome was delayed ICI (dICI), defined as ICI on the second CT scan after a first negative CT scan. We assessed the sensitivity (SE), specificity (SP), negative predictive value (NNV) and positive predictive value (PPV) of the biomarkers S100B, NSE, GFAP, UCH-L1 and Alinity TBI in order to identify dICI. Our study population was of 234 patients with a negative first CT scan who underwent a second CT scan. The rate of dICI was 4.7 %. The NPV for the detection of dICI were respectively (IC 95 %): S100B 92.7 % (86.0-96.8 %,); ubiquitin C-terminal hydrolase-L1 (UCH-L1) 91.8 % (83.8-96.6 %); glial fibrillary protein (GFP) 100 % (83.2-100 %); TBI 100 % (66.4-100 %). The AUC for the detection of dICI was 0.407 for S100B, 0.563 for neuron-specific enolase (NSE), 0.510 for UCH-L1 and 0.720 for glial fibrillary acidic protein (GFAP), respectively. The NPV of the analyzed biomarkers were high and they potentially could limit the number of head CT scan for detecting dICI in anticoagulated patients suffering MHI. GFAP and Alinity TBI seem to be effective to rule out a dCI, but future trials are needed.

Diabetic sensory neuropathy and insulin resistance are induced by loss of UCHL1 in Drosophila

Diabetic sensory neuropathy (DSN) is one of the most common complications of type 2 diabetes (T2D), however the molecular mechanistic association between T2D and DSN remains elusive. Here we identify ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinase highly expressed in neurons, as a key molecule underlying T2D and DSN. Genetic ablation of UCHL1 leads to neuronal insulin resistance and T2D-related symptoms in Drosophila. Furthermore, loss of UCHL1 induces DSN-like phenotypes, including numbness to external noxious stimuli and axonal degeneration of sensory neurons in flies’ legs. Conversely, UCHL1 overexpression improves DSN-like defects of T2D model flies. UCHL1 governs insulin signaling by deubiquitinating insulin receptor substrate 1 (IRS1) and antagonizes an E3 ligase of IRS1, Cullin 1 (CUL1). Consistent with these results, genetic and pharmacological suppression of CUL1 activity rescues T2D- and DSN-associated phenotypes. Therefore, our findings suggest a complete set of genetic factors explaining T2D and DSN, together with potential remedies for the diseases.

Altered Protein Dynamics and a More Reactive Catalytic Cysteine in a Neurodegeneration-associated UCHL1 Mutant

A mutant of ubiquitin C-terminal hydrolase L1 (UCHL1) detected in early-onset neurodegenerative patients, UCHL1R178Q, showed higher catalytic activity than wild-type UCHL1 (UCHL1WT). Lying within the active-site pocket, the arginine is part of an interaction network that holds the catalytic histidine in an inactive arrangement. However, the structural basis and mechanism of enzymatic activation upon glutamine substitution was not understood. We combined X-ray crystallography, protein nuclear magnetic resonance (NMR) analysis, enzyme kinetics, covalent inhibition analysis, and biophysical measurements to delineate activating factors in the mutant. While the crystal structure of UCHL1R178Q showed nearly the same arrangement of the catalytic residues and active-site pocket, the mutation caused extensive alteration in the chemical environment and dynamics of more than 30 residues, some as far as 15 Å away from the site of mutation. Significant broadening of backbone amide resonances in the HSQC spectra indicates considerable backbone dynamics changes in several residues, in agreement with solution small-angle X-ray scattering (SAXS) analyses which indicate an overall increase in protein flexibility. Enzyme kinetics show the activation is due to a kcat effect despite a slightly weakened substrate affinity. In line with this, the mutant shows a higher second-order rate constant (kinact/Ki) in a reaction with a substrate-derived irreversible inhibitor, Ub-VME, compared to the wild-type enzyme, an observation indicative of a more reactive catalytic cysteine in the mutant. Together, the observations underscore structural plasticity as a factor contributing to enzyme kinetic behavior which can be modulated through mutational effects.

Ubiquitin C-terminal hydrolase L1 is a regulator of tumor growth and metastasis in double-negative prostate cancer

Ubiquitin C-terminal hydrolase L1 is a regulator of tumor growth and metastasis in double-negative prostate cancer

Investigating the Association Between Extended Participation in Collision Sports and Fluid Biomarkers Among Masters Athletes.

Traumatic brain injuries (TBIs) and concussions are prevalent in collision sports, and there is evidence that levels of exposure to such sports may increase the risk of neurological abnormalities. Elevated levels of fluid-based biomarkers have been observed after concussions or among athletes with a history of participating in collision sports, and certain biomarkers exhibit sensitivity toward neurodegeneration. This study investigated a cohort of 28 male amateur athletes competing in "Masters" competitions for persons >35 years of age. The primary objective of this study was to compare the levels of blood and saliva biomarkers associated with brain injury, inflammation, aging, and neurodegeneration between athletes with an extensive history of collision sport participation (i.e., median = 27 years; interquartile range = 18-44, minimum = 8) and those with no history. Plasma proteins associated with neural damage and neurodegeneration were measured using Simoa® assays, and saliva was analyzed for markers associated with inflammation and telomere length using quantitative real-time polymerase chain reaction. There were no significant differences between collision and non-collision sport athletes for plasma levels of glial fibrillary acidic protein, neurofilament light, ubiquitin C-terminal hydrolase L1, tau, tau phosphorylated at threonine 181, and brain-derived neurotrophic factor. Moreover, salivary levels of genes associated with inflammation and telomere length were similar between groups. There were no significant differences between groups in symptom frequency or severity on the Sport Concussion Assessment Tool-5th Edition. Overall, these findings provide preliminary evidence that biomarkers associated with neural tissue damage, neurodegeneration, and inflammation may not exhibit significant alterations in asymptomatic amateur athletes with an extensive history of amateur collision sport participation.

Biofluid-based Biomarkers in Traumatic Brain Injury: A Narrative Review.

Traumatic brain injury (TBI) is a complex condition characterized by a multifaceted pathophysiology. It presents significant diagnostic and prognostic challenges in clinical settings. This narrative review explores the evolving role of biofluid biomarkers as essential tools in the diagnosis, prognosis, and treatment of TBI. In recent times, preclinical and clinical trials utilizing these biofluid biomarkers have been actively pursued internationally. Among the biomarkers for nerve tissue proteins are neuronal biomarkers like neuronal specific enolase and ubiquitin C-terminal hydrolase L1; astroglia injury biomarkers such as S100B and glial fibrillary acidic protein; axonal injury and demyelination biomarkers, including neurofilaments and myelin basic protein; new axonal injury and neurodegeneration biomarkers like total tau and phosphorylated tau; and others such as spectrin breakdown products and microtubule-associated protein 2. The interpretation of these biomarkers can be influenced by various factors, including secretion from organs other than the injury site and systemic conditions. This review highlights the potential of these biomarkers to transform TBI management and emphasizes the need for continued research to validate their efficacy, refine testing platforms, and ultimately improve patient care and outcomes.

Plasma biomarkers as prognostic markers in Alzheimer’s disease: Influence of age at onset

AbstractBackgroundLittle is known about the influence of age at onset (AAO) on plasma biomarkers and their use as prognostic biomarkers in Alzheimer’s disease (AD).MethodWe selected patients with AD diagnosis with available neuropsychological testing (NPS) at time of diagnosis and two years later, and plasma biomarkers at baseline.NPS battery included Free and Cued Selective Reminding Test (FCSRT), Landscape test (visual memory), Boston Naming Test, Semantic Fluency, BDAE auditory comprehension, Constructional and Ideomotor Praxis, Visual Object and Space Perception (VOSP) Incomplete Letters and Number Location subtests, Trail Making Test (TMT) A and B, Phonemic Fluency, and Digit Span Forward and Backward. NPS scores were compared by AAO: early‐onset AD (EOAD; <65 years) vs. late onset AD (LOAD; >65y).We analyzed plasma biomarkers phosphorilated‐tau181 (p‐tau181), total tau (t‐tau), neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP) and ubiquitin C‐terminal hydrolase L1 (UCHL‐1) using the Quanterix Simoa p‐tau181 Advantage V2 and Neurology 4‐Plex A assays. Group comparisons and linear regressions adjusted by years of education (YOE) were performed in Stata/IC 16.1.ResultForty‐two participants were included, 23 LOAD and 19 EOAD. Plasma p‐tau181 and GFAP levels were higher in LOAD (Table 1). We did not find differences between LOAD and EOAD in NPS tests at baseline or +2 years (Table 2).Plasma ptau‐181 was associated with progression in MMSE globally, VOSP‐Incomplete letters globally and in EOAD. Plasma NfL were associated to Boston Naming test globally and in EOAD, Semantic fluency test globally, VOSP‐incomplete letters in EOAD, and Free and Total Learning of FCSRT in LOAD. Plasma GFAP was associated to MMSE globally and in EOAD, Free learning of FCSRT in LOAD and VOSP‐Incomplete letters and number location globally. Plasma UCHL‐1 was associated to Semantic fluency test in LOAD (Table 3). Praxis and attention and executive function tests loss were not associated to plasma biomarkers.ConclusionPlasma p‐tau18, NfL, GFAP and UCHL‐1 were associated to pogression in memory, language and visual tests. They were predominantly associated to memory loss in LOAD and language and visual function loss in EOAD. Results need to be interpreted cautiously due to small sample size.

Serum biomarkers associated with aging and neurodegeneration in common marmosets (Callithrix jacchus)

The common marmoset (Callithrix jacchus), a small South American monkey, is an important nonhuman primate model in the study of aging and age-related neurodegenerative disease, including Alzheimer’s disease, Parkinson’s disease, and related dementias. Thorough characterization of the wild type marmoset brain agingmodel, including biomarkers of aging and neural degeneration, will further the marmoset’s utility in translational research. We measured serum concentration of four key biomarkers of neural degeneration [total tau (T-tau), glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), and ubiquitin C-terminal hydrolase-L1 (UCH-L1)] via single molecule array from 24 marmosets (female n = 13, male n = 11) ranging in age from 1.3 to 18.7 years. Aged marmosets (>7 years) had significantly higher GFAP, NfL, UCH-L1, and T-tau than adult marmosets. Sex differences were not detected for any of these biomarker concentrations. These data provide an important initial range of reference values for GFAP, NfL, T-tau, and UCH-L1 to evaluate aging and neural health in marmosets, as well as evaluation of therapeutics in clinical models of disease.

Expression of Ubiquitin C-Terminal Hydrolase-L1 in Minor Normal Salivary Gland Tissues in Perilesional Area to OSCC

Ubiquitin C-terminal hydrolase L1 (UCH-L1), is a deubiquitinating enzyme. Certain changes in ubiquitination attribute to uncontrolled growth, leading to oncogenesis. The aim of this study was to characterize UCH-L1 expression patterns in nor mal looking human minor salivary gland tissues in the perilesional area of oral squamous cell carcinoma (OSCC). A retrospective study in which UCH-L1 immunohistochemical staining performed for 27 formalin fixed paraffin embedded blocks of surgically removed primary OSCC containing perilesional minor salivary gland tissues. The expression, subcellular localization, pattern of distribution, and stain intensity were assessed. UCH-L1 was immunopositive in minor salivary gland tissues of all sections. The immunoreactivity was (70.4%) detected in the cytoplasm and (29.6%) as mixed expression (cytoplasm and nucleus). UCH-L1 was predominately expressed in the ductal system (40.7%), followed by the myoepithelial cells (33.35%), while it was weakly expressed in the acinar cells; mucous (14.9%) and serous acini (11.1%). UCH-L1 expression is detectable in minor salivary glands in a perilesional area of OSCC, Although previously approved that it was absolutely absent in normal salivary gland tissues, it may activate during the course of oncogenesis and can be classified as a marker for predicting the future invasion of OSCC to adjacent minor salivary gland tissues. This study provides evidence for additional studies on the UCH-L1 expression in normal salivary glands in patients with maxillofacial tumorigenesis.

Raman Spectroscopy Spectral Fingerprints of Biomarkers of Traumatic Brain Injury.

Traumatic brain injury (TBI) affects millions of people of all ages around the globe. TBI is notoriously hard to diagnose at the point of care, resulting in incorrect patient management, avoidable death and disability, long-term neurodegenerative complications, and increased costs. It is vital to develop timely, alternative diagnostics for TBI to assist triage and clinical decision-making, complementary to current techniques such as neuroimaging and cognitive assessment. These could deliver rapid, quantitative TBI detection, by obtaining information on biochemical changes from patient's biofluids. If available, this would reduce mis-triage, save healthcare providers costs (both over- and under-triage are expensive) and improve outcomes by guiding early management. Herein, we utilize Raman spectroscopy-based detection to profile a panel of 18 raw (human, animal, and synthetically derived) TBI-indicative biomarkers (N-acetyl-aspartic acid (NAA), Ganglioside, Glutathione (GSH), Neuron Specific Enolase (NSE), Glial Fibrillary Acidic Protein (GFAP), Ubiquitin C-terminal Hydrolase L1 (UCHL1), Cholesterol, D-Serine, Sphingomyelin, Sulfatides, Cardiolipin, Interleukin-6 (IL-6), S100B, Galactocerebroside, Beta-D-(+)-Glucose, Myo-Inositol, Interleukin-18 (IL-18), Neurofilament Light Chain (NFL)) and their aqueous solution. The subsequently derived unique spectral reference library, exploiting four excitation lasers of 514, 633, 785, and 830 nm, will aid the development of rapid, non-destructive, and label-free spectroscopy-based neuro-diagnostic technologies. These biomolecules, released during cellular damage, provide additional means of diagnosing TBI and assessing the severity of injury. The spectroscopic temporal profiles of the studied biofluid neuro-markers are classed according to their acute, sub-acute, and chronic temporal injury phases and we have further generated detailed peak assignment tables for each brain-specific biomolecule within each injury phase. The intensity ratios of significant peaks, yielding the combined unique spectroscopic barcode for each brain-injury marker, are compared to assess variance between lasers, with the smallest variance found for UCHL1 (σ2 = 0.000164) and the highest for sulfatide (σ2 = 0.158). Overall, this work paves the way for defining and setting the most appropriate diagnostic time window for detection following brain injury. Further rapid and specific detection of these biomarkers, from easily accessible biofluids, would not only enable the triage of TBI, predict outcomes, indicate the progress of recovery, and save healthcare providers costs, but also cement the potential of Raman-based spectroscopy as a powerful tool for neurodiagnostics.

UCHL1 maintains microenvironmental homeostasis in goat germline stem cells.

Spermatogonial stem cells (SSCs) play a crucial role in mammalian spermatogenesis and maintain the stable inheritance of the germline in livestock. However, stress and bacterial or viral infections can disrupt immune homeostasis of the testes, thereby leading to spermatogenesis destruction and infertility, which severely affects the health and productivity of mammals. This study aimed to explore the effect of ubiquitin C-terminal hydrolase L1 (UCHL1) knockdown (KD) in goat SSCs and mouse testes and investigate the potential anti-inflammatory function of UCHL1 in a poly(I:C)-induced inflammation model to maintain microenvironmental homeostasis. Invitro, the downregulation of UCHL1 (UCHL1 KD) in goat SSCs increased the expression levels of apoptosis and inflammatory factors and inhibited the self-renewal and proliferation of SSCs. Invivo, the structure of seminiferous tubules and spermatogenic cells was disrupted after UCHL1 KD, and the expression levels of apoptosis- and inflammation-related proteins were significantly upregulated. Furthermore, UCHL1 inhibited the TLR3/TBK1/IRF3 pathway to resist poly(I:C)-induced inflammation in SSCs by antagonizing HSPA8 and thus maintaining SSC autoimmune homeostasis. Most importantly, the results of this study showed that UCHL1 maintained immune homeostasis of SSCs and spermatogenesis. UCHL1 KD not only inhibited the self-renewal and proliferation of goat SSCs and spermatogenesis but was also involved in the inflammatory response of goat SSCs. Additionally, UCHL1 has an antiviral function in SSCs by antagonizing HSPA8, which provides an important basis for exploring the specific mechanisms of UCHL1 in goat spermatogenesis.

Enhancing hospital course and outcome prediction in patients with traumatic brain injury: A machine learning study.

We aimed to use machine learning (ML) algorithms with clinical, lab, and imaging data as input to predict various outcomes in traumatic brain injury (TBI) patients. In this retrospective study, blood samples were analyzed for glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1). The non-contrast head CTs were reviewed by two neuroradiologists for TBI common data elements (CDE). Three outcomes were designed to predict: discharged or admitted for further management (prediction 1), deceased or not deceased (prediction 2), and admission only, prolonged stay, or neurosurgery performed (prediction 3). Five ML models were trained. SHapley Additive exPlanations (SHAP) analyses were used to assess the relative significance of variables. Four hundred forty patients were used to predict predictions 1 and 2, while 271 patients were used in prediction 3. Due to Prediction 3's hospitalization requirement, deceased and discharged patients could not be utilized. The Random Forest model achieved an average accuracy of 1.00 for prediction 1 and an accuracy of 0.99 for prediction 2. The Random Forest model achieved a mean accuracy of 0.93 for prediction 3. Key features were extracranial injury, hemorrhage, UCH-L1 for prediction 1; TheGlasgow Coma Scale, age, GFAP for prediction 2; and GFAP, subdural hemorrhage volume, and pneumocephalus for prediction 3, per SHAP analysis. Combining clinical and laboratory parameters with non-contrast CT CDEs allowed our ML models to accurately predict the designed outcomes of TBI patients. GFAP and UCH-L1 were among the significant predictor variables, demonstrating the importance of these biomarkers.

Blood biomarkers of neuronal injury in paediatric cerebral malaria and severe malarial anaemia

Abstract Persistent neurodisability is a known complication in paediatric survivors of cerebral malaria and severe malarial anaemia. Tau, ubiquitin C-terminal hydrolase-L1, neurofilament-light chain, and glial fibrillary acidic protein have proven utility as biomarkers that predict adverse neurologic outcomes in adult and paediatric disorders. In paediatric severe malaria, elevated tau is associated with mortality and neurocognitive complications. We aimed to investigate whether a multi-analyte panel including ubiquitin C-terminal hydrolase-L1, neurofilament-light chain, and glial fibrillary acidic protein can serve as biomarkers of brain injury associated with mortality and neurodisability in cerebral malaria and severe malarial anaemia. In a prospective cohort study of Ugandan children, 18 months to 12 years of age with cerebral malaria (n = 182), severe malarial anaemia (n = 158), and asymptomatic community children (n = 118), we measured admission blood levels of ubiquitin C-terminal hydrolase-L1, neurofilament-light chain, and glial fibrillary acidic protein. We investigated differences in biomarker levels, associations with mortality, blood–brain barrier integrity, neurodeficits and cognitive Z-scores in survivors up to 24-month follow-up. Admission ubiquitin C-terminal hydrolase-L1 levels were elevated >95th percentile of community children in 71 and 51%, and neurofilament-light chain levels were elevated >95th percentile of community children in 40 and 37% of children with cerebral malaria and severe malarial anaemia, respectively. Glial fibrillary acidic protein was not elevated in disease groups compared with controls. In cerebral malaria, elevated neurofilament-light chain was observed in 16 children who died in hospital compared with 166 survivors (P = 0.01); elevations in ubiquitin C-terminal hydrolase-L1 levels were associated with degree of blood–brain barrier disruption (P = 0.01); and the % predictive value for neurodeficits over follow-up (discharge, 6-, 12-, and 24 months) increased for ubiquitin C-terminal hydrolase-L1 (60, 67, 72, and 83), but not neurofilament-light chain (65, 68, 60, and 67). In cerebral malaria, elevated ubiquitin C-terminal hydrolase-L1 was associated with worse memory scores in children <5 years at malaria episode who crossed to over 5 years old during follow-up cognitive testing [β −1.13 (95% confidence interval −2.05, −0.21), P = 0.02], and elevated neurofilament-light chain was associated with worse attention in children ≥5 years at malaria episode and cognitive testing [β −1.08 (95% confidence interval −2.05, −1.05), P = 0.03]. In severe malarial anaemia, elevated ubiquitin C-terminal hydrolase-L1 was associated with worse attention in children <5 years at malaria episode and cognitive testing [β −0.42 (95% confidence interval −0.76, −0.07), P = 0.02]. Ubiquitin C-terminal hydrolase-L1 and neurofilament-light chain levels are elevated in paediatric cerebral malaria and severe malarial anaemia. In cerebral malaria, elevated neurofilament-light chain is associated with mortality whereas elevated ubiquitin C-terminal hydrolase-L1 is associated with blood–brain barrier dysfunction and neurodeficits over follow-up. In cerebral malaria, both markers are associated with worse cognition, while in severe malarial anaemia, only ubiquitin C-terminal hydrolase-L1 is associated with worse cognition.

Novel Fluid Biomarkers for Mild Cognitive Impairment: A Systematic Review and Meta-Analysis.

Mild cognitive impairment (MCI) is a well-established prodromal stage of dementia (e.g., Alzheimer's disease) that is often accompanied by early signs of neurodegeneration. To facilitate a better characterization of the underlying pathophysiology, we assessed the available literature to evaluate potential fluid biomarkers in MCI. Peer-reviewed articles that measured cerebrospinal fluid (CSF) and/or peripheral biomarkers of neuronal injury (total-tau [T-tau], neurofilament light chain [NfL], heart-type fatty acid binding protein [HFABP], neuron-specific enolase, ubiquitin C-terminal hydrolase L1) and/or astroglial pathology (glial fibrillary acidic protein [GFAP], S100 calcium-binding protein B) in MCI and healthy controls were assessed. Group differences were summarized by standardized mean differences (SMDs) and 95% confidence intervals calculated using a random-effects model. Heterogeneity was quantified using I2. A total of 107 studies were included in the meta-analysis and 10 studies were qualitatively reviewed. In CSF, concentrations of NfL (SMD = 0.69 [0.56, 0.83]), GFAP (SMD = 0.41 [0.07, 0.75]), and HFABP (SMD = 0.57 [0.26, 0.89]) were elevated in MCI. In blood, increased concentrations of T-tau (SMD = 0.19 [0.09, 0.29]), NfL (SMD = 0.41 [0.32, 0.49]), and GFAP (SMD = 0.39 [0.23, 0.55]) were found in MCI. Heterogeneity that was identified in all comparisons was explored using meta-regression and subgroup analysis. Elevated NfL and GFAP can be detected in both CSF and peripheral blood. Monitoring these biomarkers in clinical settings may provide important insight into underlying neurodegenerative processes in MCI.

33 Osteopontin as a Blood Biomarker for Executive Function Outcomes in Pediatric Traumatic Brain Injury

Objective:Executive function (EF) is a self-regulatory construct well-established as a predictor of long-term academic achievement and socioemotional functioning in children (Best et al., 2009; Diamond, 2013; Zelazo & Carlson, 2020). Traumatic brain injury (TBI) in childhood frequently results in EF deficits (Beauchamp & Anderson, 2013; Levin & Hanten, 2005). In comparison to adults (Okonkwo et al., 2013), there is an absence of viable blood biomarkers for pediatric TBI to assist in diagnosis and prognosis. Osteopontin (OPN), an inflammatory cytokine, has recently been identified as a putative pediatric TBI blood biomarker (Gao et al., 2020). However, more work is needed to establish OPN’s utility in predicting functional outcomes. Thus, the present study aimed to test relations between OPN measured during the first 72 hours of hospitalization and EF 6-12 months post injury among a sample of pediatric TBI patients.Participants and Methods:Sample consisted of 38 children (age at injury = 4.60-16.67 years, M age = 10.61 years, 65.8% male, lowest Glasgow Coma Scale [GCS] score = 3-15, M gcs= 9.97) with TBI whose parents completed the Behavior Rating Inventory of Executive Function, Second Edition (BRIEF-2; Gioia et al., 2015) 6-12 months post injury. Plasma OPN was measured at hospital admission, 24 hours after admission, 48 hours after admission, and 72 hours after admission. 7-scores for each BRIEF-2 clinical scale (Inhibit, Self-Monitor, Shift, Emotional Control, Initiate, Working Memory, Plan/Organize, Task-Monitor, Organization of Materials) and composite index (Behavior Regulation Index, Emotion Regulation Index, Cognitive Regulation Index, Global Executive Composite) were used in analyses.Results:Correlation analyses revealed large positive associations (rs = .50-.73, ps = <.001.039) between 48-hour OPN and all BRIEF-2 scales/indices except Initiate. OPN at 24 hours positively correlated with Task-Monitor (r = .40, p = .037). Bivariate logistic regression analyses testing whether OPN predicted at least mildly elevated BRIEF-2 t-scores (>60) did not yield significant associations. Additional supplementary analyses testing whether alternative injury markers - glial fibrillary acidic protein (GFAP), ubiquitin C-terminal Hydrolase-L1 (UCH-L1), S100 calcium binding protein B (S100B) - measured at all time points as well as lowest GCS score correlated with EF revealed the following: admission S100B positively correlated with Inhibit (r = .34, p = .045), 48-hour UCH-L1 negatively correlated with Initiate (r = -.49, p = .041) and Cognitive Regulation Index (r = -.48, p = .044), and 72-hour UCH-L1 negatively correlated with Initiate (r = -.47, p = .048).Conclusions:Findings showed higher OPN at 48 hours post admission was broadly related to worse parent-reported EF 6-12 months later, with 24-hour OPN also showing limited associations. Higher levels of alternative injury markers likewise showed limited associations with EF outcomes. Null logistic regression findings may be due to few participants having elevated BRIEF-2 scores. Disrupted EF development may be more noticeable after longer time periods as children age and self-regulatory demands increase. Overall, OPN was found to more consistently predict EF outcomes than GCS score and other injury markers. This could be because OPN is a marker of inflammation, which may be particularly predictive of TBI cognitive outcomes.

Use of Biostatistical Models to Manage Replicate Error in Concussion Biomarker Research

Advancing research on fluid biomarkers associated with sport-related concussion (SRC) highlights the importance of detecting low concentrations using ultrasensitive platforms. However, common statistical practices may overlook replicate errors and specimen exclusion, emphasizing the need to explore robust modeling approaches that consider all available replicate data for comprehensive understanding of sample variation and statistical inferences. To evaluate the impact of replicate error and different biostatistical modeling approaches on SRC biomarker interpretation. This cross-sectional study within the Surveillance in High Schools to Reduce the Risk of Concussions and Their Consequences study used data from healthy youth athletes (ages 11-18 years) collected from 3 sites across Canada between September 2019 and November 2021. Data were analyzed from November 2022 to February 2023. Demographic variables included age, sex, and self-reported history of previous concussion. Outcomes of interest were preinjury plasma glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase-L1 (UCH-L1), neurofilament-light (NFL), total tau (t-tau) and phosphorylated-tau-181 (p-tau-181) assayed in duplicate. Bland-Altman analysis determined the 95% limits of agreement (LOAs) for each biomarker. The impact of replicate error was explored using 3 biostatistical modeling approaches assessing the associations of age, sex, and previous concussion on biomarker concentrations: multilevel regression using all available replicate data, single-level regression using the means of replicate data, and single-level regression with replicate means, excluding specimens demonstrating more than 20% coefficient variation (CV). The sample included 149 healthy youth athletes (78 [52%] male; mean [SD] age, 15.74 [1.41] years; 51 participants [34%] reporting ≥1 previous concussions). Wide 95% LOAs were observed for GFAP (-17.74 to 18.20 pg/mL), UCH-L1 (-13.80 to 14.77 pg/mL), and t-tau (65.27% to 150.03%). GFAP and UCH-L1 were significantly associated with sex in multilevel regression (GFAP: effect size, 15.65%; β = -0.17; 95% CI, -0.30 to -0.04]; P = .02; UCH-L1: effect size, 17.24%; β = -0.19; 95% CI, -0.36 to -0.02]; P = .03) and single-level regression using the means of replicate data (GFAP: effect size, 15.56%; β = -0.17; 95% CI, -0.30 to -0.03]; P = .02; UCH-L1: effect size, 18.02%; β = -0.20; 95% CI, -0.37 to -0.03]; P = .02); however, there was no association for UCH-L1 after excluding specimens demonstrating more than 20% CV. Excluding specimens demonstrating more than 20% CV resulted in decreased differences associated with sex in GFAP (effect size, 12.29%; β = -0.14; 95% CI, -0.273 to -0.004]; P = .04) and increased sex differences in UCH-L1 (effect size, 23.59%; β = -0.27; 95% CI, -0.55 to 0.01]; P = .06), with the widest 95% CIs (ie, least precision) found in UCH-L1. In this cross-sectional study of healthy youth athletes, varying levels of agreement between SRC biomarker technical replicates suggested that means of measurements may not optimize precision for population values. Multilevel regression modeling demonstrated how incorporating all available biomarker data could capture replicate variation, avoiding challenges associated with means and percentage of CV exclusion thresholds to produce more representative estimates of association.

UCHL1 Regulates Radiation Lung Injury via Sphingosine Kinase-1.

GADD45a is a gene we previously reported as a mediator of responses to acute lung injury. GADD45a-/- mice express decreased Akt and increased Akt ubiquitination due to the reduced expression of UCHL1 (ubiquitin c-terminal hydrolase L1), a deubiquitinating enzyme, while GADD45a-/- mice have increased their susceptibility to radiation-induced lung injury (RILI). Separately, we have reported a role for sphingolipids in RILI, evidenced by the increased RILI susceptibility of SphK1-/- (sphingosine kinase 1) mice. A mechanistic link between UCHL1 and sphingolipid signaling in RILI is suggested by the known polyubiquitination of SphK1. Thus, we hypothesized that the regulation of SphK1 ubiquitination by UCHL1 mediates RILI. Initially, human lung endothelial cells (EC) subjected to radiation demonstrated a significant upregulation of UCHL1 and SphK1. The ubiquitination of EC SphK1 after radiation was confirmed via the immunoprecipitation of SphK1 and Western blotting for ubiquitin. Further, EC transfected with siRNA specifically for UCHL1 or pretreated with LDN-5744, as a UCHL1 inhibitor, prior to radiation were noted to have decreased ubiquitinated SphK1 in both conditions. Further, the inhibition of UCHL1 attenuated sphingolipid-mediated EC barrier enhancement was measured by transendothelial electrical resistance. Finally, LDN pretreatment significantly augmented murine RILI severity. Our data support the fact that the regulation of SphK1 expression after radiation is mediated by UCHL1. The modulation of UCHL1 affecting sphingolipid signaling may represent a novel RILI therapeutic strategy.

A-094 Performance Evaluation of GFAP and UCH-L1 Biomarkers for Traumatic Brain Injury in the Alinity i TBI Test (in development)

Abstract Background Traumatic Brain Injury (TBI) has been recognized as an important cause of death and disability and is a growing public health problem. An estimated 69 million people globally experience a TBI annually. Blood-based biomarkers such as glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1) have shown utility to predict acute traumatic intracranial injury on head CT scan after TBI. Methods The Alinity i TBI test is a panel of in vitro diagnostic chemiluminescent microparticle immunoassays for the measurement of GFAP and UCH-L1 in plasma and serum. Performance characteristics such as detection limits, imprecision, linearity, measuring interval, expected values, and interferences were established following CLSI guidance. A feasibility cohort of 354 subjects was used to establish cutoffs which assist in determining the need for a CT (computed tomography) scan of the head. A prospective study was conducted to assess the clinical performance of the TBI test. Fresh plasma specimens from 97 mild TBI subjects presenting to Level I trauma centers were collected within 12 h of injury from subjects ≥18 years of age who presented with suspected TBI and had a head CT scan performed. The TBI interpretation is considered positive if either GFAP or UCH-L1 is greater than or equal to the cutoffs. The TBI interpretation is considered negative if both GFAP and UCH-L1 are less than the cutoffs. Results The analytical measuring interval (AMI) extends from the limit of quantitation (LoQ) to the upper LoQ and is determined by the range that demonstrates acceptable performance for linearity, imprecision, and bias. The AMI is 6.1 to 42 000.0 pg/mL for GFAP and 26.3 to 25 000.0 pg/mL for UCH-L1. Within-laboratory imprecision (20 day) ranged from 2.8% to 5.3% CV for GFAP and 2.1% to 5.6% CV for UCH-L1. Cutoffs for GFAP and UCH-L1 of 35.0 pg/mL and 400.0 pg/mL, respectively, were selected using both 10-fold cross validation and bootstrapping methods and applying a selection criterion of negative predictive value (NPV) (prevalence adjusted) ≥99% and Sensitivity ≥96%. Of the 97 mild TBI subjects evaluated, 14 had positive head CT scan results. All 14 of the subjects with positive head CT results had a positive TBI interpretation (Sensitivity 100.0%; 95% CI:78.5%,100.0%). There were 23 subjects with a negative TBI interpretation, all 23 had a negative head CT scan result. The NPV of the test was 100% (95% CI:85.7%,100.0%). Conclusion The Alinity i TBI test shows robust analytical performance across a broad concentration range of GFAP and UCH-L1 and may serve as a valuable tool to help evaluate TBI patients across the spectrum of mild to severe injury. The established cutoffs were evaluated in a prospective cohort of mild TBI (Glasgow Coma Scale score of 13–15) subjects and demonstrated high sensitivity and NPV relative to CT scan results, supporting the utility to rule out the need for CT scan in mild TBI subjects presenting to the emergency department. FUNDING: This work is in collaboration with the US Army Medical Materiel Development Activity, US Army Medical Research and Development Command CRADA 20-1266-CRA.

The association of circulating endocannabinoids with neuroimaging and blood biomarkers of neuro-injury

BackgroundPreclinical studies highlight the importance of endogenous cannabinoids (endocannabinoids; eCBs) in neurodegeneration. Yet, prior observational studies focused on limited outcome measures and assessed only few eCB compounds while largely ignoring the complexity of the eCB system. We examined the associations of multiple circulating eCBs and eCB-like molecules with early markers of neurodegeneration and neuro-injury and tested for effect modification by sex.MethodsThis exploratory cross-sectional study included a random sample of 237 dementia-free older participants from the Framingham Heart Study Offspring cohort who attended examination cycle 9 (2011–2014), were 65 years or older, and cognitively healthy. Forty-four eCB compounds were quantified in serum, via liquid chromatography high-resolution mass spectrometry. Linear regression models were used to examine the associations of eCB levels with brain MRI measures (i.e., total cerebral brain volume, gray matter volume, hippocampal volume, and white matter hyperintensities volume) and blood biomarkers of Alzheimer’s disease and neuro-injury (i.e., total tau, neurofilament light, glial fibrillary acidic protein and Ubiquitin C-terminal hydrolase L1). All models were adjusted for potential confounders and effect modification by sex was examined.ResultsParticipants mean age was 73.3 ± 6.2 years, and 40% were men. After adjustment for potential confounders and correction for multiple comparisons, no statistically significant associations were observed between eCB levels and the study outcomes. However, we identified multiple sex-specific associations between eCB levels and the various study outcomes. For example, high linoleoyl ethanolamide (LEA) levels were related to decreased hippocampal volume among men and to increased hippocampal volume among women (β ± SE = − 0.12 ± 0.06, p = 0.034 and β ± SE = 0.08 ± 0.04, p = 0.026, respectively).ConclusionsCirculating eCBs may play a role in neuro-injury and may explain sex differences in susceptibility to accelerated brain aging. Particularly, our results highlight the possible involvement of eCBs from the N-acyl amino acids and fatty acid ethanolamide classes and suggest specific novel fatty acid compounds that may be implicated in brain aging. Furthermore, investigation of the eCBs contribution to neurodegenerative disease such as Alzheimer’s disease in humans is warranted, especially with prospective study designs and among diverse populations, including premenopausal women.

UCHL1 alleviates apoptosis in chondrocytes via upregulation of HIF‑1α‑mediated mitophagy.

Stem cell‑based tissue engineering has shown significant potential for rapid restoration of injured cartilage tissues. Stem cells frequently undergo apoptosis because of the prevalence of oxidative stress and inflammation in the microenvironment at the sites of injury. Our previous study demonstrated that stabilization of hypoxia‑inducible factor1α (HIF‑1α) is key to resisting apoptosis in chondrocytes. Recently, it was reported that Ubiquitin C‑terminal hydrolaseL1 (UCHL1) can stabilize HIF‑1α by abrogating the ubiquitination process. However, the effect of UCHL1 on apoptosis in chondrocytes remains unclear. Herein, adipose‑derived stem cells were differentiated into chondrocytes. Next, the CRISPR activation (CRISPRa) system, LDN‑57444 (LDM; a specific inhibitor for UCHL1), KC7F2 (a specific inhibitor for HIF‑1α), and 3‑methyladenine (a specific inhibitor for mitophagy) were used to activate or block UCHL1, HIF‑1α, and mitophagy. Mitophagy, apoptosis, and mitochondrial function in chondrocytes were detected using immunofluorescence, TUNEL staining, and flow cytometry. Moreover, the oxygen consumption rate of chondrocytes was measured using the Seahorse XF 96 Extracellular Flux Analyzer. UCHL1 expression was increased in hypoxia, which in turn regulated mitophagy and apoptosis in the chondrocytes. Further studies revealed that UCHL1 mediated hypoxia‑regulated mitophagy in the chondrocytes. The CRISPRa module was utilized to activate UCHL1 effectively for 7days; endogenous activation of UCHL1 accelerated mitophagy, inhibited apoptosis, and maintained mitochondrial function in the chondrocytes, which was mediated by HIF‑1α. Taken together, UCHL1 could block apoptosis in chondrocytes via upregulation of HIF‑1α-mediated mitophagy and maintain mitochondrial function. These results indicate the potential of UCHL1 activation using the CRISPRa system for the regeneration of cartilage tissue.