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1.
Res Pract Thromb Haemost ; 8(6): 102557, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39318773

RESUMEN

Background: Inhibition of platelet responsiveness is important for controlling thrombosis. It is well established that platelet endothelial cell adhesion molecule-1 (PECAM-1) serves as a physiological negative regulator of platelet-collagen interactions. We recently demonstrated that leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a negative regulator of platelet production and reactivity. It is however not known if LAIR-1 and PECAM-1 function in the same or different inhibitory pathways. Objectives: In this study, we investigated the role of LAIR-1 alongside PECAM-1 in megakaryocyte development and platelet production and determined the functional redundancy through characterization of a LAIR-1/PECAM-1 double knockout (DKO) mouse model. Methods: LAIR-1 and PECAM-1 expression in megakaryocytes were evaluated by western blotting. Megakaryocyte ploidy and proplatelet formation were evaluated by flow cytometry and fluorescent microscopy. Platelet function and signalling were compared in wild-type, LAIR-1 -/- , PECAM-1 -/-  and DKO mice using aggregometry, flow cytometry and western blotting. Thrombosis was evaluated using the FeCl 3  carotid artery model. Results: We show that LAIR-1/PECAM-1 DKO mice exhibit a 17% increase in platelet count. Bone marrow-derived megakaryocytes from all 3 mouse models had normal ploidy in vitro, suggesting that neither LAIR-1 nor PECAM-1 regulates megakaryocyte development. Furthermore, relative to wild-type platelets, platelets derived from LAIR-1, PECAM-1, and DKO mice were equally hyperresponsive to collagen in vitro, indicating that LAIR-1 and PECAM-1 participate in the same inhibitory pathway. Interestingly, DKO mice exhibited normal thrombus formation in vivo due to DKO mouse platelets lacking the enhanced Src family kinase activation previously shown in platelets from LAIR-1-deficient mice. Conclusion: Findings from this study reveal that LAIR-1 and PECAM-1 act to inhibit GPVI-mediated platelet activation via the same signaling pathway. Mice lacking LAIR-1 and PECAM-1 do not however exhibit an increase in thrombus formation despite minor increase in platelet count and reactivity to collagen. This study adds to the growing evidence that immunoreceptor tyrosine-based inhibition motif-containing receptors are important regulators of platelet count and function.

2.
Cardiovasc Res ; 2024 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-39248180

RESUMEN

AIMS: Differentiated Vascular Smooth Muscle Cells (VSMCs) express a unique network of mRNA isoforms via smooth muscle specific alternative splicing (SM-AS) in functionally critical genes, including those comprising the contractile machinery. We previously described RNA Binding Protein Multiple Splicing (RBPMS) as a potent driver of differentiated SM-AS in the rat PAC1 VSMC cell line. What is unknown is how RBPMS affects VSMC phenotype and behaviour. Here, we aimed to dissect the role of RBPMS in SM-AS in human cells and determine the impact on VSMC phenotypic properties. METHODS AND RESULTS: We used human embryonic stem cell-derived VSMCs (hESC-VSMCs) as our platform. hESC-VSMCs are inherently immature and we found that they display only partially differentiated SM-AS patterns while RBPMS protein levels are low. We found that RBPMS overexpression induces SM-AS patterns in hESC-VSMCs akin to the contractile tissue VSMC splicing patterns. We present in silico and experimental findings that support RBPMS' splicing activity as mediated through direct binding and via functional cooperativity with splicing factor RBFOX2 on a significant subset of targets. We also demonstrate that RBPMS can alter the motility and the proliferative properties of hESC-VSMCs to mimic a more differentiated state. CONCLUSIONS: Overall, this study emphasizes a critical role for RBPMS in establishing the contractile phenotype splicing program of human VSMCs.

3.
JAMA Surg ; 2024 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-39141362

RESUMEN

Importance: Identifying disparities in health outcomes related to modifiable patient factors can improve patient care. Objective: To compare likelihood of withdrawal of life-supporting treatment (WLST) and mortality in patients with complete cervical spinal cord injury (SCI) with different types of insurance. Design, Setting, and Participants: This retrospective cohort study collected data between 2013 and 2020 from 498 trauma centers participating in the Trauma Quality Improvement Program. Participants included adult patients (older than 16 years) with complete cervical SCI. Data were analyzed from November 1, 2023, through May 18, 2024. Exposure: Uninsured or public insurance compared with private insurance. Main Outcomes and Measures: Coprimary outcomes were WLST and mortality. The adjusted odds ratio (aOR) of each outcome was estimated using hierarchical logistic regression. Propensity score matching was used as an alternative analysis to compare public and privately insured patients. Process of care outcomes, including the occurrence of a hospital complication and length of stay, were compared between matched patients. Results: The study included 8421 patients with complete cervical SCI treated across 498 trauma centers (mean [SD] age, 49.1 [20.2] years; 6742 male [80.1%]). Among the 3524 patients with private insurance, 503 had WLST (14.3%) and 756 died (21.5%). Among the 3957 patients with public insurance, 906 had WLST (22.2%) and 1209 died (30.6%). Among the 940 uninsured patients, 156 had WLST (16.6%) and 318 died (33.8%). A significant difference was found between uninsured and privately insured patients in the adjusted odds of WLST (aOR, 1.49; 95% CI, 1.11-2.01) and mortality (aOR, 1.98; 95% CI, 1.50-2.60). Similar results were found in subgroup analyses. Matched public compared with private insurance patients were found to have significantly greater odds of hospital complications (odds ratio, 1.27; 95% CI, 1.14-1.42) and longer hospital stay (mean difference 5.90 days; 95% CI, 4.64-7.20), which was redemonstrated on subgroup analyses. Conclusions and Relevance: Health insurance type was associated with significant differences in the odds of WLST, mortality, hospital complications, and days in hospital among patients with complete cervical SCI in this study. Future work is needed to incorporate patient perspectives and identify strategies to close the quality gap for the large number of patients without private insurance.

4.
Nat Commun ; 15(1): 6618, 2024 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-39103350

RESUMEN

A mechanistic understanding of host-microbe interactions in the gut microbiome is hindered by poorly annotated bacterial genomes. While functional genomics can generate large gene-to-phenotype datasets to accelerate functional discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut-derived genes have been performed in Escherichia coli and assayed in a small number of conditions. To address these challenges, we develop Barcoded Overexpression BActerial shotgun library sequencing (Boba-seq). We demonstrate the power of this approach by assaying genes from diverse gut Bacteroidales overexpressed in Bacteroides thetaiotaomicron. From hundreds of experiments, we identify new functions and phenotypes for 29 genes important for carbohydrate metabolism or tolerance to antibiotics or bile salts. Highlights include the discovery of a D-glucosamine kinase, a raffinose transporter, and several routes that increase tolerance to ceftriaxone and bile salts through lipid biosynthesis. This approach can be readily applied to develop screens in other strains and additional phenotypic assays.


Asunto(s)
Ácidos y Sales Biliares , Carbono , Microbioma Gastrointestinal , Carbono/metabolismo , Microbioma Gastrointestinal/genética , Ácidos y Sales Biliares/metabolismo , Antibacterianos/farmacología , Estrés Fisiológico/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteroides thetaiotaomicron/genética , Bacteroides thetaiotaomicron/metabolismo , Regulación Bacteriana de la Expresión Génica , Bacteroidetes/genética , Bacteroidetes/metabolismo , Metabolismo de los Hidratos de Carbono/genética , Humanos , Genes Bacterianos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano
5.
Blood Adv ; 2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-38968150

RESUMEN

Platelet CLEC-2 is a hemITAM-containing receptor which has a critical role in venous thrombosis, but minimal involvement in haemostasis. CLEC-2 can be blocked by Btk inhibitors. Treatment with ibrutinib is associated with increased bleeding due to off-target inhibition of Src family kinases (SFKs). Patients with X-linked agammaglobulinemia (XLA) who lack Btk however do not bleed, suggesting selective Btk inhibition is a viable antithrombotic strategy. We assessed the effects of selective Btk inhibitors PRN1008 (rilzabrutinib) and PRN473 on platelet signalling and function mediated by CLEC-2 and GPVI. We used healthy donor and XLA platelets to determine off-target inhibitor effects. Inferior vena cava (IVC) stenosis and Salmonella infection mouse models were used to assess antithrombotic effects of PRN473 in vivo. PRN1008 and PRN473 potently inhibited CLEC-2-mediated platelet activation to rhodocytin. No off-target inhibition of SFKs was seen. PRN1008 treatment of Btk-deficient platelets resulted in minor additional inhibition of aggregation and tyrosine phosphorylation, likely reflecting inhibition of Tec. No effect on GPCR-mediated platelet function was observed. PRN473 significantly reduced the number of thrombi in podoplanin positive vessels following Salmonella infection and the presence of IVC thrombosis following vein stenosis. The potent inhibition of human platelet CLEC-2, and reduced thrombosis in in vivo models, together with the lack of off-target SFK inhibition and absence of bleeding reported in rilzabrutinib treated immune thrombocytopenia patients, suggest Btk inhibition as a promising antithrombotic strategy.

6.
Adv Healthc Mater ; 13(22): e2400508, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38683016

RESUMEN

Salmonella, the most prevalent food-borne pathogen, poses significant medical and economic threats. Swift and accurate on-site identification and serotyping of Salmonella is crucial to curb its spread and contamination. Here, a synthetic biology cascade reaction is presented on a paper substrate using CRISPR-Cas12a and recombinase polymerase amplification (RPA), enabling the programming of a standard toehold RNA switch for a genome of choice. This approach employs just one toehold RNA switch design to differentiate between two different Salmonella serotypes, i.e., S. Typhimurium and S. Enteritidis, without the need for reengineering the toehold RNA switch. The sensor exhibits high sensitivity, capable of visually detecting as few as 100 copies of the whole genome from a model Salmonella pathogen on a paper substrate. Furthermore, this robust assay is successfully applied to detect whole genomes in contaminated milk and lettuce samples, demonstrating its potential in real sample analysis. Due to its versatility and practical features, genomes from different organisms can be detected by merely changing a single RNA element in this universal cell-free cascade reaction.


Asunto(s)
Sistemas CRISPR-Cas , Genoma Bacteriano , Salmonella , Sistemas CRISPR-Cas/genética , Genoma Bacteriano/genética , Salmonella/genética , Papel , Lactuca/microbiología , Lactuca/genética , Animales , Leche/microbiología , Proteínas Bacterianas , Endodesoxirribonucleasas , Proteínas Asociadas a CRISPR
7.
Neurosurgery ; 2024 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-38289070

RESUMEN

BACKGROUND AND OBJECTIVES: Withdrawal of life-sustaining treatment (WLST) in severe traumatic brain injury (TBI) is complex, with a paucity of standardized guidelines. We aimed to assess the variability in WLST practices between trauma centers in North America. METHODS: This retrospective study used data from trauma centers through the American College of Surgeons Trauma Quality Improvement Program between 2017 and 2020. We included adult patients (>16 years) with severe TBI and a documented decision for WLST. We constructed a series of hierarchical logistic regression models to adjust for patient, injury, and hospital attributes influencing WLST; residual between-center variability was characterized using the median odds ratio. The impact of disparate WLST practices was further assessed by ranking centers by their conditional random intercept and assessing mortality, length of stay, and WLST between quartiles. RESULTS: We identified a total of 85 511 subjects with severe TBI treated across 510 trauma centers, of whom 20 300 (24%) had WLST. Patient-level factors associated with increased likelihood of WLST were advanced age, White race, self-pay, or Medicare insurance status (compared with private insurance). Black race was associated with reduced tendency for WLST. Treatment in nonprofit centers and higher-severity intracranial and extracranial injuries, midline shift, and pupil asymmetry also increased the likelihood for WLST. After adjustment for patient and hospital attributes, the median odds ratio was 1.45 (1.41-1.49 95% CI), suggesting residual variation in WLST between centers. When centers were grouped into quartiles by their propensity for WLST, there was increased adjusted mortality and shorter length of stay in fourth compared with first quartile centers. CONCLUSION: We highlighted the presence of contextual phenomena associated with disparate WLST practice patterns between trauma centers after adjustment for case-mix and hospital attributes. These findings highlight a need for standardized WLST guidelines to improve equity of care provision for patients with severe TBI.

8.
Radiol Artif Intell ; 6(2): e230088, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38197796

RESUMEN

Purpose To develop an automated triage tool to predict neurosurgical intervention for patients with traumatic brain injury (TBI). Materials and Methods A provincial trauma registry was reviewed to retrospectively identify patients with TBI from 2005 to 2022 treated at a specialized Canadian trauma center. Model training, validation, and testing were performed using head CT scans with binary reference standard patient-level labels corresponding to whether the patient received neurosurgical intervention. Performance and accuracy of the model, the Automated Surgical Intervention Support Tool for TBI (ASIST-TBI), were also assessed using a held-out consecutive test set of all patients with TBI presenting to the center between March 2021 and September 2022. Results Head CT scans from 2806 patients with TBI (mean age, 57 years ± 22 [SD]; 1955 [70%] men) were acquired between 2005 and 2021 and used for training, validation, and testing. Consecutive scans from an additional 612 patients (mean age, 61 years ± 22; 443 [72%] men) were used to assess the performance of ASIST-TBI. There was accurate prediction of neurosurgical intervention with an area under the receiver operating characteristic curve (AUC) of 0.92 (95% CI: 0.88, 0.94), accuracy of 87% (491 of 562), sensitivity of 87% (196 of 225), and specificity of 88% (295 of 337) on the test dataset. Performance on the held-out test dataset remained robust with an AUC of 0.89 (95% CI: 0.85, 0.91), accuracy of 84% (517 of 612), sensitivity of 85% (199 of 235), and specificity of 84% (318 of 377). Conclusion A novel deep learning model was developed that could accurately predict the requirement for neurosurgical intervention using acute TBI CT scans. Keywords: CT, Brain/Brain Stem, Surgery, Trauma, Prognosis, Classification, Application Domain, Traumatic Brain Injury, Triage, Machine Learning, Decision Support Supplemental material is available for this article. © RSNA, 2024 See also commentary by Haller in this issue.


Asunto(s)
Lesiones Traumáticas del Encéfalo , Lesiones Encefálicas , Masculino , Humanos , Persona de Mediana Edad , Femenino , Estudios Retrospectivos , Canadá , Lesiones Traumáticas del Encéfalo/diagnóstico por imagen , Procedimientos Neuroquirúrgicos
9.
JAMA Surg ; 159(3): 287-296, 2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-38117514

RESUMEN

Importance: The decision to withdraw life-sustaining treatment for pediatric patients with severe traumatic brain injury (TBI) is challenging for clinicians and families with limited evidence quantifying existing practices. Given the lack of standardized clinical guidelines, variable practice patterns across trauma centers seem likely. Objective: To evaluate the factors influencing decisions to withdraw life-sustaining treatment across North American trauma centers for pediatric patients with severe TBI and to quantify any existing between-center variability in withdrawal of life-sustaining treatment practices. Design, Setting, and Participants: This retrospective cohort study used data collected from 515 trauma centers through the American College of Surgeons Trauma Quality Improvement Program between 2017 and 2020. Pediatric patients younger than 19 years with severe TBI and a documented decision for withdrawal of life-sustaining treatment were included. Data were analyzed from January to May 2023. Main Outcomes and Measures: A random intercept multilevel logistic regression model was used to quantify patient, injury, and hospital characteristics associated with the decision to withdraw life-sustaining treatment; the median odds ratio was used to characterize residual between-center variability. Centers were ranked by their conditional random intercepts and quartile-specific adjusted mortalities were computed. Results: A total of 9803 children (mean [SD] age, 12.6 [5.7]; 2920 [29.8%] female) with severe TBI were identified, 1003 of whom (10.2%) had a documented decision to withdraw life-sustaining treatment. Patient-level factors associated with an increase in likelihood of withdrawal of life-sustaining treatment were young age (younger than 3 years), higher severity intracranial and extracranial injuries, and mechanism of injury related to firearms. Following adjustment for patient and hospital attributes, the median odds ratio was 1.54 (95% CI, 1.46-1.62), suggesting residual variation in withdrawal of life-sustaining treatment between centers. When centers were grouped into quartiles by their propensity for withdrawal of life-sustaining treatment, adjusted mortality was higher for fourth-quartile compared to first-quartile centers (odds ratio, 1.66; 95% CI, 1.45-1.88). Conclusions and Relevance: Several patient and injury factors were associated with withdrawal of life-sustaining treatment decision-making for pediatric patients with severe TBI in this study. Variation in withdrawal of life-sustaining treatment practices between trauma centers was observed after adjustment for case mix; this variation was associated with differences in risk-adjusted mortality rates. Taken together, these findings highlight the presence of inconsistent approaches to withdrawal of life-sustaining treatment in children, which speaks to the need for guidelines to address this significant practice pattern variation.


Asunto(s)
Lesiones Traumáticas del Encéfalo , Humanos , Niño , Femenino , Preescolar , Masculino , Estudios Retrospectivos , Oportunidad Relativa , Mortalidad Hospitalaria , Centros Traumatológicos/estadística & datos numéricos
10.
Crit Care ; 27(1): 448, 2023 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-37980485

RESUMEN

BACKGROUND: Traumatic spinal cord injury (SCI) leads to profound neurologic sequelae, and the provision of life-supporting treatment serves great importance among this patient population. The decision for withdrawal of life-supporting treatment (WLST) in complete traumatic SCI is complex with the lack of guidelines and limited understanding of practice patterns. We aimed to evaluate the individual and contextual factors associated with the decision for WLST and assess between-center differences in practice patterns across North American trauma centers for patients with complete cervical SCI. METHODS: This retrospective multicenter observational cohort study utilized data derived from the American College of Surgeons Trauma Quality Improvement Program database between 2017 and 2020. The study included adult patients (> 16 years) with complete cervical SCI. We constructed a multilevel mixed effect logistic regression model to adjust for patient, injury and hospital factors influencing WLST. Factors associated with WLST were estimated through odds ratios with 95% confidence intervals. Hospital variability was characterized using the median odds ratio. Unexplained residual variability was assessed through the proportional change in variation between models. RESULTS: We identified 5070 patients with complete cervical SCI treated across 477 hospitals, of which 960 (18.9%) had WLST. Patient-level factors associated with significantly increased likelihood of WLST were advanced age, male sex, white race, prior dementia, low presenting Glasgow Coma Scale score, having a pre-hospital cardiac arrest, SCI level of C3 or above, and concurrent severe injury to the head or thorax. Patient-level factors associated with significantly decreased likelihood of WLST included being racially Black or Asian. There was significant variability across hospitals in the likelihood for WLST while accounting for case-mix, hospital size, and teaching status (MOR 1.51 95% CI 1.22-1.75). CONCLUSIONS: A notable proportion of patients with complete cervical SCI undergo WLST during their in-hospital admission. We have highlighted several factors associated with this decision and identified considerable variability between hospitals. Further work to standardize WLST guidelines may improve equity of care provided to this patient population.


Asunto(s)
Médula Cervical , Traumatismos de la Médula Espinal , Adulto , Femenino , Humanos , Masculino , Modelos Logísticos , Estudios Retrospectivos , Traumatismos de la Médula Espinal/terapia , Privación de Tratamiento
11.
J Neurosurg Pediatr ; 32(6): 701-709, 2023 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-37877947

RESUMEN

OBJECTIVE: In this study, the authors aimed to quantify the frequency of in-hospital major adverse events (AEs) in a multicenter cohort of pediatric patients with spinal cord injury (SCI) managed at North American trauma centers. They also sought to identify patient and injury factors associated with the occurrence of major and immobility-related AEs. METHODS: Data derived from the American College of Surgeons (ACS) Trauma Quality Improvement Program (TQIP) were used to identify a cohort of pediatric patients (age < 19 years) with traumatic SCI. The authors identified individuals with major and immobility-related AEs following injury. They used mixed-effects multivariable logistic regression to identify clinical variables associated with AEs after injury. This analytical approach allowed them to account for similarities in care delivery between patients managed in the same trauma settings during the study period while also adjusting for patient-level confounders. The adjusted impact of AEs on in-hospital mortality and length of stay (LOS) were also assessed through further multivariable regression analysis. Additional subgroup analyses were performed to reduce bias associated with competing risks and explore the age-specific risk factor associations with AEs. RESULTS: A total of 1853 pediatric patients who sustained either cervical or thoracic SCI were managed at ACS TQIP trauma centers between 2017 and 2020. The most frequently encountered AE types were pressure ulcer, unplanned intubation, cardiac arrest requiring cardiopulmonary resuscitation, and ventilator-associated pneumonia. The crude rate of major in-hospital and immobility-related AEs significantly differed between subgroups, with higher proportions of AEs in complete injuries compared with incomplete injuries. The adjusted risk for major AE following injury was significantly elevated for cervical complete SCI, patients with severe concomitant abdominal injuries, and for those presenting with depressed Glasgow Coma Scale scores less than 13. These same risk factors were associated with major AEs in children older than 8 years but were not significant for younger children (age ≤ 8 years). Complication occurrence was not associated with difference in risk-adjusted mortality (OR 0.72, 95% CI 0.45-1.14), but did increase LOS by 2.2 days (95% CI 1.4-2.7 days). CONCLUSIONS: The authors outlined the prevalence of in-hospital AEs in a large multicenter cohort of North American pediatric SCI patients. Important risk factors predisposing this population to AEs include cervical complete injuries, simultaneous abdominal trauma, and Glasgow Coma Scale scores < 13 at presentation. Postinjury complications impacted health resource utilization by increased LOS but did not affect postinjury mortality. These findings have important implications for pediatric SCI providers and future care quality benchmarking.


Asunto(s)
Traumatismos de la Médula Espinal , Traumatismos Vertebrales , Niño , Humanos , Estudios de Cohortes , Hospitales , Estudios Retrospectivos , Factores de Riesgo , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/epidemiología , Adolescente
12.
Nucleic Acids Res ; 51(18): 9961-9982, 2023 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-37548402

RESUMEN

Alternative pre-mRNA splicing decisions are regulated by RNA binding proteins (RBPs) that can activate or repress regulated splice sites. Repressive RBPs typically harness multivalent interactions to bind stably to target RNAs. Multivalency can be achieved by homomeric oligomerization and heteromeric interactions with other RBPs, often mediated by intrinsically disordered regions (IDRs), and by possessing multiple RNA binding domains. Cell-specific splicing decisions often involve the action of widely expressed RBPs, which are able to bind multivalently around target exons, but without effect in the absence of a cell-specific regulator. To address how cell-specific regulators can collaborate with constitutive RBPs in alternative splicing regulation, we used the smooth-muscle specific regulator RBPMS. Recombinant RBPMS is sufficient to confer smooth muscle cell specific alternative splicing of Tpm1 exon 3 in cell-free assays by preventing assembly of ATP-dependent splicing complexes. This activity depends upon a C-terminal IDR that facilitates dynamic higher-order self-assembly, cooperative binding to multivalent RNA and interactions with widely expressed splicing co-regulators, including MBNL1 and RBFOX2, allowing cooperative assembly of stable cell-specific regulatory complexes.


Asunto(s)
Empalme Alternativo , Empalme del ARN , Proteínas de Unión al ARN , Exones , ARN/genética , ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Humanos , Animales , Ratas
13.
Artículo en Inglés | MEDLINE | ID: mdl-37599416

RESUMEN

BACKGROUND: There is conflicting evidence regarding the relationship between trauma center type and mortality for children with traumatic brain injuries. Identification of mortality differences following brain injury across differing trauma center types may result in actionable quality improvement initiatives to standardize care for these children. METHODS: We utilized Trauma Quality Improvement Program data from 2017-2020 to identify children with severe traumatic brain injury managed at level I and II state- or American College of Surgeon-verified trauma centers. We used a random intercept multilevel logistic regression model to assess the relationship between exposure (trauma center type either adult, pediatric or mixed) and outcome (in-hospital mortality). Several secondary analyses were performed to assess the influence of trauma center volume, age strata and traumatic brain injury heterogeneity. RESULTS: There were 10,105 patients identified across 512 trauma centers. Crude mortality was 25.2%, 36.2% and 28.9% for pediatric, adult, and mixed trauma centers respectively. After adjustment for confounders, odds of mortality were higher for children managed at adult trauma centers (OR 1.67; 95% CI: 1.30 - 2.13) compared to pediatric trauma centers. Male sex, self-pay insurance status, and interfacility transfers, motor vehicle, pedestrian/ cyclist and firearm injury mechanisms, presence of concomitant abdomen, lower extremity, or chest injuries, midline shift >5 mm within 24 hours, presence of age-adjusted hypotension and either pupil asymmetry or non-reactivity were all associated with a greater odds of death. Adjustment for trauma volume and subgroup analysis using a homogenous traumatic brain injury subgroup did not change the demonstrated associations. CONCLUSIONS: Our results suggest mortality was higher at adult trauma centers compared to mixed and pediatric trauma centers for children with traumatic brain injuries. Importantly, there exists the potential for unmeasured confounding. We aim for these findings to direct continuing quality improvement initiatives to improve outcomes for brain injured children. LEVEL OF EVIDENCE: III; Type of study: Prognostic/ epidemiological.

14.
J Thromb Haemost ; 21(8): 2260-2267, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37150294

RESUMEN

BACKGROUND: Collagen-induced platelet activation is predominantly mediated by glycoprotein (GP) VI through formation of receptor clusters that coincide with the accumulation of signaling molecules and are hypothesized to drive strong and sustained platelet activation. OBJECTIVES: To determine the importance of GPVI clusters for thrombus formation in whole blood under shear. METHODS: We utilized whole blood microfluidics and an anti-GPVI nanobody (Nb), Nb28, labeled with AlexaFluor 488, to assess the distribution of GPVI on the surface of platelets adhering to a range of collagen-like substrates with different platelet activation potentials. RESULTS: Automated analysis of GPVI surface distribution on platelets supported the hypothesis that there is a relationship between GPVI cluster formation, thrombus size, and phosphatidylserine (PS) exposure. Substrates that supported the formation of macroclusters also induced significantly bigger aggregates, with increased amounts of PS-exposing platelets in comparison to substrates where no GPVI clusters were detected. Furthermore, we demonstrate that only direct inhibition of GPVI binding, but not of downstream signaling, is able to disrupt cluster formation. CONCLUSION: Labeled anti-GPVI Nb28 permits visualization of GPVI clustering under flow conditions. Furthermore, whilst inhibition of downstream signaling does not affect clustering, it does prevent thrombus formation. Therefore, GPVI macroclustering is a prerequisite for thrombus formation and platelet activation, namely, PS exposure, on highly GPVI-dependent collagen surfaces.


Asunto(s)
Plaquetas , Trombosis , Humanos , Plaquetas/metabolismo , Fosfatidilserinas/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Activación Plaquetaria , Colágeno/metabolismo , Agregación Plaquetaria
15.
Sci Rep ; 13(1): 6276, 2023 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-37072405

RESUMEN

Odontoid fractures are increasingly prevalent in older adults and associated with high morbidity and mortality. Optimal management remains controversial. Our study aims to investigate the association between surgical management of odontoid fractures and in-hospital mortality in a multi-center geriatric cohort. We identified patients 65 years or older with C2 odontoid fractures from the Trauma Quality Improvement Program database. The primary study outcome was in-hospital mortality. Secondary outcomes were in-hospital complications and hospital length of stay. Generalized estimating equation models were used to compare outcomes between operative and non-operative cohorts. Among the 13,218 eligible patients, 1100 (8.3%) were treated surgically. The risk of in-hospital mortality did not differ between surgical and non-surgical groups, after patient and hospital-level adjustment (OR: 0.94, 95%CI: 0.55-1.60). The risks of major complications and immobility-related complications were higher in the operative cohort (adjusted OR: 2.12, 95%CI: 1.53-2.94; and OR: 2.24, 95%CI: 1.38-3.63, respectively). Patients undergoing surgery had extended in-hospital length of stay compared to the non-operative group (9 days, IQR: 6-12 days vs. 4 days, IQR: 3-7 days). These findings were supported by secondary analyses that considered between-center differences in rates of surgery. Among geriatric patients with odontoid fractures surgical management was associated with similar in-hospital mortality, but higher in-hospital complication rates compared to non-operative management. Surgical management of geriatric patients with odontoid fractures requires careful patient selection and consideration of pre-existing comorbidities.


Asunto(s)
Fracturas Óseas , Apófisis Odontoides , Fracturas de la Columna Vertebral , Humanos , Anciano , Estudios Retrospectivos , Fracturas de la Columna Vertebral/cirugía , Resultado del Tratamiento , Apófisis Odontoides/cirugía
16.
J Thromb Haemost ; 21(1): 101-116, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36695374

RESUMEN

BACKGROUND: Platelet endothelial aggregation receptor 1 (PEAR1) is a single-transmembrane orphan receptor primarily expressed on platelets and endothelial cells. Genetic variants of PEAR1 have repeatedly and independently been identified to be associated with cardiovascular diseases, including coronary artery disease. OBJECTIVES: We have identified sulfated fucoidans and their mimetics as ligands for PEAR1 and proposed that its endogenous ligand is a sulfated proteoglycan. The aim of this study was to test this hypothesis. METHODS: A heparin proteoglycan-mimetic (HPGM) was created by linking unfractionated heparin (UFH) to albumin. The ability of the HPGM, UFH and selectively desulfated heparins to stimulate platelet aggregation and protein phosphorylation was investigated. Nanobodies against the 12th to 13th epidermal growth factor-like repeat of PEAR1 and phosphoinositide 3-kinase (PI3K) isoform-selective inhibitors were tested for the inhibition of platelet activation. RESULTS: We show that HPGM, heparin conjugated to an albumin protein core, stimulates aggregation and phosphorylation of PEAR1 in washed platelets. Platelet aggregation was abolished by an anti-PEAR1 nanobody, Nb138. UFH stimulated platelet aggregation in washed platelets, but desulfated UFH did not. Furthermore, HPGM, but not UFH, stimulated maximal aggregation in platelet-rich plasma. However, both HPGM and UFH increased integrin αIIbß3 activation in whole blood. By using PI3K isoform-selective inhibitors, we show that PEAR1 activates PI3Kß, leading to Akt phosphorylation. CONCLUSION: Our findings reveal that PEAR1 is a receptor for heparin and HPGM and that PI3Kß is a key signaling molecule downstream of PEAR1 in platelets. These findings may have important implications for our understanding of the role of PEAR1 in cardiovascular disease.


Asunto(s)
Heparina , Fosfatidilinositol 3-Quinasas , Humanos , Heparina/farmacología , Heparina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Células Endoteliales/metabolismo , Receptores de Superficie Celular/metabolismo , Plaquetas/metabolismo , Agregación Plaquetaria , Proteoglicanos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Ligandos , Albúminas
17.
Blood Adv ; 7(1): 46-59, 2023 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-36269841

RESUMEN

Mice lacking the immunoreceptor tyrosine-based inhibition motif-containing co-inhibitory receptor G6b-B (Mpig6b, G6b knockout, KO) are born with a complex megakaryocyte (MK) per platelet phenotype, characterized by severe macrothrombocytopenia, expansion of the MK population, and focal myelofibrosis in the bone marrow and spleen. Platelets are almost completely devoid of the glycoprotein VI (GPVI)-FcRγ-chain collagen receptor complex, have reduced collagen integrin α2ß1, elevated Syk tyrosine kinase activity, and a subset has increased surface immunoglobulins. A similar phenotype was recently reported in patients with null and loss-of-function mutations in MPIG6B. To better understand the cause and treatment of this pathology, we used pharmacological- and genetic-based approaches to rescue platelet counts and function in G6b KO mice. Intravenous immunoglobulin resulted in a transient partial recovery of platelet counts, whereas immune deficiency did not affect platelet counts or receptor expression in G6b KO mice. Syk loss-of-function (R41A) rescued macrothrombocytopenia, GPVI and α2ß1 expression in G6b KO mice, whereas treatment with the Syk kinase inhibitor BI1002494 partially rescued platelet count but had no effect on GPVI and α2ß1 expression or bleeding. The Src family kinase inhibitor dasatinib was not beneficial in G6b KO mice. In contrast, treatment with the thrombopoietin mimetic romiplostim rescued thrombocytopenia, GPVI expression, and platelet reactivity to collagen, suggesting that it may be a promising therapeutic option for patients lacking functional G6b-B. Intriguingly, GPVI and α2ß1 expression were significantly downregulated in romiplostim-treated wild-type mice, whereas GPVI was upregulated in romiplostim-treated G6b KO mice, suggesting a cell intrinsic feedback mechanism that autoregulates platelet reactivity depending on physiological needs.


Asunto(s)
Plaquetas , Trombocitopenia , Ratones , Animales , Plaquetas/metabolismo , Megacariocitos/metabolismo , Trombocitopenia/genética , Familia-src Quinasas/metabolismo , Colágeno/metabolismo
18.
Nucleic Acids Res ; 50(20): 11895-11915, 2022 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-36408906

RESUMEN

We previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs). RBPMS is transcriptionally downregulated during SMC dedifferentiation, but we hypothesized that RBPMS protein activity might be acutely downregulated by post-translational modifications. Publicly available phosphoproteomic datasets reveal that Thr113 and Thr118 immediately adjacent to the RRM domain are commonly both phosphorylated. An RBPMS T113/118 phosphomimetic T/E mutant showed decreased splicing regulatory activity both in transfected cells and in a cell-free in vitro assay, while a non-phosphorylatable T/A mutant retained full activity. Loss of splicing activity was associated with a modest reduction in RNA affinity but significantly reduced RNA binding in nuclear extract. A lower degree of oligomerization of the T/E mutant might cause lower avidity of multivalent RNA binding. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, we identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, our data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.


Asunto(s)
Miocitos del Músculo Liso , Empalme del ARN , Fosforilación , Miocitos del Músculo Liso/metabolismo , Músculo Liso/metabolismo , ARN/metabolismo , Células Cultivadas
19.
J Thromb Haemost ; 20(12): 2939-2952, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36239466

RESUMEN

BACKGROUND: New antithrombotic therapies with less effect on bleeding are needed for coronary artery disease. The Btk inhibitor ibrutinib blocks atherosclerotic plaque-mediated thrombus formation. However, it is associated with increased bleeding, possibly due to non-Btk-mediated effects. Btk-deficient patients do not have bleeding issues, suggesting selective Btk inhibition as a promising antithrombotic strategy. OBJECTIVES: To compare the antithrombotic effects of the highly selective Btk inhibitor AB-95-LH34 (LH34) with ibrutinib. METHODS: Glycoprotein VI and G-protein coupled receptor-mediated platelet function and signaling were analyzed in healthy human donor platelets by lumi-aggregometry, flow adhesion, and western blot following 1 h treatment with inhibitors in vitro. RESULTS: LH34 showed similar inhibition of Btk-Y223 phosphorylation as ibrutinib, but had no off-target inhibition of Src-Y418 phosphorylation. Similar dose-dependent inhibition of aggregation to atherosclerotic plaque material was observed for both. However, in response to Horm collagen, which also binds integrin α2ß1, LH34 exhibited less marked inhibition than ibrutinib. Both LH34 and ibrutinib inhibited platelet adhesion and aggregation to plaque material at arterial shear. Ibrutinib demonstrated the most potent effect, with complete blockade at high concentrations. Platelet activation (P-selectin) and procoagulant activity (phosphatidylserine exposure) in thrombi were inhibited by LH34 and completely blocked by ibrutinib at high concentrations. Furthermore, plaque-induced thrombin generation was reduced by higher concentrations of LH34 and ibrutinib. CONCLUSIONS: LH34 potently inhibits atherosclerotic plaque-induced thrombus formation and procoagulant platelet activity in vitro, with less off-target inhibition of Src than ibrutinib, suggesting it is a promising antiplatelet therapy with the potential for reduced bleeding side effects.


Asunto(s)
Aterosclerosis , Placa Aterosclerótica , Trombosis , Humanos , Placa Aterosclerótica/complicaciones , Fibrinolíticos/uso terapéutico , Plaquetas/metabolismo , Activación Plaquetaria , Inhibidores de Proteínas Quinasas/efectos adversos , Trombosis/tratamiento farmacológico , Aterosclerosis/complicaciones , Hemorragia/inducido químicamente , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/uso terapéutico
20.
Nanoscale ; 14(37): 13500-13504, 2022 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-36102688

RESUMEN

We have developed a 'recombinase amplified CRISPR enhanced chain reaction' (RACECAR) assay that can detect as little as 40 copies of hepatitis B virus (HBV) genome using a benchtop spectrofluorometer. The limit of detection was determined to be 3 copies of HBV genome. The specificity of RACECAR was confirmed against hepatitis A virus (HAV). This assay can detect the genomic targets directly in serum samples without an extraction step. The 4 h-long fluorometric assay was developed by combining three tiers of isothermal amplification processes and can be repurposed for any target of choice. This highly modular reaction setup is an untapped resource that can be incorporated into the front-runners of molecular diagnostics.


Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Recombinasas , ADN Viral/genética , Genoma Viral , Virus de la Hepatitis B/genética , Técnicas de Amplificación de Ácido Nucleico , Recombinasas/genética , Sensibilidad y Especificidad
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