Interrogating a Compound Library in Search of an Inhibitor for TREM-like Transcript-1 to Fibrinogen Binding.

Cardiovascular disease (CVD) remains one of leading causes of death worldwide. Aberrant platelet function mediate fibrin(ogen) rich thrombi that lead to occlusive thrombi associated with mortality. The receptor, TREM-like transcript-1 (TLT-1), stored in the platelet a-granules and released upon platelet activation, binds fibrinogen and von Willebrand factor. Once it is released from platelets TLT-1 is a potential therapeutic target to prevent the thrombosis associated with CVD. Here we design an assay to screen a compound library of small molecules inhibitors. HEK-293 cells stably transfected with a full length human treml-1 construct were used to screen library of 800 compounds, for inhibition of TLT-1 to fibrinogen binding in an attachment assay using crystal violet staining. The possible cytotoxicity of the best compounds was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide MTT and calcein AM staining assays. Here we demonstrate that the addition of TLT-1 to HEK-293 cells increases cell adhesion by more than 2-fold. We identified ~80 compounds that inhibit binding by more than 80%. We further tested the top compounds and confirmed that reduction of hTLT-1 to fibrinogen bound in the top compounds was not caused by cytotoxicity, as per colorimetric and fluorescent viability assays. Four compounds were identified as potential small molecule inhibitors one of which, BM-8372, demonstrated significant effect in platelet aggregation assays. Significance Statement TLT-1 is a key platelet receptor that binds fibrinogen and mediates clot formation The developed assay successfully screens 800 small molecules, pinpointing ~80 potent inhibitors that reduce TLT-1 binding by over 80%. Importantly, the study rigorously rules out cytotoxicity concerns, affirming the therapeutic potential of the identified compounds. By elucidating TLT-1's role and presenting promising inhibitors, this research offers a significant stride toward developing novel strategies to combat CVD-related thrombosis.

Whole Blood Aggregometry in Mice.

Aggregometry plays a crucial role in both clinical diagnostics and research within hematology, serving as a fundamental tool for understanding platelet function and its implications in physiological and pathological processes. In research, aggregometry provides insights into platelet aggregation dynamics and aids in understanding the underlying mechanisms of hemostasis, thrombosis, and related disorders. Light transmission aggregometry (LTA) and lumi-aggregometry, as well as whole blood aggregometry, are commonly employed methods. While LTA and lumi-aggregometry allow for specific platelet function assessment under controlled conditions, whole blood aggregometry provides a more physiologically relevant approach by evaluating platelet aggregation within the context of whole blood. Although both methodologies offer unique advantages, whole blood aggregometry allows for preservation of the native cellular environment, simplicity, and potential for better clinical correlation. In a clinical setting, with human blood samples, protocols are established for both LTA and whole blood aggregometry as they are frequently used diagnostic tools. A protocol for LTA and lumi-aggregometry in murine models has been described; however, to date, there is no standardized protocol for whole blood aggregometry in murine models accessible to hematology researchers. This article aims to outline a simple, basic protocol for murine whole blood aggregometry, offering an alternative method to the commonly used LTA aggregometry in research settings. Standardizing whole blood aggregometry protocols in murine models could enhance experimental reliability and facilitate translational research efforts in hematology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Whole blood aggregometry in mice Support Protocol: Phenylhydrazine-induced anemia in wild-type mice Basic Protocol 2: Hematocrit percentage in mice.

High Levels of Triggering Receptor Expressed in Myeloid Cells-Like Transcript-1 Positive, but Not Glycoprotein 1b+, Microparticles Are Associated With Poor Outcomes in Acute Respiratory Distress Syndrome.

OBJECTIVES: To identify triggering receptor expressed in myeloid cells-like transcript-1 positive (TLT-1+) microparticles (MPs) and evaluate if their presence is associated with clinical outcomes and/or disease severity in acute respiratory distress syndrome (ARDS). DESIGN: Retrospective cohort study. SETTING: ARDS Network clinical trials. PATIENTS: A total of 564 patients were diagnosed with ARDS. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Using flow cytometry, we demonstrated the presence of TLT-1+ platelet-derived microparticles (PMP) that bind fibrinogen in plasma samples from fresh donors. We retrospectively quantified TLT-1, glycoprotein (Gp) 1b, or αIIbßIIIa immunopositive microparticles in plasma samples from patients with ARDS enrolled in the ARMA, KARMA, and LARMA (Studies 01 and 03 lower versus higher tidal volume, ketoconazole treatment, and lisofylline treatment Clincial Trials) ARDS Network clinical trials and evaluated the relationship between these measures and clinical outcomes. No associations were found between Gp1b+ MPs and clinical outcomes for any of the cohorts. When stratified by quartile, associations were found for survival, ventilation-free breathing, and thrombocytopenia with αIIbßIIIa+ and TLT-1+ MPs (χ2p < 0.001). Notably, 63 of 64 patients in this study who failed to achieve unassisted breathing had TLT+ PMP in the 75th percentile. In all three cohorts, patients whose TLT+ MP counts were higher than the median had higher Acute Physiology and Chronic Health Evaluation III scores, were more likely to present with thrombocytopenia and were 3.7 times (p < 0.001) more likely to die than patients with lower TLT+ PMP after adjusting for other risk factors. CONCLUSIONS: Although both αIIbßIIIa+ and TLT+ microparticles (αIIbßIIIa, TLT-1) were associated with mortality, TLT-1+ MPs demonstrated stronger correlations with Acute Physiology and Chronic Health Evaluation III scores, unassisted breathing, and multiple system organ failure. These findings warrant further exploration of the mechanistic role of TLT-1+ PMP in ARDS or acute lung injury progression.

Affordable Method for Hematocrit Determination in Murine Models.

Hematocrit (Hct) is a powerful tool often used in a clinical setting for the diagnosis of blood conditions such as anemia. It is also used in the research field as a hematological parameter in both human and mouse models. Measuring Hct, however, involves the use of expensive standardized equipment (such as a CritSpin™ Microhematocrit Centrifuge). Here, we describe a novel, simple, and affordable method to determine the Hct in untreated wild-type (WT) mice and phenylhydrazine (PHZ)-induced anemic mice with reasonable accuracy, using a benchtop centrifuge commonly available in laboratories. Hct of murine samples processed with a benchtop centrifuge, when compared to the standardized method CritSpin™, showed comparable results. This approach for determining Hct of murine can prove useful to research laboratories that cannot afford specialized equipment for Hct studies. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Affordable Method for Hematocrit Determination in Murine Models Basic Protocol 2: Murine Sample Validation Support Protocol: Phenylhydrazine-induced anemia in wild-type (WT) mice.

Accelerating Climate Action Through Academic Health Systems: Leadership's Call to Duty.

This Viewpoint makes the case for academic health systems to lead the way on climate change action in the US, including planning to reduce greenhouse gas emissions, educating current and future clinicians, and communicating with their patients and communities.

Cryo-EM Structure of AAV2 Rep68 bound to integration site AAVS1: Insights into the mechanism of DNA melting.

The Rep68 protein from Adeno-Associated Virus (AAV) is a multifunctional SF3 helicase that performs most of the DNA transactions required for the viral life cycle. During AAV DNA replication, Rep68 assembles at the origin and catalyzes the DNA melting and nicking reactions during the hairpin rolling replication process to complete the second-strand synthesis of the AAV genome. Here, we report the Cryo-EM structures of Rep68 bound to double-stranded DNA (dsDNA) containing the sequence of the AAVS1 integration site in different nucleotide-bound states. In the apo state, Rep68 forms a heptameric complex around DNA, with three Origin Binding Domains (OBDs) bound to the Rep Binding Site (RBS) sequence and three other OBDs forming transient dimers with them. The AAA+ domains form an open ring with no interactions between subunits and with DNA. We hypothesize the heptameric quaternary structure is necessary to load onto dsDNA. In the ATPγS-bound state, a subset of three subunits binds the nucleotide, undergoing a large conformational change, inducing the formation of intersubunit interactions interaction and interaction with three consecutive DNA phosphate groups. Moreover, the induced conformational change positions three phenylalanine residues to come in close contact with the DNA backbone, producing a distortion in the DNA. We propose that the phenylalanine residues can potentially act as a hydrophobic wedge in the DNA melting process.

Not so once upon a time.

This story reflects a personal narrative of a graduate student who experienced a disturbing incident during Mardi Gras in New Orleans, shedding light on the enduring issue of racism. The author's journey to graduate school and the challenges faced along the way provide context for the pivotal moment of racial discrimination. The narrative then shifts to the author's decision to prioritize education over anger, highlighting the sacrifices made to protect their future as a graduate student. The incident serves as a stark reminder that, despite personal achievements and aspirations, racial prejudice persists. In conclusion, the author calls for resilience and focus in the pursuit of personal goals while acknowledging the ongoing struggle against racism and other forms of discrimination in society. This personal story serves as a poignant reminder of the challenges faced by individuals and the need for continued efforts to combat systemic intolerance.

The Characterization and Evaluation of the Soluble Triggering Receptor Expressed on Myeloid Cells-like Transcript-1 in Stable Coronary Artery Disease.

Platelets play crucial roles in the development and progression of coronary artery disease (CAD). The triggering receptor expressed in myeloid cells-like transcript-1 (TLT-1) is stored in platelet α granules, and activated platelets release a soluble fragment (sTLT-1). We set out to better characterize the constituent amino acids of sTLT-1 and to evaluate sTLT-1 for use as a biomarker in patients with stable CAD. We evaluated sTLT-1 release using immunoprecipitation and mass spectrometry and employed statistical methods to retrospectively correlate sTLT-1 concentrations, utilizing ELISA in plasma samples from 1510 patients with documented stable CAD. We identified TLT-1 residues to 133 in platelet releasates. ADAM17 cuts TLT-1, suggesting that S136 is the C-terminal amino acid in sTLT-1. Our results revealed that for CAD patients, sTLT-1 levels did not differ significantly according to primary outcomes of death or major cardiac event; however, patients with left ventricular (LV) dysfunction had significantly lower plasma sTLT-1 levels as compared to those with normal LV function (981.62 ± 1141 pg/mL vs. 1247.48 ± 1589 pg/mL; p = 0.003). When patients were stratified based on sTLT-1 peak frequency distribution (544 pg/mL), a significant association with congestive heart failure was identified (OR = 2.94; 1.040-8.282; p = 0.042), which could be explained by LV dysfunction.

Early laboratory hematological parameters associated with COVID-19 mortality in an Amazonian population of Peru

Introduction: COVID-19 has a significant impact on the hematopoietic system and hemostasis. Leukocytosis, lymphopenia, and thrombocytopenia are associated with increased severity and even death in COVID-19 cases. Objective: The aim is to examine the laboratory results of COVID-19 patients from a hospital in the Peruvian Amazon and their clinical prognosis. Material and Methods: An analytical cross-sectional study was carried out whose purpose was to identify the laboratory tests of patients with COVID-19 and mortality in a hospital in Ucayali, Peru during the period from March 13 to May 9, 2020, selecting a total of 127 with Covid-19. Mean and the standard deviation was described for age, leukocytes, neutrophils, platelets, RDW-SD; median and interquartile range for the variables lymphocyte, RN / L, fibrinogen, CRP, D-dimer, DHL, hematocrit, monocytes, eosinophils. Results: No differences were observed in this population regarding death and sex (OR: 1.31; 95% CI 0.92 to 1.87), however, it was observed that, for each one-year increase, the probability of death increased by 4% (PR: 1.04, 95% CI 1.03 to 1.05). The IRR (Incidence Risk Ratio) analysis for the numerical variables showed results strongly associated with hematological values such as Leukocytes (scaled by 2500 units) (IRR: 1.08, 95% CI 1.03 to 1.13), neutrophils (scaled by 2500 units) (IRR: 1.08; 95% CI 1.03 to 1.13), on the contrary, it is observed that the increase of 1000 units in lymphocytes, the probability of dying decreased by 48% (IRR: 0.52; 95% CI 0.38 to 071). Conclusions: Parameters such as leukocytes,neutrophils and D-dimer were statistically much higher in patients who died.

Introducción: COVID-19 tiene un impacto significativo en el sistema hematopoyético y la hemostasia. La leucocitosis, la linfopenia y la trombocitopenia se asocian con una mayor gravedad e incluso la muerte en los casos de COVID-19. Objetivo: examinar los resultados de laboratorio de pacientes con COVID-19 de un hospital de la Amazonía peruana y su pronóstico clínico. Material y métodos: Se realizó un estudio transversal analítico cuyo propósito fue identificar las pruebas de laboratorio de pacientes con COVID-19 y mortalidad en un hospital de Ucayali, Perú durante el periodo del 13 de marzo al 9 de mayo del 2020, seleccionando un total de 127 con COVID-19. Se describió la media y la desviación estándar para edad, leucocitos, neutrófilos, plaquetas, RDW-SD; mediana y rango intercuartílico para las variables linfocito, RN/L, fibrinógeno, PCR, dímero D, DHL, hematocrito, monocitos, eosinófilos. Resultados: No se observaron diferencias en esta población en cuanto a muerte y sexo (OR: 1,31; IC 95% 0,92 a 1,87), sin embargo, se observó que, por cada aumento de un año, la probabilidad de muerte aumentaba un 4% (RP: 1,04). , IC del 95%: 1,03 a 1,05). El análisis de RIR (Razón de incidencia de riesgos) para las variables numéricas mostró resultados fuertemente asociados con valores hematológicos como Leucocitos (escala de 2500 unidades) (RRI: 1.08, 95% CI 1.03 a 1.13), neutrófilos (escala de 2500 unidades) (RRI: 1.08; IC 95% 1.03 a 1.13), por el contrario, se observa que al aumento de 1000 unidades en linfocitos, la probabilidad de morir disminuyó en un 48% (TIR: 0.52; IC 95% 0.38 a 071). Conclusiones: Parámetros tales como los leucocitos, los neutrófilos y el dímero D fueron estadísticamente mucho más altos en los pacientes que fallecieron.

Electronic and structural study of T315I mutated form in DFG-out conformation of BCR-ABL inhibitors.

In this work, the four main drugs for the treatment of chronic myeloid leukemia were analyzed, being imatinib, dasatinib, nilotinib and ponatinib followed by four derivative molecules of nilotinib and ponatinib. For these derivative molecules, the fluorine atoms were replaced by hydrogen and chlorine atoms in order to shade light to the structural effects on this set of inhibitors. Electronic studies were performed at density functional theory level with the B3LYP functional and 6-311+G(d,p) basis set. The frontier molecular orbitals, gap HOMO-LUMO, and NBO were analyzed and compared to docking studies for mutant T315I tyrosine kinase protein structure code 3IK3, in the DFG-out conformation. Structural similarities were pointed out, such as the presence of groups common to all inhibitors and modifications raised up on new generations of imatinib-based inhibitors. One of them is the trifluoromethyl group present in nilotinib and later included in ponatinib, in addition to the 1-methylpiperazin-1-ium group that is present in imatinib and ponatinib. The frontier molecular orbitals of imatinib and ponatinib are contributing to the same amino acid residues, and the ineffectiveness of imatinib against the T315I mutation was discussed.Communicated by Ramaswamy H. Sarma.

The enigmatic nature of the triggering receptor expressed in myeloid cells -1 (TLT- 1).

Receptors are important pharmacological targets on cells. The Triggering Receptor Expressed on Myeloid Cells (TREM) - Like Transcript - 1 is an abundant, yet little understood, platelet receptor. It is a single Ig domain containing receptor isolated in the α-granules of resting platelets and brought to the platelet surface upon activation. On platelets, the integrin αIIbß3 is the major receptor having roughly 80,000 copies. αIIbß3 is a heterodimeric multidomain structure that mediates platelet aggregation through its interaction with the plasma protein fibrinogen. Anti-platelet drugs have successfully targeted αIIbß3 to control thrombosis. Like αIIbß3, TLT-1 also binds fibrinogen, making its role in platelet function somewhat obscure. In this review, we highlight the known structural features of TLT-1 and present the challenges of understanding TLT-1 function. In our analysis of the dynamics of the platelet surface after activation we propose a model in which TLT-1 supports αIIbß3 function as a mechanoreceptor that may direct platelets toward immune function.

Prognosis and hematological findings in patients with COVID-19 in an Amazonian population of Peru

ObjectiveThis study examined the laboratory results of COVID-19 patients from a hospital in the Peruvian Amazon and their clinical prognosis. MethodsAn analytical cross-sectional study was carried out whose purpose was to identify the laboratory tests of patients with COVID-19 and mortality in a hospital in Ucayali, Peru during the period from March 13 to May 9, 2020, selecting a total of 127 with Covid-19. Mean and the standard deviation was described for age, leukocytes, neutrophils, platelets, RDW-SD; median and interquartile range for the variables lymphocyte, RN / L, fibrinogen, CRP, D-dimer, DHL, hematocrit, monocytes, eosinophils. ResultsNo differences were observed in this population regarding death and sex (OR: 1.31; 95% CI 0.92 to 1.87), however, it was observed that, for each one-year increase, the probability of death increased by 4% (PR: 1.04, 95% CI 1.03 to 1.05). The IRR (Incidence Risk Ratio) analysis for the numerical variables showed results strongly associated with hematological values such as Leukocytes (scaled by 2500 units) (IRR: 1.08, 95% CI 1.03 to 1.13), neutrophils (scaled by 2500 units) (IRR: 1.08; 95% CI 1.03 to 1.13), on the contrary, it is observed that the increase of 1000 units in lymphocytes, the probability of dying decreased by 48% (IRR: 0.52; 95% CI 0.38 to 071). ConclusionParameters such as leukocytes and neutrophils were statistically much higher in patients who died.

Concern about malignancy contributes to the psychosocial toll of dermatomyositis.

Objective: Dermatomyositis (DM) is associated with increased morbidity and mortality, decreased quality of life, and an increased incidence of psychiatric illness. We performed a pilot study to investigate whether concern about malignancy contributes to the psychosocial toll of DM.Method: Patients with a recent DM diagnosis at our institution between 2013 and 2018 and no history of DM-associated malignancy completed standardized questionnaires and a novel survey generated by our group, and participated in focus groups.Results: Seventeen patients (14 females and three males) completed the surveys. The mean Dermatology Life Quality Index (DLQI) score was 2.75. The mean score on the Patient Health Questionnaire-9 (PHQ-9) was 5.35, with a range of 0-20. Our independent DM-specific questionnaire revealed a mean of 17.41 (range 2-40).Conclusion: Concern about the increased risk of malignancy contributes to the psychosocial toll of DM. Individual impacts are highly variable and patient specific. Clinicians should assess for depression and anxiety in patients with DM, understanding that concern for malignancy adds to the total psychosocial burden in some patients.

The Effects of the Alkaloid Tambjamine J on Mice Implanted with Sarcoma 180 Tumor Cells.

The tambjamines are a small group of bipyrrolic alkaloids that, collectively, display a significant range of biological activities including antitumor, antimicrobial and immunosuppressive properties. The key objective of the present study was to undertake preclinical assessments of tambjamine J (T-J) so as to determine its in vivo antitumor effects. To that end, sarcoma 180 cells were transplanted in mice and the impacts of the title compound then evaluated using a range of protocols including hematological, biochemical, histopathological, genotoxic and clastogenic assays. As a result it was established that this alkaloid has a significant therapeutic window and effectively reduces tumor growth (by 40 % and 79 % at doses of 10 and 20 mg/kg/day, respectively). In this regard it displays similar antitumor activity to the anticancer agent cyclophosphamide and alters animal weight in an analogous manner.

Developing a Workforce for Health in North Carolina: Planning for the Future.

Among the many trends influencing health and health care delivery over the next decade, three are particularly important: the transition to value-based care and increased focus on population health; the shift of care from acute to community-based settings; and addressing the vulnerability of rural health care systems in North Carolina.

Unveiling contextual realities by microscopically entangling a neutron.

The development of qualitatively new measurement capabilities is often a prerequisite for critical scientific and technological advances. Here we introduce an unconventional quantum probe, an entangled neutron beam, where individual neutrons can be entangled in spin, trajectory and energy. The spatial separation of trajectories from nanometers to microns and energy differences from peV to neV will enable investigations of microscopic magnetic correlations in systems with strongly entangled phases, such as those believed to emerge in unconventional superconductors. We develop an interferometer to prove entanglement of these distinguishable properties of the neutron beam by observing clear violations of both Clauser-Horne-Shimony-Holt and Mermin contextuality inequalities in the same experimental setup. Our work opens a pathway to a future of entangled neutron scattering in matter.

Defining the TLT-1 interactome from resting and activated human platelets.

The triggering receptor expressed on myeloid cells (TREM) protein family forms a class of type I transmembrane proteins expressed in immune cells that play important roles in innate and adaptive immune responses. The TREM family member TREM-like transcript 1 (TLT-1, also TREML1) is expressed in megakaryocytes and packaged into platelet granules. TLT-1 binds fibrinogen and plays a role in bleeding initiated by inflammatory insults. Here, we describe a proteomics screen that maps the TLT-1 interactome in resting and activated human platelets. Several identified TLT-1 interactors are involved in cell adhesion and migration, as well as platelet activation. Select interactors, including ß3-integrin, RACK1, GRB2, and Rabs 5A, 7, and 11A, were additionally characterized in co-immunoprecipitation/immunoblotting experiments. Finally, several phosphorylation sites were found on immunoprecipitated TLT-1, including Thr280, a novel, regulated site on a conserved residue near the TLT-1 ITIM regulatory sequence. SIGNIFICANCE: Platelet function relies on the secretion of active molecules from intracellular vesicles, or granules, which contain soluble and membrane-bound proteins that are essential for platelet aggregation, coagulation reactions, and pathogen defense mechanisms. TLT-1 is sequestered in α-granules and transported to the plasma membrane, where it plays a unique role in hemostasis after inflammatory insults. Despite the known importance of TLT-1 in platelet biology, our knowledge of TLT-1 mechanistic signaling is limited. This study defines the TLT-1 interactome in resting and active human platelets, identifying several novel TLT-1 interactors, as well as TLT-1 phosphorylation sites, all with likely signaling implications in platelet aggregation dynamics.

Platelet biology of the rapidly failing lung.

Acute respiratory distress syndrome (ARDS) is characterized by a rapid-onset respiratory failure with a mortality rate of approximately 40%. This physiologic inflammatory process is mediated by disruption of the alveolar-vascular interface, leading to pulmonary oedema and impaired oxygen exchange, which often warrants mechanical ventilation to increase survival in the acute setting. One of the least understood aspects of ARDS is the role of the platelets in this process. Platelets, which protect vascular integrity, play a pivotal role in the progression and resolution of ARDS. The recent substantiation of the age-old theory that megakaryocytes are found in the lungs has rejuvenated interest in and raised new questions about the importance of platelets for pulmonary function. In addition to primary haemostasis, platelets provide a myriad of inflammatory functions that are poised to aid the innate immune system. This review focuses on the evidence for regulatory roles of platelets in pulmonary inflammation, with an emphasis on two receptors, CLEC-2 and TLT-1. Studies of these receptors identify novel pathways through which platelets may regulate vascular integrity and inflammation in the lungs, thereby influencing the development of ARDS.