260 research outputs found
Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood
Calibrated Automated Thrombography (CAT) is a versatile and sensitive
method for analyzing coagulation reactions culminating in thrombin
generation (TG). Here, we present a CAT method for analyzing TG in
murine whole blood by adapting the CAT assay used for measuring TG
in human plasma. The diagnostically used artificial and physiologic factor
XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP)
stimulated TG in murine blood in a dose-dependent manner resulting in
a gradual increase in endogenous thrombin potential and peak thrombin,
with shortened lag times and times to peak. The activated FXII inhibitor
rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG
triggered by kaolin, ellagic acid and polyP and TG was completely attenuated
in blood of FXII- (F12â/â) and FXI-deficient (F11â/â) mice. Moreover,
reconstitution of blood from F12â/â mice with human FXII restored impaired
contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven
TG in mouse whole blood and addition of the selective inhibitor PPX_112
ablated natural polyP-stimulated TG. In conclusion, the data provide a method
for analysis of contact activation-mediated TG in murine whole blood. As the
FXII-driven intrinsic pathway of coagulation has emerged as novel target for
antithrombotic agents that are validated in mouse thrombosis and bleeding
models, our novel assay could expedite therapeutic drug development
Targeting NETs using dual-active DNase1 variants
Background: Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively.
Methods: Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences.
Results: We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine.
Conclusion: Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states
Validation of epidermal AMBRA1 and loricrin (AMBLor) as a prognostic biomarker for nonulcerated American Joint Committee on Cancer stage I/II cutaneous melanoma
\ua9 2023 The Author(s). Published by Oxford University Press on behalf of British Association of Dermatologists.Background: Combined expression of the autophagy-regulatory protein AMBRA1 (activating molecule in Beclin1-regulated autophagy) and the terminal differentiation marker loricrin in the peritumoral epidermis of stage I melanomas can identify tumour subsets at low risk of -metastasis. Objectives: To validate the combined expression of peritumoral AMBRA1 and loricrin (AMBLor) as a prognostic biomarker able to identify both stage I and II melanomas at low risk of tumour recurrence. Methods: Automated immunohistochemistry was used to analyse peritumoral AMBRA1 and loricrin expression in geographically distinct discovery (n = 540) and validation (n = 300) cohorts of nonulcerated American Joint Committee on Cancer (AJCC) stage I and II melanomas. AMBLor status was correlated with clinical outcomes in the discovery and validation cohorts separately and combined. Results: Analysis of AMBLor in the discovery cohort revealed a recurrence-free survival (RFS) rate of 95.5% in the AMBLor low-risk group vs. 81.7% in the AMBLor at-risk group (multivariate log-rank, P < 0.001) and a negative predictive value (NPV) of 96.0%. In the validation cohort, AMBLor analysis revealed a RFS rate of 97.6% in the AMBLor low-risk group vs. 78.3% in the at-risk group (multivariate log-rank, P < 0.001) and a NPV of 97.6%. In a multivariate model considering AMBLor, Breslow thickness, age and sex, analysis of the combined discovery and validation cohorts showed that the estimated effect of AMBLor was statistically significant, with a hazard ratio of 3.469 (95% confidence interval 1.403-8.580, P = 0.007) and an overall NPV of 96.5%. Conclusions: These data provide further evidence validating AMBLor as a prognostic biomarker to identify nonulcerated AJCC stage I and II melanoma tumours at low risk of disease recurrence
We Were Never Cool:Investigating knowledge production and discourses of cool in the sociology of music
Crack formation and prevention in colloidal drops
Crack formation is a frequent result of residual stress release from colloidal films made by the evaporation of colloidal droplets containing nanoparticles. Crack prevention is a significant task in industrial applications such as painting and inkjet printing with colloidal nanoparticles. Here, we illustrate how colloidal drops evaporate and how crack generation is dependent on the particle size and initial volume fraction, through direct visualization of the individual colloids with confocal laser microscopy. To prevent crack formation, we suggest use of a versatile method to control the colloid-polymer interactions by mixing a nonadsorbing polymer with the colloidal suspension, which is known to drive gelation of the particles with short-range attraction. Gelation-driven crack prevention is a feasible and simple method to obtain crack-free, uniform coatings through drying-mediated assembly of colloidal nanoparticlesopen0
More than just a bracelet: the use of material symbolism to communicate love
There is growing recognition of the place of love in residential care for children (Smith, 2009). This paper is a critical analysis of a range of existing research on residential child care as well as studies of material culture and of care relationships more broadly. It argues that, despite increasing regulation and surveillance, adults and children find ways to show and feel love in the context of residential care. Whilst love may be regarded as something to be avoided or indeed prohibited in an adult/child care setting these deep bonds find expression in the everyday life of the children's home. By looking at love in this embodied way, the 'realness' of material things to assert connection and recognition of love (Layne, 2000) is examined. As Gorenstein (1996, p.8) suggests 'objects...[are] the perfect vehicles for conveying themes that are not commonly accepted in a community'. The paper emphasises the recognition of these symbolic and metaphorical forms of communication in practice
Comparative ileal amino acid digestibility and growth performance in growing pigs fed different level of canola meal
Relating reactions to service disruptions and transit use frequency: the mediating role of perceived service quality and transit system resilience
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