Water’s ability to donate and accept hydrogen bonds leads to unique and complex collective dynamical phenomena associated with its hydrogen-bond network. It is appreciated that the vibrations ...governing liquid water’s molecular dynamics are delocalized, with nuclear motion evolving coherently over the span of several molecules. Using two-dimensional infrared spectroscopy, we have found that the nuclear motions of heavy water, D2O, are qualitatively different than those of H2O. The nonlinear spectrum of liquid D2O reveals distinct O–D stretching resonances, in contrast to H2O. Furthermore, our data indicates that condensed-phase O–D vibrations have a different character than those in the gas phase, which we understand in terms of weakly delocalized symmetric and antisymmetric stretching vibrations. This difference in molecular dynamics reflects the shift in the balance between intra- and intermolecular couplings upon deuteration, an effect which can be understood in terms of the anharmonicity of the nuclear potential energy surface.
We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture ...functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.
•Natural silk protein sponge and vascular tubes reproduce human bone marrow niche environments for functional platelet generation ex vivo.•Programmable bioengineered model for the investigation and therapeutic targeting of altered platelet formation.
Fibronectin (FN) is a major extracellular matrix protein implicated in cell adhesion and differentiation in the bone marrow (BM) environment. Alternative splicing of FN gene results in the generation ...of protein variants containing an additional EIIIA domain that sustains cell proliferation or differentiation during physiological or pathological tissue remodeling. To date its expression and role in adult hematopoiesis has not been explored. In our research, we demonstrate that during physiological hematopoiesis a small fraction of BM derived FN contains the EIIIA domain and that mice constitutively including (EIIIA(+/+) ) or excluding (EIIIA(-/-) ) the EIIIA exon present comparable levels of hematopoietic stem cells, myeloid and lymphoid progenitors within BM. Moreover, only minor alterations were detected in blood parameters and in hematopoietic frequencies of BM granulocytes/monocytes and B cells. As opposed to other tissues, unique compensatory mechanisms, such as increased FN accumulation and variable expression of the EIIIA receptors, Toll like receptor-4 and alpha9 integrin subunit, characterized the BM of these mice. Our data demonstrate that FN is a fundamental component of the hematopoietic tissue and that the EIIIA exon may play a key role in modulating hematopiesis in conditions of BM stress or diseases. Stem Cells 2016;34:2263-2268.
We report a new pathogenic mechanism in von Willebrand disease involving the use of a non-canonical splicing site. The proband, carrying the homozygous c.2269_2270del mutation previously classified ...as a type 3 mutation, showed severely reduced plasma and platelet von Willebrand factor antigen levels and functions, and no factor VIII binding capacity. A particular von Willebrand factor multimer pattern emerged in plasma, characterized by the presence of only two oligomers: the dimer and an unusually large band, with no intermediate components. There were von Willebrand factor multimers in platelets, but each band ran more slowly than the normal counterpart. No anti-von Willebrand factor antibodies were detectable. The proband was classified as having severe type 1 von Willebrand disease. Seeking the relationship between phenotype and genotype, we found the c.2269_2270del mutation associated with three different RNA: r.2269_2270del (RNAI), giving a truncated von Willebrand factor protein; r.2269_2270del;2282_2288del (RNAII), resulting from activation of a cryptic "AG" splicing site; and r.2269_2270del;2281_2282insAG (RNAIII), where the wild-type "AG" acceptor of exon 18 was retained due to the non-canonical 2279-2280 "CG" acceptor splicing site being used. The aberrant RNAII and RNAIII caused the alteration of the furin cleavage and binding sites, respectively, both resulting in a von Willebrand factor protein characterized by the persistence of von Willebrand factor propeptide, as confirmed by western blot analysis of the recombinant mutated von Willebrand factor molecules produced
Taken together, these findings explain the residual von Willebrand factor synthesis, slower-running multimers, and absent factor VIII binding capacity. The apparently pure gene null mutation c.2269_2270del profoundly alters von Willebrand factor gene splicing, inducing a complex RNA expression pattern.
Platelets contain and release matrix metalloproteinase-2 (MMP-2) that in turn potentiates platelet aggregation. Platelet deposition on a damaged vascular wall is the first, crucial, step leading to ...thrombosis. Little is known about the effects of MMP-2 on platelet activation and adhesion under flow conditions. We studied the effect of MMP-2 on shear-dependent platelet activation using the O'Brien filtration system, and on platelet deposition using a parallel-plate perfusion chamber. Preincubation of human whole blood with active MMP-2 (50 ng/ml, i.e. 0.78 nM) shortened filter closure time (from 51.8 ± 3.6 sec to 40 ± 2.7 sec, p<0.05) and increased retained platelets (from 72.3 ± 2.3% to 81.1 ± 1.8%, p<0.05) in the O'Brien system, an effect prevented by a specific MMP-2 inhibitor. High shear stress induced the release of MMP-2 from platelets, while TIMP-2 levels were not significantly reduced, therefore, the MMP-2/TIMP-2 ratio increased significantly showing enhanced MMP-2 activity. Preincubation of whole blood with active MMP-2 (0.5 to 50 ng/ml, i.e 0.0078 to 0.78 nM) increased dose-dependently human platelet deposition on collagen under high shear-rate flow conditions (3000 sec⁻¹) (maximum +47.0 ± 11.9%, p<0.05, with 50 ng/ml), while pre-incubation with a MMP-2 inhibitor reduced platelet deposition. In real-time microscopy studies, increased deposition of platelets on collagen induced by MMP-2 started 85 sec from the beginning of perfusion, and was abolished by a GPIIb/IIIa antagonist, while MMP-2 had no effect on platelet deposition on fibrinogen or VWF. Confocal microscopy showed that MMP-2 enhances thrombus volume (+20.0 ± 3.0% vs control) rather than adhesion. In conclusion, we show that MMP-2 potentiates shear-induced platelet activation by enhancing thrombus formation.
Abstract Objectives Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. ...Methods Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Results Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately −35% and −25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α−ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47phox translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Conclusion Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation.
: Acquired nasopharyngeal stenosis is a rare and heterogeneous pathological condition that has different causes, generally resulting as a complication of a pharyngeal surgery, especially in patients ...affected by obstructive sleep apnea (OSA). Different approaches have been proposed for the treatment of nasopharyngeal stenosis but a unique and standardized management has not yet been presented. The aim of our paper is to evaluate the efficacy of our surgical technique, describing its steps and results with the aim to consider it as a possible solution for the treatment of this condition.
: This is a retrospective cohort study. Eight patients (mean age 27.25 years old (yo), range 8-67 yo; Male/Female ratio 4/4; mean body mass index (BMI) 26.1) affected by OSA (mean apnea hypopnea index (AHI) before OSA surgery was 22.1) and acquired nasopharyngeal stenosis as a consequence of different pharyngeal surgeries were treated with our modified approach in the Department of Otolaryngology, Morgagni Pierantoni Hospital, Forlì, Italy. Resolution of stenosis and complication rate were the main outcome measures.
: Complete resolution of the stenosis was achieved in all cases and no complications were recorded at three weeks, six months, and 2 years follow-up.
: Our technique appears to be a promising method for the management of nasopharyngeal stenosis in OSA patients. However, further studies comparing different techniques and reporting on larger series and longer follow up time are needed to prove the efficacy of the proposed technique.
The fibronectin EDA isoform (EDA FN) is instrumental in fibrogenesis but, to date, its expression and function in bone marrow (BM) fibrosis have not been explored. We found that mice constitutively ...expressing the EDA domain (EIIIA
), but not EDA knockout mice, are more prone to develop BM fibrosis upon treatment with the thrombopoietin (TPO) mimetic romiplostim (TPO
). Mechanistically, EDA FN binds to TLR4 and sustains progenitor cell proliferation and megakaryopoiesis in a TPO-independent fashion, inducing LPS-like responses, such as NF-κB activation and release of profibrotic IL-6. Pharmacological inhibition of TLR4 or TLR4 deletion in TPO
mice abrogated Mk hyperplasia, BM fibrosis, IL-6 release, extramedullary hematopoiesis, and splenomegaly. Finally, developing a novel ELISA assay, we analyzed samples from patients affected by primary myelofibrosis (PMF), a well-known pathological situation caused by altered TPO signaling, and found that the EDA FN is increased in plasma and BM biopsies of PMF patients as compared with healthy controls, correlating with fibrotic phase.
Sickle cell disease is an autosomal recessive genetic red cell disorder with a worldwide distribution. Growing evidence suggests a possible involvement of complement activation in the severity of ...clinical complications of sickle cell disease. In this study we found activation of the alternative complement pathway with microvascular deposition of C5b-9 on skin biopsies from patients with sickle cell disease. There was also deposition of C3b on sickle red cell membranes, which is promoted locally by the exposure of phosphatidylserine. In addition, we showed for the first time a peculiar "stop-and-go" motion of sickle cell red blood cells on tumor factor-α-activated vascular endothelial surfaces. Using the C3b/iC3b binding plasma protein factor Has an inhibitor of C3b cell-cell interactions, we found that factor H and its domains 19-20 prevent the adhesion of sickle red cells to the endothelium, normalizing speed transition times of red cells. We documented that factor H acts by preventing the adhesion of sickle red cells to P-selectin and/or the Mac-1 receptor (CD11b/CD18), supporting the activation of the alternative pathway of complement as an additional mechanism in the pathogenesis of acute sickle cell related vaso-occlusive crises. Our data provide a rationale for further investigation of the potential contribution of factor H and other modulators of the alternative complement pathway with potential implications for the treatment of sickle cell disease.
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