RESUMEN
Self-assembled fibrinogen nanofibers are promising candidates for skin tissue engineering due to their biocompatibility and ability to mimic the native blood clot architecture. Here, we studied the structure-property relationship and degradation of rehydrated fibrinogen nanofibers prepared by salt-induced self-assembly, focusing on the effect of scaffold layering, cross-linking time and freeze-drying. Optimal fiber stability was achieved with cross-linking by formaldehyde (FA) vapor, while treatment with liquid aldehydes, genipin, EDC, and transglutaminase failed to preserve the nanofibrous architecture upon rehydration. Scaffold layering did not significantly influence the mechanical properties but changed the scaffold architecture, with bulk fiber scaffolds being more compact than layered scaffolds. Freeze-drying maintained the mechanical properties and interconnected pore network with average pore diameters around 20 µm, which will enhance the storage stability of self-assembled fibrinogen scaffolds. Varying cross-linking times altered the scaffold mechanics without affecting the swelling behavior, indicating that scaffold hydration can be controlled independently of the mechanical characteristics. Cross-linking times of 240 min increased scaffold stiffness and decreased elongation, while 30 min resulted in mechanical properties similar to native skin. Cross-linking for 120 min was found to reduce scaffold degradation by various enzymes in comparison to 60 min. Overall, after 35 days of incubation, plasmin and a combination of urokinase and plasminogen exhibited the strongest degradative effect, with nanofibers being more susceptible to enzymatic degradation than planar fibrinogen due to their higher specific surface area. Based on these results, self-assembled fibrinogen fiber scaffolds show great potential for future applications in soft tissue engineering that require controlled structure-function relationships and degradation characteristics.
Asunto(s)
Materiales Biocompatibles , Fibrinógeno , Ensayo de Materiales , Nanofibras , Andamios del Tejido , Nanofibras/química , Fibrinógeno/química , Fibrinógeno/metabolismo , Andamios del Tejido/química , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Ingeniería de Tejidos , Tamaño de la Partícula , Estructura MolecularRESUMEN
The survival and proliferation of pathogenic Leptospira within a host are complex phenomena that require careful consideration. The ErpY-like lipoprotein, found on the outer membrane surface of Leptospira, plays a crucial role in enhancing the bacterium's pathogenicity. The rErpY-like protein, in its recombinant form, contributes significantly to spirochete virulence by interacting with various host factors, including host complement regulators. This interaction facilitates the bacterium's evasion of the host complement system, thereby augmenting its overall pathogenicity. The rErpY-like protein exhibits a robust binding affinity to soluble fibrinogen, a vital component of the host coagulation system. In this study, we demonstrate that the rErpY-like protein intervenes in the clotting process of the platelet-poor citrated plasma of bovines and humans in a concentration-dependent manner. It significantly reduces clot density, alters the viscoelastic properties of the clot, and diminishes the average clotting rate in plasma. Furthermore, the ErpY-like protein inhibits thrombin-catalyzed fibrin formation in a dose-dependent manner and exhibits saturable binding to thrombin, suggesting its significant role in leptospiral infection. These findings provide compelling evidence for the anticoagulant effect of the ErpY-like lipoprotein and its significant role in leptospiral infection.
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Coagulación Sanguínea , Fibrinógeno , Trombina , Fibrinógeno/metabolismo , Fibrinógeno/química , Humanos , Trombina/metabolismo , Animales , Bovinos , Unión Proteica , Leptospira/metabolismo , Leptospirosis/microbiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Interacciones Huésped-PatógenoRESUMEN
The molecular mechanism of the formation of protein corona by the interaction of gold nanorods (AuNRs) with fibrinogen and transferrin was studied by spectroscopic methods and molecular docking. Studies have shown that AuNRs can be used as quencher to quench the fluorescence of fibrinogen/transferrin. The quenching mechanism mainly comes from static quenching. Fibrinogen has two different binding sites on the longitudinal and the transverse plane of AuNRs respectively, while transferrin has only one binding site on the surface of AuNRs. The adsorption process conforms to Freundlich adsorption isotherm and the pseudo-second-order reaction. The chemisorption is the rate-limiting step. Fibrinogen/transferrin may be a component of the "hard corona" because they bind AuNRs with high binding affinity. The formation of protein corona leads to a decrease in the hydrophobicity of the microenvironment around transferrin tryptophan (Trp) residues and an increase in the hydrophobicity of the microenvironment around fibrinogen/transferrin tyrosine (Tyr) residues, affecting the tertiary and secondary structure of fibrinogen/transferrin. Molecular docking can clearly see the specific amino acid residues of fibrinogen and transferrin adsorbed on AuNRs, and verify the experimental results.
Asunto(s)
Fibrinógeno , Oro , Simulación del Acoplamiento Molecular , Nanotubos , Unión Proteica , Corona de Proteínas , Transferrina , Oro/química , Transferrina/química , Transferrina/metabolismo , Nanotubos/química , Fibrinógeno/química , Fibrinógeno/metabolismo , Corona de Proteínas/química , Corona de Proteínas/metabolismo , Adsorción , Sitios de Unión , Interacciones Hidrofóbicas e Hidrofílicas , HumanosRESUMEN
Examining the critical role of anticoagulants in medical practice, particularly their central function in preventing abnormal blood clotting, is of the utmost importance. However, the study of interactions between blood proteins and alternative anticoagulant nano-surfaces is still understood poorly. In this study, novel approach involving direct functionalisation of magnetic iron oxide nanoparticles (MNPs) as carriers with sulphated dextran (s-dext) is presented, with the aim of evaluating the potential of magnetically-responsive MNPs@s-dext as anticoagulants. The physicochemical characterisation of the synthesised MNPs@s-dext includes crystal structure analysis, morphology study, surface and electrokinetic properties, thermogravimetric analysis and magnetic properties` evaluation, which confirms the successful preparation of the nanocomposite with sulfonate groups. The anticoagulant potential of MNPs@s-dext was investigated using a standardised activated partial thromboplastin time (APTT) test and a modified APTT test with a quartz crystal microbalance with dissipation (QCM-D) which confirmed the anticoagulant effect. Time-resolved solid-liquid interactions between the MNPs@s-dext and model blood proteins bovine serum albumin and fibrinogen were also investigated, to gain insight into their hemocompatibility, and revealed protein-repellence of MNPs@s-dext against blood proteins. The study also addressed comprehensive cytotoxicity studies of prepared nanocomposites, and provided valuable insights into potential applicability of MNPs@s-dext as a promising magnetic anticoagulant in biomedical contexts.
Asunto(s)
Anticoagulantes , Sulfato de Dextran , Nanocompuestos , Anticoagulantes/farmacología , Anticoagulantes/química , Humanos , Nanocompuestos/química , Nanocompuestos/toxicidad , Sulfato de Dextran/química , Albúmina Sérica Bovina/química , Coagulación Sanguínea/efectos de los fármacos , Nanopartículas Magnéticas de Óxido de Hierro/química , Nanopartículas Magnéticas de Óxido de Hierro/toxicidad , Animales , Compuestos Férricos/química , Compuestos Férricos/farmacología , Fibrinógeno/química , Supervivencia Celular/efectos de los fármacos , Tiempo de Tromboplastina Parcial , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/toxicidadRESUMEN
Liquid-infused polymers are recognized for their ability to repel foulants, making them promising for biomedical applications including catheter-associated urinary tract infections (CAUTIs). However, the impact of the quantity of free liquid layer covering the surface on protein and bacterial adhesion is not well understood. Here, we explore how the amount of free silicone liquid layer in infused silicone catheter materials influences the adhesion of bacteria and proteins relevant to CAUTIs. To alter the quantity of the free liquid layer, we either physically removed excess liquid from fully infused catheter materials or partially infused them. We then evaluated the impact on bacterial and host protein adhesion. Physical removal of the free liquid layer from the fully infused samples reduced the height of the liquid layer from 60 µm to below detection limits and silicone liquid loss into the environment by approximately 64% compared to controls, without significantly increasing the deposition of protein fibrinogen or the adhesion of the common uropathogen Enterococcus faecalis. Partially infused samples showed even greater reductions in liquid loss: samples infused to 70%-80% of their maximum capacity exhibited about an 85% decrease in liquid loss compared to fully infused controls. Notably, samples with more than 70% infusion did not show significant increases in fibrinogen or E. faecalis adhesion. These findings suggest that adjusting the levels of the free liquid layer in infused polymers can influence protein and bacterial adhesion on their surfaces. Moreover, removing the free liquid layer can effectively reduce liquid loss from these polymers while maintaining their functionality.
Asunto(s)
Adhesión Bacteriana , Enterococcus faecalis , Adhesión Bacteriana/efectos de los fármacos , Enterococcus faecalis/fisiología , Enterococcus faecalis/efectos de los fármacos , Polímeros/química , Siliconas/química , Propiedades de Superficie , Fibrinógeno/química , Fibrinógeno/metabolismo , HumanosRESUMEN
Staphylococcus aureus contamination continues to be a harmful foodborne pathogen threatening of human health, and there is a growing need for rapid detection technologies. This study proposed a novel paper biosensor based on a polydiacetylene (PDA) polymer functionalized fibrinogen (Fg) for the detection of S. aureus in food sources. The fluorophore was developed based on the high binding ability of fibrinogen-binding proteins on the surface of S. aureus. This binding caused twisting in the PDA backbone, leading to changes in chromatic and fluorescent. The detection limit of this method was 50.1 CFU/mL for S. aureus-contaminated foodstuffs and 65.0 CFU/mL for the pure S. aureus culture, and the novelty came from its rapidity and selectivity for S. aureus compared to other foodborne bacteria. In summary, the present work provides a rapid detection method for S. aureus detection, which will help in addressing food safety-related issues.
Asunto(s)
Técnicas Biosensibles , Fibrinógeno , Contaminación de Alimentos , Papel , Polímero Poliacetilénico , Staphylococcus aureus , Staphylococcus aureus/aislamiento & purificación , Técnicas Biosensibles/instrumentación , Polímero Poliacetilénico/química , Contaminación de Alimentos/análisis , Fibrinógeno/química , Fibrinógeno/metabolismo , Microbiología de Alimentos , Límite de Detección , Polímeros/químicaRESUMEN
Trauma is the leading cause of death for adults under the age of 44. Internal bleeding remains a significant challenge in medical emergencies, necessitating the development of effective hemostatic materials that could be administered by paramedics before a patient is in the hospital and treated by surgeons. In this study, we introduce a graphene oxide (GO)-based PEGylated synthetic hemostatic nanomaterial with an average size of 211 ± 83 nm designed to target internal bleeding by mimicking the role of fibrinogen. Functionalization of GO-g-PEG with peptides derived from the α-chain of fibrinogen, such as GRGDS, or the γ-chain of fibrinogen, such as HHLGGAKQAGDV:H12, was achieved with peptide loadings of 72 ± 6 and 68 ± 15 µM, respectively. In vitro studies with platelet-rich plasma (PRP) under confinement demonstrated aggregation enhancement of 39 and 24% for GO-g-PEG-GRGDS and GO-g-PEG-H12, respectively, compared to buffer, while adenosine diphosphate (ADP) alone induced a 5% aggregation. Compared to the same materials in the absence of ADP, GO-g-PEG-GRGDS achieved a 47% aggregation enhancement, while GO-g-PEG-H12 a 25% enhancement. This is particularly important for injectable hemostats and highlights the fact that our nanographene-based materials can only act as hemostats in the presence of agonists, reducing the possibility of unwanted clotting during circulation. Further studies on collagen-coated wells under dynamic flow revealed statistically significant augmentation of PRP fluorescence signal using GRGDS- or H12-coated GO-g-PEG compared to controls. Hemolysis studies showed <1% lysis of red blood cells (RBCs) at the highest PEGylated nanographene concentration. Finally, whole human blood coagulation studies reveal faster and more pronounced clotting using our nanohemostats vs PBS control from 3 min and below (blood is clotted with 10% CaCl2 within 4-5 min), with the biggest differences to be shown at 2 and 1 min. At 1 min, the clot weight was found to be â¼45% of that between 4 and 5 min, while no clot was formed in PBS-treated blood. Reduction of CaCl2 to 5 and 3%, or utilization of prostaglandin E1, an anticoagulant, still leads to clots but of smaller weight. The findings highlight the potential of our fibrinogen-mimic PEGylated nanographene as a promising non-hemolytic injectable scaffold for targeting internal bleeding, offering insights into its platelet aggregation capabilities under confinement and under dynamic flow as well as its pronounced coagulation abilities.
Asunto(s)
Fibrinógeno , Grafito , Hemostáticos , Grafito/química , Hemostáticos/química , Hemostáticos/farmacología , Humanos , Fibrinógeno/química , Fibrinógeno/metabolismo , Polietilenglicoles/química , Coagulación Sanguínea/efectos de los fármacos , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Hemorragia/tratamiento farmacológicoRESUMEN
In this paper, we propose a model that connects two standard inflammatory responses to viral infection, namely, elevation of fibrinogen and the lipid drop shower, to the initiation of non-thrombin-generated clot formation. In order to understand the molecular basis for the formation of non-thrombin-generated clots following viral infection, human epithelial and Madin-Darby Canine Kidney (MDCK, epithelial) cells were infected with H1N1, OC43, and adenovirus, and conditioned media was collected, which was later used to treat human umbilical vein endothelial cells and human lung microvascular endothelial cells. After direct infection or after exposure to conditioned media from infected cells, tissue surfaces of both epithelial and endothelial cells, exposed to 8 mg/mL fibrinogen, were observed to initiate fibrillogenesis in the absence of thrombin. No fibers were observed after direct viral exposure of the endothelium or when the epithelium cells were exposed to SARS-CoV-2 isolated spike proteins. Heating the conditioned media to 60 °C had no effect on fibrillogenesis, indicating that the effect was not enzymatic but rather associated with relatively thermally stable inflammatory factors released soon after viral infection. Spontaneous fibrillogenesis had previously been reported and interpreted as being due to the release of the alpha C domains due to strong interactions of the interior of the fibrinogen molecule in contact with hydrophobic material surfaces rather than cleavage of the fibrinopeptides. Contact angle goniometry and immunohistochemistry were used to demonstrate that the lipids produced within the epithelium and released in the conditioned media, probably after the death of infected epithelial cells, formed a hydrophobic residue responsible for fibrillogenesis. Hence, the standard inflammatory response constitutes the ideal conditions for surface-initiated clot formation.
Asunto(s)
Fibrinógeno , Humanos , Perros , Animales , Fibrinógeno/química , Fibrinógeno/metabolismo , Trombina/metabolismo , Trombina/farmacología , Células de Riñón Canino Madin Darby , Células Endoteliales de la Vena Umbilical Humana , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Coagulación Sanguínea , COVID-19/virología , COVID-19/metabolismo , Medios de Cultivo Condicionados/farmacología , Medios de Cultivo Condicionados/química , Células Endoteliales/metabolismo , Células Endoteliales/virología , Células Epiteliales/virología , Células Epiteliales/metabolismoRESUMEN
Tyrosine sulfation, an understudied but crucial post-translational modification, cannot be directly detected in conventional nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) due to the extreme sulfate lability. Here, we report the detection of sulfate-retaining fragments from LC-electron capture dissociation (ECD) and nanoLC-electron transfer higher energy collision dissociation (EThcD). Sulfopeptide candidates were identified by Proteome Discoverer and MSFragger analysis of nanoLC-HCD MS/MS data and added to inclusion lists for LC-ECD or nanoLC-EThcD MS/MS. When this approach failed, targeted LC-ECD with fixed m/z isolation windows was performed. For the plasma protein fibrinogen, the known pyroglutamylated sulfopeptide QFPTDYDEGQDDRPK from the beta chain N-terminus was identified despite a complete lack of sulfate-containing fragment ions. The peptide QVGVEHHVEIEYD from the gamma-B chain C-terminus was also identified as sulfated or phosphorylated. This sulfopeptide is not annotated in Uniprot but was previously reported. MSFragger further identified a cysteine-containing peptide from the middle of the gamma chain as sulfated and deamidated. NanoLC-EThcD and LC-ECD MS/MS confirmed the two former sulfopeptides via sulfate-retaining fragment ions, whereas an unexpected fragmentation pattern was observed for the third sulfopeptide candidate. Manual interpretation of the LC-ECD spectrum revealed two additional isobaric identifications: a trisulfide-linked cysteinyl-glycine or a carbamidomethyl-dithiothreiotol covalent adduct. Synthesis of such adducts confirmed the latter identity.
Asunto(s)
Fibrinógeno , Espectrometría de Masas en Tándem , Tirosina , Tirosina/química , Tirosina/análogos & derivados , Espectrometría de Masas en Tándem/métodos , Fibrinógeno/química , Fibrinógeno/metabolismo , Cromatografía Liquida/métodos , Humanos , Procesamiento Proteico-Postraduccional , Tripsina/química , Tripsina/metabolismo , Sulfatos/química , Secuencia de Aminoácidos , Péptidos/química , Péptidos/análisis , ElectronesRESUMEN
This research pioneers a sustainable approach to wound healing by developing a bio sheet for dressings. It ingeniously incorporates healing properties of reduced graphene oxide synthesized via an eco-friendly method, using phytoextracts to eliminate toxic chemicals. Collagen extracted from fish waste and fibrinogen from post-animal waste contribute to the bio sheet's green profile. The study emphasizes the global shift towards utilizing natural materials in therapeutic applications for efficient wound healing while minimizing toxic side effects. The formulation involves a simple muffle treatment for the conversion of carbon to graphene oxide, followed by a greener reduction technique to synthesize rGO. Characterization- by peaks that obtained at 255 nm, 240 nm, 280 nm, 360 nm using UV-Vis analysis were confirmed the presents of desired carbon and glycoproteins, In X-ray diffraction (2θ value) we found peaks at 24.37°, 27.84°, 17.43° which indicated the presents of rGO, Fibrinogen, and Collagen, and FT-IR methods confirms the functional groups like OH, CH2, CC, CHO and NC in the derived materials and Zeta potential distribution were done for Compounds. To add up purity and efficiency analysis via GCMS were done for proteins. The resulting bio sheet, created in a controlled environment with a gelling agent, Undergoes morphological analysis through FESEM-EDX. Additional assessments, including antioxidant, anti-inflammatory, and hemocompatibility assays, illuminate the nature of the formulated bio sheet. To addition, biosheet is analyzed for physical and mechanical properties. Further insight is gained through the analysis of surface plots of the EDX images of both the composite and bio sheet using ImageJ software. This comprehensive approach underscores the potential of sustainable and naturally derived materials in advancing wound care technologies.
Asunto(s)
Grafito , Nanopartículas , Cicatrización de Heridas , Cicatrización de Heridas/efectos de los fármacos , Grafito/química , Animales , Nanopartículas/química , Fibrinógeno/química , Colágeno/química , Vendajes , Materiales Biocompatibles/química , Humanos , Antioxidantes/química , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Thrombosis is associated with various fatal arteriovenous syndromes including ischemic stroke, myocardial infarction, and pulmonary embolism. However, current clinical thrombolytic treatment strategies still have many problems in targeting and safety to meet the thrombolytic therapy needs. Understanding the molecular mechanism that underlies thrombosis is critical in developing effective thrombolytic strategies. It is well known that platelets play a central role in thrombosis and the binding of fibrinogen to activated platelets is a common pathway in the process of clot formation. Based on this, a concept of biomimetic thrombus-targeted thrombolytic strategy inspired from fibrinogen binding to activated platelets in thrombosis was proposed, which could selectively bind to activated platelets at a thrombus site, thus enabling targeted delivery and local release of thrombolytic agents for effective thrombolysis. In this review, we first summarized the main characteristics of platelets and fibrinogen, and then introduced the classical molecular mechanisms of thrombosis, including platelet adhesion, platelet activation and platelet aggregation through the interactions of activated platelets with fibrinogen. In addition, we highlighted the recent advances in biomimetic thrombus-targeted thrombolytic strategies which inspired from fibrinogen binding to activated platelets in thrombosis. The possible future directions and perspectives in this emerging area are briefly discussed.
Asunto(s)
Biomimética , Plaquetas , Fibrinógeno , Activación Plaquetaria , Trombosis , Humanos , Fibrinógeno/metabolismo , Fibrinógeno/química , Trombosis/tratamiento farmacológico , Trombosis/metabolismo , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Terapia Trombolítica/métodos , Unión Proteica , Animales , Fibrinolíticos/farmacología , Fibrinolíticos/uso terapéutico , Fibrinolíticos/químicaRESUMEN
Due to its structural and functional complexity the heart imposes immense physical, physiological and electromechanical challenges on the engineering of a biological replacement. Therefore, to come closer to clinical translation, the development of a simpler biological assist device is requested. Here, we demonstrate the fabrication of tubular cardiac constructs with substantial dimensions of 6 cm in length and 11 mm in diameter by combining human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and human foreskin fibroblast (hFFs) in human fibrin employing a rotating mold technology. By centrifugal forces employed in the process a cell-dense layer was generated enabling a timely functional coupling of iPSC-CMs demonstrated by a transgenic calcium sensor, rhythmic tissue contractions, and responsiveness to electrical pacing. Adjusting the degree of remodeling as a function of hFF-content and inhibition of fibrinolysis resulted in stable tissue integrity for up to 5 weeks. The rotating mold device developed in frame of this work enabled the production of tubes with clinically relevant dimensions of up to 10 cm in length and 22 mm in diameter which-in combination with advanced bioreactor technology for controlled production of functional iPSC-derivatives-paves the way towards the clinical translation of a biological cardiac assist device.
Asunto(s)
Fibrinógeno , Células Madre Pluripotentes Inducidas , Miocitos Cardíacos , Ingeniería de Tejidos , Humanos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Fibrinógeno/metabolismo , Fibrinógeno/química , Ingeniería de Tejidos/métodos , Fibroblastos/metabolismo , Diferenciación Celular , Células Cultivadas , Reactores Biológicos , Fibrina/metabolismo , Fibrina/química , Andamios del Tejido/químicaRESUMEN
Bacterial adhesins are cell-surface proteins that anchor to the cell wall of the host. The first stage of infection involves the specific attachment to fibrinogen (Fg), a protein found in human blood. This attachment allows bacteria to colonize tissues causing diseases such as endocarditis. The study of this family of proteins is hence essential to develop new strategies to fight bacterial infections. In the case of the Gram-positive bacterium Staphylococcus aureus, there exists a class of adhesins known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Here, we focus on one of them, the clumping factor A (ClfA), which has been found to bind Fg through the dock-lock-latch mechanism. Interestingly, it has recently been discovered that MSCRAMM proteins employ a catch-bond to withstand forces exceeding 2 nN, making this type of interaction as mechanically strong as a covalent bond. However, it is not known whether this strength is an evolved feature characteristic of the bacterial protein or is typical only of the interaction with its partner. Here, we combine single-molecule force spectroscopy, biophysical binding assays, and molecular simulations to study the intrinsic mechanical strength of ClfA. We find that despite the extremely high forces required to break its interactions with Fg, ClfA is not by itself particularly strong. Integrating the results from both theory and experiments we dissect contributions to the mechanical stability of this protein.
Asunto(s)
Coagulasa , Fibrinógeno , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/química , Coagulasa/metabolismo , Coagulasa/química , Fibrinógeno/química , Fibrinógeno/metabolismo , Unión Proteica , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/química , Humanos , Estabilidad ProteicaRESUMEN
BACKGROUND: Peroxynitrite (ONOO-) is an oxidant linked with several human pathologies. Apigenin, a natural flavonoid known for its health benefits, remains unexplored in relation to ONOO- effects. This study investigated the potential of apigenin to structurally protect fibrinogen, an essential blood clotting factor, from ONOO--induced damage. METHODS: Multi-approach analyses were carried out where fibrinogen was exposed to ONOO- generation while testing the efficacy of apigenin. The role of apigenin against ONOO--induced modifications in fibrinogen was investigated using UV spectroscopy, tryptophan or tyrosine fluorescence, protein hydrophobicity, carbonylation, and electrophoretic analyses. RESULTS: The findings demonstrate that apigenin significantly inhibits ONOO--induced oxidative damage in fibrinogen. ONOO- caused reduced UV absorption, which was reversed by apigenin treatment. Moreover, ONOO- diminished tryptophan and tyrosine fluorescence, which was effectively restored by apigenin treatment. Apigenin also reduced the hydrophobicity of ONOO--damaged fibrinogen. Moreover, apigenin exhibited protective effects against ONOO--induced protein carbonylation. SDS-PAGE analyses revealed that ONOO-treatment eliminated bands corresponding to fibrinogen polypeptide chains Aα and γ, while apigenin preserved these changes. CONCLUSIONS: This study highlights, for the first time, the role of apigenin in structural protection of human fibrinogen against peroxynitrite-induced nitrosative damage. Our data indicate that apigenin offers structural protection to all three polypeptide chains (Aα, Bß, and γ) of human fibrinogen. Specifically, apigenin prevents the dislocation or breakdown of the amino acids tryptophan, tyrosine, lysine, arginine, proline, and threonine and also prevents the exposure of hydrophobic sites in fibrinogen induced by ONOO-.
Asunto(s)
Apigenina , Fibrinógeno , Estrés Nitrosativo , Ácido Peroxinitroso , Fibrinógeno/metabolismo , Fibrinógeno/química , Apigenina/farmacología , Apigenina/química , Humanos , Ácido Peroxinitroso/química , Estrés Nitrosativo/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Carbonilación Proteica/efectos de los fármacos , Tirosina/química , Tirosina/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
Surgical site infection (SSI) is a common issue post-surgery which often prolongs hospitalization and can lead to serious complications such as sternal wound infection following cardiac surgery via median sternotomy. Controlled release of suitable antibiotics could allow maximizing drug efficacy and safety, and therefore achieving a desired therapeutic response. In this study, we have developed a vancomycin laden PEGylated fibrinogen-polyethylene glycol diacrylate (PF-PEGDA) hydrogel system that can release vancomycin at a controlled and predictable rate to be applied in SSI prevention. Two configurations were developed to study effect of the hydrogel on drug release, namely, vancomycin laden hydrogel and vancomycin solution on top of blank hydrogel. The relationship between the rigidity of the hydrogel and drug diffusion was found to comply with a universal power law, i.e., softer hydrogels result in a greater diffusion coefficient hence faster release rate. Besides, vancomycin laden hydrogels exhibited burst release, whereas the vancomycin solution on top of blank hydrogels exhibited lag release. A mathematical model was developed to simulate vancomycin permeation through the hydrogels. The permeation of vancomycin can be predicted accurately by using the mathematical model, which provided a useful tool to customize drug loading, hydrogel thickness and stiffness for personalized medication to manage SSI. To evaluate the potential of hydrogels for bone healing applications in cardiovascular medicine, we performed a proof-of-concept median sternotomy in rabbits and applied the hydrogels. The hydrogel formulations accelerated the onset of osteo-genetic processes in rabbits, demonstrating its potential to be used in human.
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Antibacterianos , Preparaciones de Acción Retardada , Fibrinógeno , Hidrogeles , Polietilenglicoles , Vancomicina , Vancomicina/administración & dosificación , Vancomicina/química , Vancomicina/farmacocinética , Polietilenglicoles/química , Fibrinógeno/química , Animales , Hidrogeles/química , Preparaciones de Acción Retardada/farmacocinética , Antibacterianos/administración & dosificación , Antibacterianos/química , Antibacterianos/farmacocinética , Liberación de Fármacos , Conejos , Infección de la Herida Quirúrgica/prevención & control , Infección de la Herida Quirúrgica/tratamiento farmacológico , HumanosRESUMEN
Hydrophilic antifouling polymers provide excellent antifouling effects under usual short-term use conditions, but the long-term accumulation of contaminants causes them to lose their antifouling properties. To overcome this drawback, surface-initiated ring-opening graft polymerization (SI-ROP) was performed on the surface of the material by applying the cyclic carbide monomer 4'-(fluorosulfonyl)benzyl-5-methyl-2-oxo-1,3-dioxane-5-carboxylate (FMC), which contains a sulfonylfluoride group on the side chain, followed by a "sulfur(IV)-fluorine exchange" (SuFEx) post click modification reaction to link the hydrophilic polyethylene glycol (PEG) to the polyFMC (PFMC) brush, and a novel antifouling strategy for self-polishing dynamic antifouling surfaces was developed. The experimental results showed that the antifouling surface could effectively prevent the adsorption of proteins such as bovine serum albumin (BSA, â¼96.4%), fibrinogen (Fg, â¼87.8%) and lysozyme (Lyz â¼69.4%) as well as the adhesion of microorganisms such as the bacteria Staphylococcus aureus (S. aureus) (â¼87.5%) and HeLa cells (â¼67.2%). Moreover, the enzymatically self-polished surface still has excellent antifouling properties. Therefore, this modification method has potential applications in the field of biosensors and novel antifouling materials.
Asunto(s)
Adhesión Bacteriana , Incrustaciones Biológicas , Cemento de Policarboxilato , Polietilenglicoles , Albúmina Sérica Bovina , Staphylococcus aureus , Propiedades de Superficie , Staphylococcus aureus/efectos de los fármacos , Cemento de Policarboxilato/química , Polietilenglicoles/química , Incrustaciones Biológicas/prevención & control , Adhesión Bacteriana/efectos de los fármacos , Humanos , Albúmina Sérica Bovina/química , Adsorción , Polimerizacion , Bovinos , Animales , Fibrinógeno/química , Fibrinógeno/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Muramidasa/química , Muramidasa/metabolismo , Muramidasa/farmacologíaRESUMEN
Traumatic brain injury (TBI) is a critical public health concern, yet there are no therapeutics available to improve long-term outcomes. Drug delivery to TBI remains a challenge due to the blood-brain barrier and increased intracranial pressure. In this work, a chemical targeting approach to improve delivery of materials to the injured brain, is developed. It is hypothesized that the provisional fibrin matrix can be harnessed as an injury-specific scaffold that can be targeted by materials via click chemistry. To accomplish this, the brain clot is engineered in situ by delivering fibrinogen modified with strained cyclooctyne (SCO) moieties, which incorporated into the injury lesion and is retained there for days. Improved intra-injury capture and retention of diverse, clickable azide-materials including a small molecule azide-dye, 40 kDa azide-PEG nanomaterial, and a therapeutic azide-protein in multiple dosing regimens is subsequently observed. To demonstrate therapeutic translation of this approach, a reduction in reactive oxygen species levels in the injured brain after delivery of the antioxidant catalase, is achieved. Further, colocalization between azide and SCO-fibrinogen is specific to the brain over off-target organs. Taken together, a chemical targeting strategy leveraging endogenous clot formation is established which can be applied to improve therapeutic delivery after TBI.
Asunto(s)
Azidas , Lesiones Traumáticas del Encéfalo , Fibrinógeno , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/patología , Animales , Azidas/química , Fibrinógeno/metabolismo , Fibrinógeno/química , Química Clic , Fibrina/metabolismo , Fibrina/química , Especies Reactivas de Oxígeno/metabolismo , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/patología , Ratones , Catalasa/metabolismo , Polietilenglicoles/química , Ratas , Ciclooctanos/química , Sistemas de Liberación de Medicamentos , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacosRESUMEN
Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new fibrinogenolytic agents requires the separation of each component first and then checking their fibrinogenolytic activities. Currently, polyacrylamide gel electrophoresis (PAGE) and chromatography are mostly used in the separating stage. Meanwhile, the fibrinogen plate assay and reaction products based PAGE are usually adopted to display their fibrinogenolytic activities. However, because of the spatiotemporal separation of those two stages, it is impossible to separate and display the active fibrinogenolytic agents with the same gel. To simplify the separating and displaying processes of fibrinogenolytic agent identification, we constructed a new fibrinogen-PAGE method to rapidly separate and display the fibrinogenolytic agents of peanut worms (Sipunculus nudus) in this study. This method includes fibrinogen-PAGE preparation, electrophoresis, renaturation, incubation, staining, and decolorization. The fibrinogenolytic activity and molecular weight of the protein can be detected simultaneously. According to this method, we successfully detected more than one active fibrinogenolytic agent of peanut wormhomogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.
Asunto(s)
Electroforesis en Gel de Poliacrilamida , Fibrinógeno , Fibrinógeno/química , Fibrinógeno/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida/métodos , Fibrinolíticos/química , Fibrinolíticos/farmacología , Fibrinolíticos/aislamiento & purificaciónRESUMEN
Understanding nanoparticle-cell interaction is essential for advancing research in nanomedicine and nanotoxicology. Apart from the transcytotic pathway mediated by cellular recognition and energetics, nanoparticles (including nanomedicines) may harness the paracellular route for their transport by inducing endothelial leakiness at cadherin junctions. This phenomenon, termed as NanoEL, is correlated with the physicochemical properties of the nanoparticles in close association with cellular signalling, membrane mechanics, as well as cytoskeletal remodelling. However, nanoparticles in biological systems are transformed by the ubiquitous protein corona and yet the potential effect of the protein corona on NanoEL remains unclear. Using confocal fluorescence microscopy, biolayer interferometry, transwell, toxicity, and molecular inhibition assays, complemented by molecular docking, here we reveal the minimal to significant effects of the anionic human serum albumin and fibrinogen, the charge neutral immunoglobulin G as well as the cationic lysozyme on negating gold nanoparticle-induced endothelial leakiness in vitro and in vivo. This study suggests that nanoparticle-cadherin interaction and hence the extent of NanoEL may be partially controlled by pre-exposing the nanoparticles to plasma proteins of specific charge and topology to facilitate their biomedical applications.
Asunto(s)
Cadherinas , Fibrinógeno , Oro , Nanopartículas del Metal , Corona de Proteínas , Corona de Proteínas/química , Corona de Proteínas/metabolismo , Humanos , Cadherinas/metabolismo , Cadherinas/química , Oro/química , Nanopartículas del Metal/química , Fibrinógeno/química , Fibrinógeno/metabolismo , Animales , Células Endoteliales de la Vena Umbilical Humana , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Simulación del Acoplamiento Molecular , RatonesRESUMEN
A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.