RESUMO
Thrombosis can lead to a wide variety of life-threatening circumstances. As current thrombolytic drug screening models often poorly predict drug profiles, leading to failure of thrombolytic therapy or clinical translation, more representative clot substrates are necessary for drug evaluation. Utilizing a Chandler loop device to form clot analogs at high shear has gained popularity in stroke societies. However, shear-dependent clot microstructure has not been fully addressed and low shear conditions are often overlooked. We herein characterized the impact of wall shear rate (126 to 951 s-1) on clot properties in the Chandler loop. Different revolutions (20-60) per minute and tubing sizes (3.2 to 7.9 mm) were employed to create different sized clots to mimic various thrombosis applications. Increased shear resulted in decreased RBC counts (76.9 ± 4.3% to 17.6 ± 0.9%) and increased fibrin (10 to 60%) based on clot histology. Increased fibrin sheet morphology and platelet aggregates were observed at higher shear under scanning electron microscope. These results show the significant impact of shear and tubing size on resulting clot properties and demonstrate the capability of forming a variety of reproducible in-vivo-like clot analogs in the Chandler loop device controlling for simple parameters to tune clot characteristics.
Assuntos
Acidente Vascular Cerebral , Trombose , Humanos , Plaquetas , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/terapia , Fibrinolíticos , FibrinaRESUMO
BACKGROUND: We test if inhaled nitric oxide (NO) attenuates platelet functional and metabolic hyper-reactivity in subjects with submassive pulmonary embolism (PE). METHODS: Participants with PE were randomized to either 50 ppm NO + O2 or O2 only for 24 h with blood sampling at enrollment and after treatment; results were compared with healthy controls. Platelet metabolic activity was assessed by oxygen consumption (basal and uncoupled) and reactivity was assessed with agonist-stimulated thromboelastography (TEG) and fluorometric measurement of agonist-stimulated cytosolic [Ca++] without and with pharmacological soluble guanylate (sGC) modulation. RESULTS: Participants (N = 38 per group) were well-matched at enrollment for PE severity, comorbidities as well as TEG parameters and platelet O2 consumption. NO treatment doubled the mean plasma [NO3-] (P < 0.001) indicating successful delivery, but placebo treatment produced no change. After 24 h, neither TEG nor O2 consumption parameters differed significantly between treatment groups. Platelet cytosolic [Ca++] was elevated with PE versus controls, and was decreased by treatment with cinaciguat (an sGC activator), but not riociguat (an sGC stimulator). Stimulated platelet lysate sGC activity was increased with PE compared with controls. CONCLUSIONS: In patients with acute submassive PE, despite evidence of adequate drug delivery, inhaled NO had no major effect on platelet O2 consumption or agonist-stimulated parameters on TEG. Pharmacological activation, but not stimulation, of sGC effectively decreased platelet cytosolic [Ca++], and platelet sGC activity was increased with PE, confirming the viability of sGC as a therapeutic target.
Assuntos
Plaquetas/efeitos dos fármacos , Óxido Nítrico/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Embolia Pulmonar/sangue , Administração por Inalação , Adulto , Benzoatos/farmacologia , Cálcio/metabolismo , Método Duplo-Cego , Ativadores de Enzimas/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/administração & dosagem , Embolia Pulmonar/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Guanilil Ciclase Solúvel/metabolismoRESUMO
It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte-activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV+ patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial-cadherin+ endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation.
Assuntos
Morte Celular/fisiologia , Células Endoteliais/virologia , Infecções por HIV/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Endotélio/virologia , Células HEK293 , Infecções por HIV/metabolismo , Humanos , Células Jurkat , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Pulmão/metabolismo , Pulmão/virologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Proteínas de Neoplasias/metabolismo , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/virologia , Proteínas de Ligação a RNA/metabolismo , Linfócitos T/metabolismo , Linfócitos T/virologiaRESUMO
The potent fibrinolytic enzyme, plasmin has numerous clinical applications for recannulizing vessels obstructed by thrombus. Despite its diminutive size, 91 kDa, success in the recombinant expression of this serine protease has been limited. For this reason, a truncated non-glycosylated plasmin variant was developed capable of being expressed and purified from E. coli. This mutated plasmin, known as δ-plasmin, eliminates four of the five kringle domains present on native plasmin, retaining only kringle 1 fused directly to the unmodified catalytic domain of plasmin. This study demonstrates that δ-plasmin exhibits similar kinetic characteristics to full length plasmin despite its heavily mutated form; KM = 268.78 ± 19.12, 324.90 ± 8.43 µM and Kcat = 770.48 ± 41.73, 778.21 ± 1.51 1/min for plasmin and δ-plasmin, respectively. A comparative analysis was also carried out to investigate the inhibitory effects of a range of benzamidine based small molecule inhibitors: benzamidine, p-aminobenzamidine, 4-carboxybenzamidine, 4-aminomethyl benzamidine, and pentamidine. All of the small molecule inhibitors, with the exception of unmodified benzamidine, demonstrated comparable competitive inhibition constants (Ki) for both plasmin and δ-plasmin ranging from Ki < 4 µM for pentamidine to Ki > 1000 µM in the case of aminomethyl benzamidine. This result further supports that δ-plasmin retains much of the same functionality as native plasmin despite its greatly reduced size and complexity. This study serves the purpose of demonstrating the tunable inhibition of plasmin and δ-plasmin with potential applications for the improved clinical delivery of δ-plasmin to treat various thrombi.
Assuntos
Antifibrinolíticos/farmacologia , Benzamidinas/farmacologia , Fibrinolisina/antagonistas & inibidores , Antifibrinolíticos/química , Benzamidinas/química , Fibrinolisina/química , Fibrinolisina/genética , Humanos , Cinética , Modelos Moleculares , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/genética , Plasminogênio/química , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-AtividadeRESUMO
The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.
Assuntos
Biotinilação , Doença pelo Vírus Ebola/diagnóstico , Fragmentos Fab das Imunoglobulinas/metabolismo , Testes Sorológicos/métodos , Antígenos Virais/metabolismo , Sítios de Ligação , Ligação Proteica , Sensibilidade e EspecificidadeRESUMO
Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.
Assuntos
Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Antígenos/análise , Antígenos/imunologia , Biotinilação , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Nucleotídeos/imunologia , Sítios de Ligação , Estrutura Molecular , Nucleotídeos/químicaRESUMO
Described here is a UV photo-cross-linking method that utilizes the NBS (nucleotide binding site) for site-specific covalent functionalization of antibodies with reactive thiol moieties (UV-NBS(Thiol)), while preserving antibody activity. By synthesizing an indole-3-butyric acid (IBA) conjugated version of cysteine we site-specifically photo-cross-linked a reactive thiol moiety to antibodies at the NBS. This thiol moiety can then be used as an orthogonally reactive location to conjugate various types of functional ligands that possess a thiol reactive group through disulfide bond formation or reaction with a maleimide functionalized ligand. Our results demonstrate the utility of the UV-NBS(Thiol) method by successfully functionalizing a prostate specific antigen antibody (IgG(PSA)) with IBA-Thiol and subsequent reaction with maleimide-fluorescein. An optimal UV energy of 0.5-1.5 J/cm(2) was determined to yield the most efficient photo-cross-linking and resulted in 1-1.5 conjugations per antibody while preserving antibody/antigen binding activity and Fc recognition. Utilizing the IBA-Thiol ligand allows for an efficient means of site-specifically conjugating UV sensitive functionalities to antibody NBS that would otherwise not have been amenable by the previously described UV-NBS photo-cross-linking method. The UV-NBS(Thiol) conjugation strategy can be utilized in various diagnostic and therapeutic applications with nearly limitless potential for the preparation of site-specific covalent conjugation of affinity tags, fluorescent molecules, peptides, and chemotherapeutics to antibodies.
Assuntos
Anticorpos/imunologia , Anticorpos/metabolismo , Antígenos/metabolismo , Imunoglobulina G/imunologia , Nucleotídeos/metabolismo , Compostos de Sulfidrila/metabolismo , Anticorpos/química , Sítios de Ligação , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Indóis/química , Indóis/metabolismo , Modelos Moleculares , Nucleotídeos/química , Compostos de Sulfidrila/química , Raios UltravioletaRESUMO
Thromboembolism and related complications are a leading cause of morbidity and mortality worldwide and various assays have been developed to test thrombolytic drug efficiency both in vitro and in vivo. There is increasing demand for more physiologically relevant in-vitro clot models for drug development due to the complexity and cost associated with animal models in addition to their often lack of translatability to human physiology. Flow, pressure, and shear rate are important characteristics of the circulatory system, with clots that are formed under flow displaying different morphology and digestion characteristics than statically formed clots. These factors are often unrepresented in conventional in-vitro clot digestion assays, which can have pharmacological implications that impact drug translational success rates. The Real-Time Fluorometric Flowing Fibrinolysis (RT-FluFF) assay was developed as a high-fidelity thrombolysis testing platform that uses fluorescently tagged clots formed under shear flow, which are then digested using circulating plasma in the presence or absence of fibrinolytic pharmaceutical agents. Modifying the flow rates of both clot formation and clot digestion steps allows the system to imitate arterial, pulmonary, and venous conditions across highly diverse experimental setups. Measurements can be taken continuously using an in-line fluorometer or by taking discrete time points, as well as a conventional end point clot mass measurement. The RT-FluFF assay is a flexible system that allows for the real-time tracking of clot digestion under flow conditions that more accurately represent in-vivo physiological conditions while retaining the control and reproducibility of an in-vitro testing system.
Assuntos
Fibrinólise , Humanos , Fibrinólise/efeitos dos fármacos , Fibrinólise/fisiologia , Trombose , Fluorometria/métodos , Terapia Trombolítica/métodosRESUMO
This Article describes an affinity-based precipitation method for the rapid and nonchromatographic purification of bivalently active monoclonal antibodies by combining the selectivity of affinity chromatography with the simplicity of salt-induced precipitation. This procedure involves (i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; (ii) formation and selective precipitation of cyclic antibody complexes created by binding to trivalent haptens specific for the antibody; and (iii) membrane filtration of the solubilized antibody pellet to remove the trivalent hapten from the purified antibody. We applied this technique to the purification of two pharmaceutical antibodies, trastuzumab and rituximab, by synthesizing trivalent haptens specific for each antibody. Using this method, we were able to purify both antibodies from typical contaminants including CHO cell conditioned media, ascites fluid, DNA, and other antibodies with yields >85% and with >95% purity. The purified antibodies displayed native binding levels to cell lines expressing the target proteins demonstrating that the affinity-based precipitation method did not adversely affect the antibodies. The selectivity of the affinity-based precipitation method for bivalently active antibodies was established by purifying trastuzumab from a solution containing both active and chemically denatured trastuzumab. Prior to purification, the solutions displayed 20-76% reduction in binding activity, and after purification, native binding activity was restored, indicating that the purified product contained only bivalently active antibody. Taken together, the affinity-based precipitation method provides a rapid and straightforward process for the purification of antibodies with the potential to improve product quality while decreasing the purification costs at both the lab and the industrial scale.
Assuntos
Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/isolamento & purificação , Anticorpos Monoclonais Murinos/química , Anticorpos Monoclonais Murinos/isolamento & purificação , Ascite , Precipitação Química , Sulfato de Amônio/química , Animais , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Murinos/imunologia , Especificidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Meios de Cultivo Condicionados/química , DNA/isolamento & purificação , Haptenos/imunologia , Desnaturação Proteica , Rituximab , TrastuzumabRESUMO
Liposome Extruder Purification (LEP) allows for the rapid purification of diverse liposome formulations using the same extrusion apparatus employed during liposome formation. The LEP process provides a means for purifying functionalized liposomes from non-conjugated drug or protein contaminants with >93% liposome recovery and >93% contaminant removal in a single step.
Assuntos
Fracionamento Químico/métodos , Lipossomos/isolamento & purificação , Animais , Bovinos , Química Farmacêutica , Lipossomos/química , Fatores de TempoRESUMO
Cisplatin is a first line chemotherapy for most types of cancer. However, its use is dose-limited due to severe nephrotoxicity. Here we report the rational engineering of a novel nanoplatinate inspired by the mechanisms underlying cisplatin bioactivation. We engineered a novel polymer, glucosamine-functionalized polyisobutylene-maleic acid, where platinum (Pt) can be complexed to the monomeric units using a monocarboxylato and an O --> Pt coordinate bond. We show that at a unique platinum to polymer ratio, this complex self-assembles into a nanoparticle, which releases cisplatin in a pH-dependent manner. The nanoparticles are rapidly internalized into the endolysosomal compartment of cancer cells, and exhibit an IC50 (4.25 +/- 0.16 microM) comparable to that of free cisplatin (3.87 +/- 0.37 microM), and superior to carboplatin (14.75 +/- 0.38 microM). The nanoparticles exhibited significantly improved antitumor efficacy in terms of tumor growth delay in breast and lung cancers and tumor regression in a K-ras(LSL/+)/Pten(fl/fl) ovarian cancer model. Furthermore, the nanoparticle treatment resulted in reduced systemic and nephrotoxicity, validated by decreased biodistribution of platinum to the kidney as quantified using inductively coupled plasma spectroscopy. Given the universal need for a better platinate, we anticipate this coupling of nanotechnology and structure-activity relationship to rationally reengineer cisplatin could have a major impact globally in the clinical treatment of cancer.
Assuntos
Cisplatino , Nanopartículas/uso terapêutico , Nanotecnologia/métodos , Animais , Carboplatina/farmacocinética , Carboplatina/farmacologia , Cisplatino/farmacocinética , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Platina , Polienos , Polímeros , Cloridrato de Raloxifeno , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
A great need exists for the development of a more representative in-vitro model to efficiently screen novel thrombolytic therapies. We herein report the design, validation, and characterization of a highly reproducible, physiological scale, flowing clot lysis platform with real-time fibrinolysis monitoring to screen thrombolytic drugs utilizing a fluorescein isothiocyanate (FITC)-labeled clot analog. Using this Real-Time Fluorometric Flowing Fibrinolysis assay (RT-FluFF assay), a tPa-dependent degree of thrombolysis was observed both via clot mass loss as well as fluorometrically monitored release of FITC-labeled fibrin degradation products. Percent clot mass loss ranged from 33.6% to 85.9% with fluorescence release rates of 0.53 to 1.17 RFU/min in 40 and 1000 ng/mL tPa conditions, respectively. The platform is easily adapted to produce pulsatile flows. Hemodynamics of human main pulmonary artery were mimicked through matching dimensionless flow parameters calculated using clinical data. Increasing pressure amplitude range (4-40 mmHg) results in a 20% increase of fibrinolysis at 1000 ng/mL tPA. Increasing shear flow rate (205-913 s-1) significantly increases fibrinolysis and mechanical digestion. These findings suggest pulsatile level affects thrombolytic drug activities and the proposed in-vitro clot model offers a versatile testing platform for thrombolytic drug screening.
RESUMO
This study investigates the influence of microplastics on blood clotting. It addresses the lack of comprehensive research on the effects of microplastic size and surface modification on clotting dynamics in human whole blood. Thromboelastography was used to examine aminated (aPS), carboxylated (cPS), and non-functionalized (nPS) polystyrene particles with sizes of 50, 100, and 500 nm. Results show that cPS consistently activated the clotting cascade, demonstrating increased fibrin polymerization rates, and enhanced clot strength in a size and concentration-dependent manner. nPS had minimal effects on clotting dynamics except for 50 nm particles at the lowest concentration. The clotting effects of aPS (100 nm particles) resembled those of cPS but were diminished in the 500 nm aPS group. These findings emphasize the importance of microplastic surface modification, size, concentration, and surface area on in-vitro whole blood clotting dynamics.
Assuntos
Tromboelastografia , Trombose , Humanos , Tromboelastografia/métodos , Microplásticos , Plásticos , Coagulação SanguíneaRESUMO
The conserved nucleotide binding site (NBS), found within the Fab variable domain of antibodies, remains a not-so-widely known and underutilized site. Here we describe a novel affinity chromatography method that utilizes the NBS as a target for selectively purifying antibodies from complex mixtures. The affinity column was prepared by coupling indole butyric acid (IBA), which has a monovalent affinity for the NBS with a K(d) ranging between 1 and 8 µM, to ToyoPearl resin resulting in the NBS targeting affinity column (NBS(IBA)). The proof-of-concept studies performed using the chimeric pharmaceutical antibody rituximab demonstrated that antibodies were selectively captured and retained on the NBS(IBA) column and were successfully eluted by applying a mild NaCl gradient at pH 7.0. Furthermore, the NBS(IBA) column consistently yielded >95% antibody recovery with >98% purity, even when the antibody was purified from complex mixtures such as conditioned cell culture supernatant, hybridoma media, and mouse ascites fluid. The results presented in this study establish the NBS(IBA) column as a viable small-molecule-based affinity chromatography method for antibody purification with significant implications in industrial antibody production. Potential advantages of the NBS(IBA) platform are improved antibody batch quality, enhanced column durability, and reduced overall production cost.
Assuntos
Anticorpos/isolamento & purificação , Cromatografia de Afinidade , Nucleotídeos/metabolismo , Animais , Anticorpos Monoclonais Murinos/isolamento & purificação , Sítios de Ligação , Bovinos , Indóis/química , Camundongos , Rituximab , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Cloreto de Sódio/químicaRESUMO
The conserved nucleotide binding site (NBS), found on the Fab variable domain of all antibody isotypes, remains a not-so-widely known and unutilized site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a K(d) ranging from 1 to 8 µM for different antibody isotypes and can be covalently photo-cross-linked to the antibody at the NBS upon exposure to UV light. Using the UV-NBS method, antibody was successfully immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm(2) yielded significant antibody immobilization on the surface with maximal relative antibody activity per immobilized antibody without any detectable damage to antigen binding activity. Comparison of the UV-NBS method with two other commonly used methods, ε-NH(3)(+) conjugation and physical adsorption, demonstrated that the UV-NBS method yields surfaces with significantly enhanced antigen detection efficiency, higher relative antibody activity, and improved antigen detection sensitivity. Taken together, the UV-NBS method provides a practical, site-specific surface immobilization method, with significant implications in the development of a large array of platforms with diverse sensor and diagnostic applications.
Assuntos
Anticorpos Imobilizados/química , Anticorpos Imobilizados/imunologia , Antígenos/imunologia , Sequência Conservada , Nucleotídeos/metabolismo , Anticorpos Imobilizados/metabolismo , Sítios de Ligação , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Modelos Moleculares , Processos Fotoquímicos , Conformação Proteica , Propriedades de SuperfícieRESUMO
OBJECTIVES: There has long existed significant underrepresentation of minority students in STEM training and careers. Ongoing efforts to improve opportunities and participation for underrepresented minority students have focused on multiple areas, from increased funding to early exposure to research in STEM. We developed the novel Future Life Map career planning exercise with the goal of contributing to this multi-faceted approach. The exercise emphasizes on the consideration of multiple potential career destinations and routes to those destination. The exercise was designed with the goal of improving participant awareness of options and career planning self-efficacy to improve success and retention of underrepresented minority student participation and retention in STEM. METHODS: We implemented the Future Life Map exercise with 2 separate groups of under-represented minority undergraduate students pursuing careers in STEM. Participants then completed an anonymous survey to evaluate the exercise and describe the value they derived from completing the Future Life Map. RESULTS: The exercise presentation and its supporting documents were highly rated by participants with >81% of respondents rating it as "very informative" (4 or 5 on a 5-point Likert Scale). Participants reported that they were very likely to recommend the exercise to others (25 of 27 participants) and were likely to repeat the activity for their own future decision making (22 participants). Themes that emerged from participant reporting of the value of the exercise were: increased awareness of career and training options, improved understanding of the research required to make informed career/life decisions, and new awareness of specific information about career options under consideration. CONCLUSION: The Future Life Map exercise was successful in improving participant awareness of career options, career planning ability, and helped participants to feel more empowered. This is likely of particular benefit for improving participation and retention of under-represented minority students pursuing careers in STEM.
Assuntos
Escolha da Profissão , Grupos Minoritários/educação , Estudantes/psicologia , Orientação Vocacional/métodos , Adulto , Conscientização , Tomada de Decisões , Feminino , Humanos , Masculino , Autoeficácia , Adulto JovemRESUMO
There is an emerging interest in utilizing synthetic multivalent inhibitors that comprise of multiple inhibitor moieties linked on a common scaffold to achieve strong and selective enzyme inhibition. As multivalent inhibition is impacted by valency and linker length, in this study, we explore the effect of multivalent benzamidine inhibitors of varying valency and linker length on plasmin inhibition. Plasmin is an endogenous enzyme responsible for digesting fibrin present in blood clots. Monovalent plasmin(ogen) inhibitors are utilized clinically to treat hyperfibrinolysis-associated bleeding events. Benzamidine is a reversible inhibitor that binds to plasmin's active site. Herein, multivalent benzamidine inhibitors of varying valencies (mono-, bi- and tri-valent) and linker lengths (â¼1-12â nm) were synthesized to systematically study their effect on plasmin inhibition. Inhibition assays were performed using a plasmin substrate (S-2251) to determine inhibition constants (Ki). Pentamidine (shortest bivalent) and Tri-AMB (shortest trivalent) were the strongest inhibitors with Ki values of 2.1±0.8 and 3.9±1.7â µM, respectively. Overall, increasing valency and decreasing linker length, increases effective local concentration of the inhibitor and therefore, resulted in stronger inhibition of plasmin via statistical rebinding. This study aids in the design of multivalent inhibitors that can achieve desired enzyme inhibition by means of modulating valency and linker length.
Assuntos
Benzamidinas , Fibrinolisina , Fibrinolisina/química , Fibrinolisina/metabolismo , Benzamidinas/farmacologiaRESUMO
Acute pulmonary embolism (PE) does not always resolve after treatment and can progress to chronic thromboembolic disease (CTED) or the more severe chronic thromboembolic pulmonary hypertension (CTEPH). The mechanisms surrounding the likelihood of PE resolution or progress to CTED/CTEPH remain largely unknown. We have developed a rat model of CTEPH that closely resembles the human disease in terms of hemodynamics and cardiac manifestations. Embolization of rats with polystyrene microspheres followed by suppression of angiogenesis with the inhibitor of vascular endothelial growth factor receptor 2 (VEGF-R2) SU5416 results in transient, acute pulmonary hypertension that progresses into chronic PE with PH with sustained right ventricular systolic pressures exceeding 70 mmHg (chronic pulmonary embolism [CPE] model). This model is similar to the widely utilized hypoxia/SU5416 model with the exception that the "first hit" is PE. Rats with CPE have impaired right heart function characterized by reduced VO2 Max, reduced cardiac output, and increased Fulton index. None of these metrics are adversely affected by PE alone. Contrast-mediated CT imaging of lungs from rats with PE minus SU5416 show large increases in pulmonary vascular volume, presumably due to an angiogenic response to acute PE/PH. Co-treatment with SU5416 suppresses angiogenesis and produces the CTEPH-like phenotype. We report here that treatment of CPE rats with agonists for soluble guanylate cyclase, a source of cGMP which is in turn a signal for angiogenesis, markedly increases angiogenesis in lungs, and ameliorates the cardiac deficiencies in the CPE model. These results have implications for future development of therapies for human CTEPH.
Assuntos
Hipertensão Pulmonar , Embolia Pulmonar , Animais , Doença Crônica , Hemodinâmica , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Embolia Pulmonar/complicações , Ratos , Guanilil Ciclase Solúvel , Fator A de Crescimento do Endotélio VascularRESUMO
Micro/nanoplastics, whether manufactured or resulting from environmental degradation, can enter the body through ingestion, inhalation, or dermal pathways. Previous research has found that nanoplastics with diameters of ≤100 nm can translocate into the circulatory system in a dose-dependent manner and potentially impact thrombosis and hemostasis. To investigate the direct effects of microplastics on fibrin clot formation, a simplified ex vivo human thrombin/fibrinogen clot model was utilized. The 100 nm polystyrene particles (non-functionalized [nPS] and aminated [aPS]) were preincubated (0-200 µg/mL) with either thrombin or fibrinogen, and fibrin clot formation was characterized via turbidity and thromboelastography (TEG). When the particles were preincubated with fibrinogen, little effect was observed for aPS or nPS on turbidity or TEG up through 100 µg/mL. TEG results demonstrated a significant impact on clot formation rate and strength, in the case of nPS preincubated with thrombin exhibiting a significant dose-dependent inhibitory effect. In conclusion, the presence of microplastics can have inhibitory effects on fibrin clot formation that are dependent upon both particle surface charge and concentration. Negatively charged nPS exhibited the most significant impacts to clot strength, turbidity, and rate of fibrin formation when first incubated with thrombin, with its impact being greatly diminished when preincubated with fibrinogen in this simplified fibrin clot model.