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Although research indicates affect variability-the extent to which an individual's emotions fluctuate-is associated with behavioral outcomes related to adjustment and adaptability, it is unclear to what extent findings make important contributions to the literature when past research has failed to account for the role of mean levels of emotion. Accordingly, we conducted a repeated-measures laboratory study of college students (N = 253) learning to perform a complex computer task to examine the relative importance of affect variability indices (i.e., spin, pulse, and flux) compared to mean levels in explaining variance in off-task attention and task performance before and after changes in task demands (i.e., skill acquisition and adaptation). In doing so, we also disentangled valence and arousal (i.e., activating versus deactivating) aspects of emotion. Relative importance analyses showed mean levels of emotion were the most dominant predictors (i.e., explained the most variance)-negative deactivating emotions for off-task attention and positive activating emotions for performance. However, flux in negative activating and negative deactivating emotions also explained enough variance to be considered important, suggesting that flux has been overlooked in empirical research. Our findings also highlight that future research must account for mean levels when examining relationships between affect variability and outcomes of interest.
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The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.
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Anticorpos Biespecíficos , Receptores do Fator de Necrose Tumoral , Imunoglobulina G/genética , Engenharia de ProteínasRESUMO
Learning and adaptation are essential for success. However, human effort is inherently finite, which creates a dilemma for employees. Is it better to prioritize capitalizing on existing knowledge structures to maximize immediate performance benefits (exploitation) or develop adaptive capabilities (exploration) at the expense of short-term productivity? Understanding how employees answer this question can inform the design of evidence-based interventions for optimizing and sustaining learning amidst workplace challenges. In this article, we attempt to unpack the composition of on-task effort during performance-based learning by testing the proposition that the information-knowledge gap-a regulatory discrepancy between unknown aspects of a task and a person's perceived competence in dealing with that task-is the psychological mechanism responsible for guiding effort-allocation decisions during performance-based learning. In Study 1, we found that larger information-knowledge gaps resulted in increased subsequent investments of on-task attention within a sample of adults learning to perform a complex task (N = 121). As participants learned, information-knowledge gaps systematically shrank, resulting in a reduced emphasis on learning-oriented effort (i.e., exploration) relative to achievement-oriented effort (i.e., exploitation) over time. In Study 2 (N = 176), a task-change paradigm revealed that introducing novel demands caused information-knowledge gaps to suddenly expand, which prompted participants to increase on-task effort and shift their focus away from achievement and back toward learning as an adaptive response. Collectively, these findings support the notion that information-knowledge gaps shape how (and when) on-task effort is spent and present a framework for understanding how learners strategically structure their limited attentional resources. (PsycInfo Database Record (c) 2024 APA, all rights reserved).
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Atenção , Aprendizagem , Adulto , Humanos , Atenção/fisiologiaRESUMO
In the manufacturing of therapeutic monoclonal antibodies (mAbs), the final steps of the purification process are typically ultrafiltration/diafiltration (UF/DF), dilution, and conditioning. These steps are developed such that the final drug substance (DS) is formulated to the desired mAb, buffer, and excipient concentrations. To develop these processes, process and formulation development scientists often perform experiments to account for the Gibbs-Donnan and volume-exclusion effects during UF/DF, which affect the output pH and buffer concentration of the UF/DF process. This work describes the development of an in silico model for predicting the DS pH and buffer concentration after accounting for the Gibbs-Donnan and volume-exclusion effects during the UF/DF operation and the subsequent dilution and conditioning steps. The model was validated using statistical analysis to compare model predictions against experimental results for nine molecules of varying protein concentrations and formulations. In addition, our results showed that the structure-based in silico approach used to calculate the protein charge was more accurate than a sequence-based approach. Finally, we used the model to gain fundamental insights about the Gibbs-Donnan effect by highlighting the role of the protein charge concentration (the protein concentration multiplied with protein charge at the formulation pH) on the Gibbs-Donnan effect. Overall, this work demonstrates that the Gibbs-Donnan and volume-exclusions effects can be predicted using an in silico model, potentially alleviating the need for experiments.
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Anticorpos Monoclonais , Ultrafiltração , Ultrafiltração/métodos , Anticorpos Monoclonais/química , Excipientes/químicaRESUMO
Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations. The goal of this distillation is to help harmonize aggregate quantitation approaches across the biopharmaceutical industry. We review key considerations for sample and instrument suitability, experimental design, and data analysis best practices and conversely, highlight potential pitfalls to accurate aggregate analysis. Our goal is to provide experienced users benchmarks against which they can standardize their analyses and to provide guidance for new AUC analysts that will aid them to become proficient in this fundamental technique.
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Anticorpos Monoclonais , Anticorpos Monoclonais/química , Cromatografia em Gel , Ultracentrifugação/métodosRESUMO
PURPOSE OF REVIEW: The promise of cell and gene therapy (CGT) products for a multitude of diseases has revitalized investigators to advance novel CGT product candidates to first-in-human trials by pursuing the investigational new drug (IND) mechanism administered by the United States (US) Food and Drug Administration (FDA). This review is intended to familiarize academic investigators with the IND governing regulations set forth by the FDA. RECENT FINDINGS: CGT products are extraordinarily complex biologics and, therefore, early-stage evaluation programs must be customized to satisfactorily address their unique developmental challenges. The US FDA continues to foster the development of transformational technology that will facilitate the broad application of safe and effective gene therapy products that have the potential to alleviate many conditions previously out of reach of therapeutic intervention. FDA is committed to working with the scientific community and industry to facilitate the availability of these treatments to patients. SUMMARY: The pathway to meet regulatory compliance during early stage IND programs can be daunting to academic investigators interested in CGT product development that typically don't progress beyond phase 1/2. However, by keeping abreast of current regulatory framework and building upon FDA's supportive infrastructure, an investigator can be well-positioned to advance innovative scientific discoveries towards early stage clinical assessments.
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The port delivery system with ranibizumab (PDS) is an investigational long-acting drug delivery system for the continuous release of ranibizumab, an anti-VEGF biologic, in the vitreous humor. The efficacy of the PDS implant relies on the maintenance of long-term drug stability under physiological conditions. Herein, the long-term stability of three anti-VEGF biologics - ranibizumab, bevacizumab and aflibercept - was investigated in phosphate buffered saline (PBS) at 37 °C for several months. Comparison of stability profiles shows that bevacizumab and aflibercept are increasingly prone to aggregation whereas ranibizumab undergoes minimal aggregation. Ranibizumab also shows the smallest loss in antigen binding capacity after long-term incubation in PBS. Even though the aggregated forms of bevacizumab and aflibercept bind to VEGF, the consequences of aggregation on immunogenicity, implant function and efficacy are unknown. These results highlight the importance of maintaining long-term drug stability under physiologically relevant conditions which is necessary for achieving efficacy with an in vivo continuous drug delivery device such as the PDS implant.
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Produtos Biológicos , Fator A de Crescimento do Endotélio Vascular , Inibidores da Angiogênese , Bevacizumab , Injeções Intravítreas , Ranibizumab , Proteínas Recombinantes de FusãoRESUMO
The authors sought to determine whether consuming collagen peptides (CP) enhances musculoskeletal recovery of connective tissues following a damaging exercise bout. Resistance-trained males consumed 15 g/day of CP (n = 7) or placebo (n = 8), and after 7 days, maximal voluntary isometric contraction (MVIC), countermovement jump height, soreness, and collagen turnover were examined. Five sets of 20 drop jumps were performed and outcome measures were collected 24, 48, and 120 hr postexercise. Countermovement jump height was maintained in the CP group at 24 hr (PRE = 39.9 ± 8.8 cm vs. 24 hr = 37.9 ± 8.9 cm, p = .102), whereas the CP group experienced a significant decline at 24 hr (PRE = 40.4 ± 7.9 cm vs. 24 hr = 35.5 ± 6.4 cm, p = .001; d = 0.32). In both groups, muscle soreness was significantly higher than PRE at 24 hr (p = .001) and 48 hr (p = .018) but not at 120 hr (p > .05). MVIC in both legs showed a significant time effect (left: p = .007; right: p = .010) over the 5-day postexercise period. Neither collagen biomarker changed significantly at any time point. CP supplementation attenuated performance decline 24 hr following muscle damage. Acute consumption of CP may provide a performance benefit the day following a bout of damaging exercise in resistance-trained males.
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Colágeno/administração & dosagem , Suplementos Nutricionais , Exercício Físico/fisiologia , Mialgia/prevenção & controle , Fragmentos de Peptídeos/administração & dosagem , Treinamento Resistido/efeitos adversos , Adolescente , Adulto , Biomarcadores/metabolismo , Colágeno/metabolismo , Método Duplo-Cego , Humanos , Contração Isométrica , Perna (Membro)/fisiologia , Masculino , Força Muscular , Fragmentos de Peptídeos/metabolismo , Projetos Piloto , Adulto JovemRESUMO
Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target.
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Trifosfato de Adenosina/metabolismo , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleases/metabolismo , Trifosfato de Adenosina/química , Sítio Alostérico/efeitos dos fármacos , Cristalografia por Raios X , Retículo Endoplasmático/metabolismo , Endorribonucleases/química , Técnicas de Inativação de Genes , Humanos , Ligantes , Modelos Moleculares , Fosforilação , Conformação Proteica , Dobramento de Proteína , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Ribonucleases/química , Resposta a Proteínas não DobradasRESUMO
The purpose of this laboratory study involving repeated measures of emotion as 214 undergraduates (58.4% male) learned a complex video game was to address the need for empirical research on dynamic personality constructs by examining how two aspects of affect variability-spin and pulse-explain variance in skill acquisition and adaptive performance. Spin refers to within-person fluctuations in affect pleasantness and activation potential. Pulse refers to within-person fluctuations in affect intensity. Despite research showing high affect variability reflects a personality profile of heighted reactivity to emotionally charged events and poor adjustment, little empirical research has examined their relationships with behavioral outcomes, much less aspects of skilled performance. Compared to traditional measures of personality, which yield weak effects for predicting acquisition and adaptive performance, measures of affect variability hold considerable promise because they, like performance, reflect dynamic within-person phenomena. Accordingly, the main question addressed by this study was whether spin and pulse incrementally explain acquisition and adaptive performance beyond Big Five measures of personality. In general, we expected harmful, incremental effects for both spin and pulse, and hypothesized two mechanisms for these harmful effects: (1) by undermining effort and (2) by undermining the effort-performance relationship. Using a task-change paradigm and discontinuous growth modeling that disentangled adaptation from acquisition, results showed that affect variability, independent of the Big Five, produced harmful effects via both hypothesized mechanisms. Participants higher in affect spin and pulse showed less sustained effort across performance sessions and exhibited lower performance. Furthermore, the harmful effects of spin and pulse were stronger in adaptation compared to acquisition, with pulse showing stronger direct effects on performance during adaptation and spin moderating the effort-performance relationship such that effort was only beneficial during adaptation for those lower in spin. In light of these results, one might question the common advice "keep calm and carry on," which may not be viable for persons high in affect variability. Accordingly, results are discussed in terms of the need to better understand the specific mediating processes by which high affect variability undermines success across a variety of learning and performance contexts.
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In clinical gene transfer applications, lentiviral vectors (LV) have rapidly become the primary means to achieve permanent and stable expression of a gene of interest or alteration of gene expression in target cells. This status can be attributed primarily to the ability of the LV to (1) transduce dividing as well as quiescent cells, (2) restrict or expand tropism through envelope pseudo-typing, and (3) regulate gene expression within different cell lineages through internal promoter selection. Recent progress in viral vector design such as the elimination of unnecessary viral elements, split packaging, and self-inactivating vectors has established a significant safety profile for these vectors. The level of GMP compliance required for the manufacture of LV is dependent upon their intended use, stage of drug product development, and country where the vector will be used as the different regulatory authorities who oversee the clinical usage of such products may have different requirements. As such, successful GMP manufacture of LV requires a combination of diverse factors including: regulatory expertise, compliant facilities, validated and calibrated equipments, starting materials of the highest quality, trained production personnel, scientifically robust production processes, and a quality by design approach. More importantly, oversight throughout manufacturing by an independent Quality Assurance Unit who has the authority to reject or approve the materials is required. We describe here the GMP manufacture of LV at our facility using a four plasmid system where 293T cells from an approved Master Cell Bank (MCB) are transiently transfected using polyethylenimine (PEI). Following transfection, the media is changed and Benzonase added to digest residual plasmid DNA. Two harvests of crude supernatant are collected and then clarified by filtration. The clarified supernatant is purified and concentrated by anion exchange chromatography and tangential flow filtration. The final product is then diafiltered directly into the sponsor defined final formulation buffer and aseptically filled.
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Centros Médicos Acadêmicos , Terapia Genética , Vetores Genéticos/biossíntese , Vetores Genéticos/normas , Lentivirus , Técnicas de Cultura de Células , Meios de Cultura , Arquitetura de Instituições de Saúde , Terapia Genética/normas , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , TransfecçãoRESUMO
Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.
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Domínio Catalítico , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Animais , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Células Sf9 , SpodopteraRESUMO
Subvisible particle formation in monoclonal antibody drug product resulting from mixing and filling operations represents a significant processing risk that can lead to filter fouling and thereby lead to process delays or failures. Several previous studies from our lab and others demonstrated the formation of subvisible particulates in mAb formulations resulting from mixing operations using some bottom-mounted mixers or stirrer bars. It was hypothesized that the stress (e.g., shear/cavitation) derived from tight clearance and/or close contact between the impeller and shaft was responsible for protein subvisible particulate generation. These studies, however, could not distinguish between the two surfaces without contact (tight clearance) or between two contacting surfaces (close contact). In the present study we expand on those findings and utilize small-scale mixing models that are able to, for the first time, distinguish between tight clearances and tight contact. In this study we evaluated different mixer types including a top-mounted mixer, several impeller-based bottom-mounted mixers, and a rotary piston pump. The impact of tight clearance/close contact on subvisible particle formation in at-scale mixing platforms was demonstrated in the gap between the impeller and drive unit as well as between the piston and the housing of the pump. Furthermore, small-scale mixing models based on different designs of magnetic stir bars that mimic the tight clearance/close contact of the manufacturing-scale mixers also induced subvisible particles in mAb formulations. Additional small-scale models that feature tight clearance but no close contact (grinding) suggested that it is the repeated grinding/contacting of the moving parts and not the presence of tight clearance in the processing equipment that is the root cause of protein subvisible particulate formation. When multiple mAbs, Fabs (fragment antigen binding), or non-antibody related proteins were mixed in the small-scale mixing model, for molecules investigated, it was observed that mAbs and Fabs appear to be more susceptible to particle formation than non-antibody-related proteins. In the grinding zone, mAb/Fab molecules aggregated into insoluble particles with neither detectable soluble aggregates nor fragmented species. This investigation represents a step closer to the understanding of the underlying stress mechanism leading to mAb subvisible particulate formation as the result of drug product processing.LAY ABSTRACT: Mixing and fill finish are important unit operations in drug product manufacturing for compounding (dilution, pooling, homogenization, etc.) and filling into primary packaging containers (vials, pre-filled syringes, etc.), respectively. The current trend in adopting bottom-mounted mixers as well as low fill-volume filling systems has raised concerns about their impact on drug product quality and process performance. However, investigations into the effects of their use for biopharmaceutical products, particularly monoclonal antibody formulations, are rarely published. The purpose of this study is three-fold: (1) to revisit the impact of bottom-mounted mixer design on monoclonal antibody subvisible particle formation; (2) to identify the root cause for subvisible particle formation; and (3) to fully utilize available particle analysis tools to demonstrate the correlation between particle count in the solution and filter fouling during sterile filtration. The outcomes of this study will benefit scientists and engineers who develop biologic product manufacturing processes by providing a better understanding of drug product process challenges.
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Anticorpos Monoclonais/química , Fragmentos Fab das Imunoglobulinas/química , Preparações Farmacêuticas/química , Tecnologia Farmacêutica/métodos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Composição de Medicamentos , Desenho de Equipamento , Agregados Proteicos , Estresse Mecânico , Tecnologia Farmacêutica/instrumentação , UltracentrifugaçãoRESUMO
OBJECTIVE: The aim of this laboratory experiment was to demonstrate how taking a longitudinal, multilevel approach can be used to examine the dynamic relationship between subjective workload and performance over a given period of activity involving shifts in task demand. BACKGROUND: Subjective workload and conditions of the performance environment are oftentimes examined via cross-sectional designs without distinguishing within-from between-person effects. Given the dynamic nature of performance phenomena, multilevel designs coupled with manipulations of task demand shifts are needed to better model the dynamic relationships between state and trait components of subjective workload and performance. METHOD: With a sample of 75 college students and a computer game representing a complex decision-making environment, increases and decreases in task demand were counterbalanced and subjective workload and performance were measured concurrently in regular intervals within performance episodes. Data were analyzed using hierarchical linear modeling. RESULTS: Both between- and especially within-person effects were dynamic. Nevertheless, at both levels of analysis, higher subjective workload reflected performance problems, especially more downstream from increases in task demand. CONCLUSION: As a function of cognitive-energetic processes, shifts in task demand are associated with changes in how subjective workload is related to performance over a given period of activity. Multilevel, longitudinal approaches are useful for distinguishing and examining the dynamic relationships between state and trait components of subjective workload and performance. APPLICATION: The findings of this research help to improve the understanding of how a sequence of demands can exceed a performer's capability to respond to further demands.
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Tomada de Decisões , Análise e Desempenho de Tarefas , Carga de Trabalho/psicologia , Adolescente , Feminino , Humanos , Masculino , Estresse Psicológico , Jogos de Vídeo , Adulto JovemRESUMO
The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function.
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Anticorpos Monoclonais Humanizados/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Bevacizumab , Bovinos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Ligação Proteica , Ranibizumab , Ultracentrifugação , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
We describe a general Biacore method for measuring equilibrium binding affinities and stoichiometries for interactions between unmodified proteins and their unmodified ligands free in solution. Mixtures of protein and ligand are preequilibrated at different ratios in solution and then analyzed by Biacore using a sensor chip surface that detects only unbound analyte. Performing the Biacore analysis under mass transport limited conditions allows the concentration of unbound analyte to be determined from the initial velocity of binding. Plots of initial velocity versus the concentration of the varied binding partner are fitted to a quadratic binding equation to give the affinity and stoichiometry of binding. We demonstrate the method using soluble Her2 extracellular domain binding to monovalent, bivalent, and trivalent forms of an anti-Her2 antibody. The affinity we measured agrees with that obtained from conventional Biacore kinetic analysis, and the stoichiometries for the resulting 1:1, 1:2, and 1:3 complexes were confirmed by gel filtration with in-line light scattering. The method is applicable over an affinity range of approximately 100 pM to 1 µM and is particularly useful when there is concern that covalently modifying one or the other binding partner might affect its binding properties or where multivalency might otherwise complicate a quantitative analysis of binding.
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Cromatografia em Gel/métodos , Proteínas/análise , Receptor ErbB-2/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Animais , Cricetinae , Cricetulus , Humanos , Cinética , Ligação Proteica , Proteínas/química , Receptor ErbB-2/análiseRESUMO
TWEAK (TNF homologue with weak apoptosis-inducing activity) and Fn14 (fibroblast growth factor-inducible protein 14) are members of the tumor necrosis factor (TNF) ligand and receptor super-families. Having observed that Xenopus Fn14 cross-reacts with human TWEAK, despite its relatively low sequence homology to human Fn14, we examined the conservation in tertiary fold and binding interfaces between the two species. Our results, combining NMR solution structure determination, binding assays, extensive site-directed mutagenesis and molecular modeling, reveal that, in addition to the known and previously characterized ß-hairpin motif, the helix-loop-helix motif makes an essential contribution to the receptor/ligand binding interface. We further discuss the insight provided by the structural analyses regarding how the cysteine-rich domains of the TNF receptor super-family may have evolved over time. DATABASE: Structural data are available in the Protein Data Bank/BioMagResBank databases under the accession codes 2KMZ, 2KN0 and 2KN1 and 17237, 17247 and 17252. STRUCTURED DIGITAL ABSTRACT: TWEAK binds to hFn14 by surface plasmon resonance (View interaction) xeFn14 binds to TWEAK by enzyme linked immunosorbent assay (View interaction) TWEAK binds to xeFn14 by surface plasmon resonance (View interaction) hFn14 binds to TWEAK by enzyme linked immunosorbent assay (View interaction).
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Receptores do Fator de Necrose Tumoral/química , Fatores de Necrose Tumoral/química , Proteínas de Xenopus/química , Sequência de Aminoácidos , Animais , Citocina TWEAK , Espaço Extracelular/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor de TWEAK , Xenopus laevisRESUMO
An inherent aspect of learner-controlled instructional environments is the ability of learners to affect the degree of difficulty faced during training. However, research has yet to examine how learner-controlled practice difficulty affects learning. Based on the notion of desirable difficulties (Bjork, 1994), this study examined the cognitive and motivational antecedents and outcomes of learner-controlled practice difficulty in relation to learning a complex task. Using a complex videogame involving both strong cognitive and psychomotor demands, 112 young adult males were given control over their practice difficulty, which was reflected in the complexity of the training task. Results show that general mental ability, prior experience, pre-training self-efficacy, and error encouragement were positively related to learner-controlled practice difficulty. In turn, practice difficulty was directly related to task knowledge and post-training performance, and it was related to adaptive performance through the mediating influences of task knowledge and post-training performance. In general, this study supports the notion that training difficulty operationalized in terms of task complexity is positively related to both knowledge and performance outcomes. Results are discussed with respect to the need for more research examining how task complexity and other forms of difficulty could be leveraged to advance learner-controlled instructional practices.
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Cognição/fisiologia , Individualidade , Aprendizagem/fisiologia , Motivação/fisiologia , Prática Psicológica , Desempenho Psicomotor/fisiologia , Adolescente , Adulto , Humanos , Masculino , Autoeficácia , Estudantes/psicologia , Jogos de Vídeo/psicologia , Adulto JovemRESUMO
We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPIs), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for binding of the large receptor TNFR1 to its ligands TNFα (K(D) = 1.4 ± 0.4 nM) and lymphotoxin-α (K(D) = 50 ± 10 nM), and also for binding of the small receptor Fn14 to TWEAK (K(D) = 70 ± 10 nM). We additionally assembled data for all other TNF-TNFR family complexes for which reliable single-site binding affinities have been reported. We used these values to calculate the binding efficiencies, defined as binding energy per square angstrom of surface area buried at the contact interface, for nine of these complexes for which cocrystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinities. The results show that the most efficient PPI complexes generate ~20 cal mol(-1) Å(-2) of binding energy. A minimal contact area of ~500 Å(2) is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of colocalizing two proteins from 1 M solution. The most compact and efficient TNF-TNFR complex was the BAFF-BR3 complex, which achieved ~80% of the maximal achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method for predicting how large a natural or engineered contact interface must be to achieve a given level of binding affinity.
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Linfotoxina-alfa/metabolismo , Ligação Proteica , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fatores de Necrose Tumoral/metabolismo , Citocina TWEAK , Cinética , Ligantes , Receptores do Fator de Necrose Tumoral/metabolismo , Ressonância de Plasmônio de Superfície , Receptor de TWEAKRESUMO
TWEAK, a TNF family ligand with pleiotropic cellular functions, was originally described as capable of inducing tumor cell death in vitro. TWEAK functions by binding its receptor, Fn14, which is up-regulated on many human solid tumors. Herein, we show that intratumoral administration of TWEAK, delivered either by an adenoviral vector or in an immunoglobulin Fc-fusion form, results in significant inhibition of tumor growth in a breast xenograft model. To exploit the TWEAK-Fn14 pathway as a therapeutic target in oncology, we developed an anti-Fn14 agonistic antibody, BIIB036. Studies described herein show that BIIB036 binds specifically to Fn14 but not other members of the TNF receptor family, induces Fn14 signaling, and promotes tumor cell apoptosis in vitro. In vivo, BIIB036 effectively inhibits growth of tumors in multiple xenograft models, including colon (WiDr), breast (MDA-MB-231), and gastric (NCI-N87) tumors, regardless of tumor cell growth inhibition response observed to BIIB036 in vitro. The anti-tumor activity in these cell lines is not TNF-dependent. Increasing the antigen-binding valency of BIB036 significantly enhances its anti-tumor effect, suggesting the contribution of higher order cross-linking of the Fn14 receptor. Full Fc effector function is required for maximal activity of BIIB036 in vivo, likely due to the cross-linking effect and/or ADCC mediated tumor killing activity. Taken together, the anti-tumor properties of BIIB036 validate Fn14 as a promising target in oncology and demonstrate its potential therapeutic utility in multiple solid tumor indications.