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Bioinks for cell-based bioprinting face availability limitations. Furthermore, the bioink development process needs comprehensive printability assessment methods and a thorough understanding of rheological factors' influence on printing outcomes. To bridge this gap, our study aimed to investigate the relationship between rheological properties and printing outcomes. We developed a specialized bioink artifact specifically designed to improve the quantification of printability assessment. This bioink artifact adhered to established criteria from extrusion-based bioprinting approaches. Seven hydrogel-based bioinks were selected and tested using the bioink artifact and rheological measurement. Rheological analysis revealed that the high-performing bioinks exhibited notable characteristics such as high storage modulus, low tan(δ), high shear-thinning capabilities, high yield stress, and fast, near-complete recovery abilities. Although rheological data alone cannot fully explain printing outcomes, certain metrics like storage modulus and tan(δ) correlated well (R2 > 0.9) with specific printing outcomes, such as gap-spanning capability and turn accuracy. This study provides a comprehensive examination of bioink shape fidelity across a wide range of bioinks, rheological measures, and printing outcomes. The results highlight the importance of considering the holistic view of bioink's rheological properties and directly measuring printing outcomes. These findings underscore the need to enhance bioink availability and establish standardized methods for assessing printability.
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The pathogenic processes driving Alzheimer's disease (AD) are complex. An incomplete understanding of underlying disease mechanisms has presented insurmountable obstacles for developing effective disease-modifying therapies. Advanced chronological age is the greatest risk factor for developing AD. Intervening on biological aging may alter disease progression and represents a novel, complementary approach to current strategies. Toward this end, cellular senescence has emerged as a promising target. This complex stress response harbors damaged cells in a cell cycle arrested, apoptosis-resistant cell state. Senescent cells accumulate with age where they notoriously secrete molecules that contribute to chronic tissue dysfunction and disease. Thus, benefits of cell survival in a senescent fate are countered by their toxic secretome. The removal of senescent cells improves brain structure and function in rodent models at risk of developing AD, and in those with advanced Aß and tau pathology. The present review describes the path to translating this promising treatment strategy to AD clinical trials. We review evidence for senescent cell accumulation in the human brain, considerations and strategies for senescence-targeting trials specific to AD, approaches to detect senescent brain cells in biofluids, and summarize the goals of the first senolytic trials for the treatment of AD (NCT04063124 and NCT04685590). This article is part of the Special Issue - Senolytics - Edited by Joao Passos and Diana Jurk.
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Envejecimiento , Enfermedad de Alzheimer , Senoterapéuticos/farmacología , Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Senescencia Celular/efectos de los fármacos , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , HumanosRESUMEN
In 1960, Rita Levi-Montalcini and Barbara Booker made an observation that transformed neuroscience: as neurons mature, they become apoptosis resistant. The following year Leonard Hayflick and Paul Moorhead described a stable replicative arrest of cells in vitro, termed "senescence". For nearly 60 years, the cell biology fields of neuroscience and senescence ran in parallel, each separately defining phenotypes and uncovering molecular mediators to explain the 1960s observations of their founding mothers and fathers, respectively. During this time neuroscientists have consistently observed the remarkable ability of neurons to survive. Despite residing in environments of chronic inflammation and degeneration, as occurs in numerous neurodegenerative diseases, often times the neurons with highest levels of pathology resist death. Similarly, cellular senescence (hereon referred to simply as "senescence") now is recognized as a complex stress response that culminates with a change in cell fate. Instead of reacting to cellular/DNA damage by proliferation or apoptosis, senescent cells survive in a stable cell cycle arrest. Senescent cells simultaneously contribute to chronic tissue degeneration by secreting deleterious molecules that negatively impact surrounding cells. These fields have finally collided. Neuroscientists have begun applying concepts of senescence to the brain, including post-mitotic cells. This initially presented conceptual challenges to senescence cell biologists. Nonetheless, efforts to understand senescence in the context of brain aging and neurodegenerative disease and injury emerged and are advancing the field. The present review uses pre-defined criteria to evaluate evidence for post-mitotic brain cell senescence. A closer interaction between neuro and senescent cell biologists has potential to advance both disciplines and explain fundamental questions that have plagued their fields for decades.
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Cellular stress responses influence cell fate decisions. Apoptosis and proliferation represent opposing reactions to cellular stress or damage and may influence distinct health outcomes. Clinical and epidemiological studies consistently report inverse comorbidities between age-associated neurodegenerative diseases and cancer. This review discusses how one particular stress response, cellular senescence, may contribute to this inverse correlation. In mitotically competent cells, senescence is favorable over uncontrolled proliferation, i.e., cancer. However, senescent cells notoriously secrete deleterious molecules that drive disease, dysfunction and degeneration in surrounding tissue. In recent years, senescent cells have emerged as unexpected mediators of neurodegenerative diseases. The present review uses pre-defined criteria to evaluate evidence of cellular senescence in mitotically competent brain cells, highlights the discovery of novel molecular regulators and discusses how this single cell fate decision impacts cancer and degeneration in the brain. We also underscore methodological considerations required to appropriately evaluate the cellular senescence stress response in the brain.
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Bioink printability persists as a limiting factor toward many bioprinting applications. Printing parameter selection is largely user-dependent, and the effect of cell density on printability has not been thoroughly investigated. Recently, methods have been developed to give greater insight into printing outcomes. This study aims to further advance those methods and apply them to study the effect of printing parameters (feedrate and flowrate) and cell density on printability. Two printed structures, a crosshatch and five-layer tube, were established as printing standards and utilized to determine the printing outcomes. Acellular bioinks were printed using a testing matrix of feedrates of 37.5, 75, 150, 300, and 600 mm/min and flowrates of 21, 42, 84, 168, and 336 mm3/min. Structures were also printed with cell densities of 5, 10, 20, and 40 × 106 cell/mL at 150 mm/min and 84 mm3/min. Only speed ratios (defined as flowrate divided by feedrate) from 0.07 to 2.24 mm2 were suitable for analysis. Increasing speed ratio dramatically increased the height, width, and wall thickness of tubular structures, but did not influence radial accuracy. For crosshatch structures, the area of pores and the frequency of broken filaments were decreased without impacting pore shape (Pr). Within speed ratios, feedrate and flowrate had negligible, inconsistent effects. Cell density did not affect any printing outcomes despite slight rheological changes. Printing outcomes were dominated by the speed ratio, with feedrate, flowrate, and cell density having little impact on printing outcomes when controlling for speed ratio within the ranges tested. The relevance of these results to other bioinks and printing conditions requires continued investigation by the bioprinting community, as well as highlight speed ratio as a key variable to report and suggest that rheology is a more sensitive measure than printing outcomes.
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Bioimpresión , Impresión Tridimensional , Recuento de Células , ReologíaRESUMEN
Bioprinting researchers agree that "printability" is a key characteristic for bioink development, but neither the meaning of the term nor the best way to experimentally measure it has been established. Furthermore, little is known with respect to the underlying mechanisms which determine a bioink's printability. A thorough understanding of these mechanisms is key to the intentional design of new bioinks. For the purposes of this review, the domain of printability is defined as the bioink requirements which are unique to bioprinting and occur during the printing process. Within this domain, the different aspects of printability and the factors which influence them are reviewed. The extrudability, filament classification, shape fidelity, and printing accuracy of bioinks are examined in detail with respect to their rheological properties, chemical structure, and printing parameters. These relationships are discussed and areas where further research is needed, are identified. This review serves to aid the bioink development process, which will continue to play a major role in the successes and failures of bioprinting, tissue engineering, and regenerative medicine going forward.
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Bioimpresión , Hidrogeles/química , Tinta , Impresión Tridimensional , Ingeniería de Tejidos , HumanosRESUMEN
Full-thickness skin wounds are a significant clinical burden in the United States. Skin bioprinting is a relatively new technology that is under investigation as a new treatment for full-thickness injuries, and development of hydrogels with strong physical and biological characteristics are required to improve both structural integrity of the printed constructs while allowing for a more normal extracellular matrix milieu. This project aims to evaluate the physical and biological characteristics of fibrinogen hydrogel supplemented with decellularized human skin-derived extracellular matrix (dsECM). The hybrid hydrogel improves the cell viability and structural strength of bioprinted skin constructs. Scanning electron microscopy demonstrates that the hybrid hydrogel is composed of both swelling bundles interlocked in a fibrin network, similar to healthy human skin. This hybrid hydrogel has improved rheological properties and shear thinning properties. Extrusion-based printing of the fibrinogen hydrogel + dsECM demonstrates significant improvement in crosshatch pore size. These findings suggest that incorporating the properties of dsECM and fibrinogen hydrogels will improve in vivo integration of the bioprinted skin constructs and support of healthy skin wound regeneration.
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The goal of this study was to use 3D bioprinting technology to create a bioengineered dental construct containing human dental pulp stem cells (hDPSCs). To accomplish this, we first developed a novel bone morphogenetic protein (BMP) peptide-tethering bioink formulation and examined its rheological properties, its printability, and the structural stability of the bioprinted construct. Second, we evaluated the survival and differentiation of hDPSCs in the bioprinted dental construct by measuring cell viability, proliferation, and gene expression, as well as histological and immunofluorescent analyses. Our results showed that the peptide conjugation into the gelatin methacrylate-based bioink formulation was successfully performed. We determined that greater than 50% of the peptides remained in the bioprinted construct after three weeks in vitro cell culture. Human DPSC viability was >90% in the bioprinted constructs immediately after the printing process. Alizarin Red staining showed that the BMP peptide construct group exhibited the highest calcification as compared to the growth medium, osteogenic medium, and non-BMP peptide construct groups. In addition, immunofluorescent and quantitative reverse transcription-polymerase chain reaction analyses showed robust expression of dentin sialophosphoprotein and osteocalcin in the BMP peptide dental constructs. Together, these results strongly suggested that BMP peptide-tethering bioink could accelerate the differentiation of hDPSCs in 3D bioprinted dental constructs.
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Materiales Biomiméticos/farmacología , Bioimpresión , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular , Pulpa Dental/citología , Osteogénesis , Impresión Tridimensional , Células Madre/citología , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Gelatina/química , Humanos , Hidrogeles/química , Metacrilatos/química , Osteogénesis/efectos de los fármacos , Péptidos/farmacología , Células Madre/efectos de los fármacos , Porcinos , Andamios del Tejido/químicaRESUMEN
Extrusion-based bioprinting is one of the leading manufacturing techniques for tissue engineering and regenerative medicine. Its primary limitation is the lack of materials, known as bioinks, which are suitable for the bioprinting process. The degree to which a bioink is suitable for bioprinting has been described as its 'printability.' However, a lack of clarity surrounding the methodologies used to evaluate a bioink's printability, as well as the usage of the term itself, have hindered the field. This article presents a review of measures used to assess the printability of extrusion-based bioinks in an attempt to assist researchers during the bioink development process. Many different aspects of printability exist and many different measurements have been proposed as a consequence. Researchers often do not evaluate a new bioink's printability at all, while others simply do so qualitatively. Several quantitative measures have been presented for the extrudability, shape fidelity, and printing accuracy of bioinks. Different measures have been developed even within these aspects, each testing the bioink in a slightly different way. Additionally, other relevant measures which had little or no examples of quantifiable methods are also to be considered. Looking forward, further work is needed to improve upon current assessment methodologies, to move towards a more comprehensive view of printability, and to standardize these printability measurements between researchers. Better assessment techniques will naturally lead to a better understanding of the underlying mechanisms which affect printability and better comparisons between bioinks. This in turn will help improve upon the bioink development process and the bioinks available for use in bioprinting.
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Bioimpresión/instrumentación , Impresión Tridimensional/instrumentación , Animales , Bioimpresión/métodos , Humanos , Ingeniería de Tejidos/instrumentación , Andamios del Tejido/químicaRESUMEN
Introduction: Hierarchical nanofibrous scaffolds are emerging as a promising bone repair material due to their high cell adhesion activity and nutrient permeability. However, the existing method for hierarchical nanofibrous scaffolds fabrication is complicated and not perfectly suitable for further biomedical application in view of both structure and function. In this study, we constructed a hierarchical nanofibrous poly (l-lactic acid)/poly(ε-caprolactone) (PLLA/PCL) scaffold and further evaluated its bone healing ability. Methods: The hierarchical PLLA/PCL nanofibrous scaffold (PLLA/PCL) was prepared by one-pot TIPS and then rapidly mineralized at room temperature by an electrochemical deposition technique. After electrode-positioning at 2 V for 2 hrs, a scaffold coated with hydroxyapatite (M-PLLA/PCL) could be obtained. Results: The pore size of the M-PLLA/PCL scaffold was hierarchically distributed so as to match the biophysical structure for osteoblast growth. The M-PLLA/PCL scaffold showed better cell proliferation and osteogenesis activity compared to the PLLA/PCL scaffold. Further in vivo bone repair studies indicated that the M-PLLA/PCL scaffold could accelerate defect healing in 12 weeks. Conclusion: The results of this study implied that the as-prepared hydroxyapatite coated hierarchical PLLA/PCL nanofibrous scaffolds could be developed as a promising material for efficient bone tissue repair after carefully tuning the TIPS and electrodeposition parameters.
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Regeneración Ósea/fisiología , Galvanoplastia/métodos , Minerales/química , Nanofibras/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/farmacología , Andamios del Tejido/química , Fosfatasa Alcalina/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Electricidad , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/ultraestructura , Osteogénesis/efectos de los fármacos , Porosidad , Ratas Sprague-Dawley , Cráneo/diagnóstico por imagen , Cráneo/efectos de los fármacos , Cráneo/patología , Factores de Tiempo , Ingeniería de Tejidos/métodos , Microtomografía por Rayos XRESUMEN
We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.
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Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , Proteína Fluorescente RojaRESUMEN
Three-dimensional bioprinting has emerged as a promising technique in tissue engineering applications through the precise deposition of cells and biomaterials in a layer-by-layer fashion. However, the limited availability of hydrogel bioinks is frequently cited as a major issue for the advancement of cell-based extrusion bioprinting technologies. It is well known that highly viscous materials maintain their structure better, but also have decreased cell viability due to the higher forces which are required for extrusion. However, little is known about the effect of the two distinct components of dynamic modulus of viscoelastic materials, storage modulus (G') and loss modulus (Gâ³), on the printability of hydrogel-based bioinks. Additionally, 'printability' has been poorly defined in the literature, mostly consisting of gross qualitative measures which do not allow for direct comparison of bioinks. This study developed a framework for evaluating printability and investigated the effect of dynamic modulus, including storage modulus (G'), loss modulus (Gâ³), and loss tangent (Gâ³/G') on the printing outcome. Gelatin and alginate as model hydrogels were mixed at various concentrations to obtain hydrogel formulations with a wide range of storage and loss moduli. These formulations were then evaluated for the quantitatively defined values of extrudability, extrusion uniformity, and structural integrity. For extrudability, increasing either the loss or storage modulus increased the pressure required to extrude the bioink. A mathematical model relating the G' and Gâ³ to the required extrusion pressure was derived based on the data. A lower loss tangent was correlated with increased structural integrity while a higher loss tangent correlated with increased extrusion uniformity. Gelatin-alginate composite hydrogels with a loss tangent in the range of 0.25-0.45 exhibited an excellent compromise between structural integrity and extrusion uniformity. In addition to the characterization of a common bioink, the methodology introduced in this paper could also be used to evaluate the printability of other bioinks in the future.
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Alginatos/química , Bioimpresión/métodos , Gelatina/química , Ensayo de Materiales/métodos , Elasticidad , Reología , Ingeniería de Tejidos , ViscosidadRESUMEN
Hemostasis is of critical importance for damage control and the initiation of tissue repair in surgery and trauma. Mesoporous silica materials, due to their favorable biocompatibility and excellent surface properties, have attracted increasing attention for their outstanding hemostatic performance. In this study, silver nanoparticle-incorporated mesoporous silica granules (AgNP-MSG) were prepared by means of one-pot sol-gel processing, which brought about a highly blood-absorbent composite capable of prompt hemostasis. Detailed studies were performed to evaluate its structure, cellular compatibility, and adsorption capacity, emphasizing the influence of the composite on in vivo degradability and hemostasis efficacy. The as-prepared composite showed good compatibility and sustained antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Further in vivo experimentation demonstrated that hemorrhage in a rat liver injury model was effectively controlled within 7 s by 5% AgNP-MSG, much more rapidly than commercially derived hemostatic gauze. Histological analysis further demonstrated that the fabricated composites could completely degrade at the site of liver injury 2 weeks later. These results suggest that as-prepared AgNP-MSG has great potential as a hemostatic material with robust antibacterial activity for future therapeutic translation.
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OBJECTIVES: Elderly patients represent the fastest growing and most difficult to treat population sustaining acetabular fractures. When treated surgically, isolated extrapelvic or combined intrapelvic-extrapelvic constructs may be used. No biomechanical or clinical study has compared the merits of these 2 techniques in cadaveric models. This research aims to biomechanically quantify the additional benefit of intrapelvic fixation to a standard extrapelvic fixation construct. METHODS: Ten cadaveric pelves underwent standardized anterior column and quadrilateral plate fracture creation. One hemipelvis from each subject received isolated extrapelvic fixation, whereas the other received adjunctive intrapelvic fixation. Specimens were then subjected to a 50% of body weight (BW) nondestructive stiffness test followed by loading to failure. For the 50% BW test, displacement at 50% BW and stiffness were calculated. For the load to failure test, stiffness, elastic energy, and plastic energy were calculated. Yield point, force at clinical failure (defined at 2 mm of displacement), and maximum force were also identified. A Wilcoxon matched-pairs t test was used to compare fixation groups. RESULTS: The addition of an intrapelvic plate improved construct performance for all test parameters. A statistically significant difference (P < 0.05) was reached for yield force, maximum force, and plastic energy. CONCLUSIONS: These findings demonstrate that the addition of intrapelvic plating may offer distinct advantages in prevention of catastrophic construct failure in situations in which significant lateral to medial force is applied to the greater trochanter such as patient falling.
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Acetábulo/cirugía , Placas Óseas , Fuerza Compresiva/fisiología , Fijación Interna de Fracturas/métodos , Fracturas Óseas/cirugía , Acetábulo/lesiones , Anciano , Anciano de 80 o más Años , Fenómenos Biomecánicos , Densidad Ósea , Cadáver , Disección , Femenino , Fijación Interna de Fracturas/instrumentación , Humanos , Masculino , Tomografía Computarizada por Rayos X/métodosRESUMEN
Using time-resolved fluorescence resonance energy transfer (FRET), we have developed and validated the first high-throughput screening (HTS) method to discover compounds that modulate an intracellular Ca2+ channel, the ryanodine receptor (RyR), for therapeutic applications. Intracellular Ca2+ regulation is critical for striated muscle function, and RyR is a central player. At resting [Ca2+], an increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, diabetes, and neurological disorders. This leaky state of the RyR is an attractive target for pharmacological agents to treat such pathologies. Our FRET-based HTS detects RyR binding of accessory proteins calmodulin (CaM) or FKBP12.6. Under conditions that mimic a pathological state, we carried out a screen of the 727-compound NIH Clinical Collection, which yielded six compounds that reproducibly changed FRET by >3 SD. Dose-response of FRET and [3H]ryanodine binding readouts reveal that five hits reproducibly alter RyR1 structure and activity. One compound increased FRET and inhibited RyR1, which was only significant at nM [Ca2+], and accentuated without CaM present. These properties characterize a compound that could mitigate RyR1 leak. An excellent Z' factor and the tight correlation between structural and functional readouts validate this first HTS method to identify RyR modulators.
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Calmodulina/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/genética , Calmodulina/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/patología , Unión Proteica , Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/genética , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/genéticaRESUMEN
We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.
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Técnicas Biosensibles , Descubrimiento de Drogas/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento , Citometría de Imagen/métodos , Alcanosulfonatos/farmacología , Compuestos Azo/farmacología , Descubrimiento de Drogas/instrumentación , Inhibidores Enzimáticos/farmacología , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Citometría de Imagen/instrumentación , Indoles/farmacología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tapsigargina/farmacología , Proteína Fluorescente RojaRESUMEN
A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA's distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA's ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.
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Técnicas Biosensibles , Descubrimiento de Drogas/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento , Citometría de Imagen/métodos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Descubrimiento de Drogas/instrumentación , Inhibidores Enzimáticos/farmacología , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Citometría de Imagen/instrumentación , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tapsigargina/farmacología , Proteína Fluorescente RojaRESUMEN
Weight bearing asymmetry is common in patients with unilateral lower limb musculoskeletal pathologies. The Nintendo Wii Balance Board (WBB) has been suggested as a low-cost and widely-available tool to measure weight bearing asymmetry in a clinical environment; however no study has evaluated the validity of this tool during dynamic tasks. Therefore, the purpose of this study was to determine the concurrent validity of force measurements acquired from the WBB as compared to laboratory force plates. Thirty-five individuals before, or within 1 year of total joint arthroplasty performed a sit-to-stand and return-to-sit task in two conditions. First, subjects performed the task with both feet placed on a single WBB. Second, the task was repeated with each foot placed on an individual laboratory force plate. Peak vertical ground reaction force (VGRF) under each foot and the inter-limb symmetry ratio were calculated. Validity was examined using Intraclass Correlation Coefficients (ICC), regression analysis, 95% limits of agreement and Bland-Altman plots. Force plates and the WBB exhibited excellent agreement for all outcome measurements (ICC=0.83-0.99). Bland-Altman plots showed no obvious relationship between the difference and the mean for the peak VGRF, but there was a consistent trend in which VGRF on the unaffected side was lower and VGRF on the affected side was higher when using the WBB. However, these consistent biases can be adjusted for by utilizing regression equations that estimate the force plate values based on the WBB force. The WBB may serve as a valid, suitable, and low-cost alternative to expensive, laboratory force plates for measuring weight bearing asymmetry in clinical settings.
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Prueba de Esfuerzo/instrumentación , Pie/fisiología , Actividad Motora/fisiología , Equilibrio Postural/fisiología , Soporte de Peso/fisiología , Anciano , Femenino , Humanos , MasculinoRESUMEN
We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications.
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Fluorescencia , Colorantes Fluorescentes/química , Modelos Químicos , Espectrometría de Fluorescencia/instrumentación , Espectrometría de Fluorescencia/métodosRESUMEN
We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.