RESUMEN
Multiplexed analysis of multiple biomarkers in a tissue sample requires use of reporter dyes with specific spectral properties that enable discrimination of signals. Conventional chromogens with broad absorbance spectra, widely used in immunohistochemistry (IHC), offer limited utility for multiplexed detection. Many dyes with narrow absorbance spectra, eg rhodamines, fluoresceins, and cyanines, potentially useful for multiplexed detection are well-characterized; however, generation of a chromogenic reagent useful for IHC analysis has not been demonstrated. Studies reported herein demonstrate utility of tyramine-chemistry for synthesis of a wide variety of new chromogenic dye conjugates useful for multiplexed in situ analysis using conventional light microscopes. The dyes, useful individually or in blends to generate new colors, provide signal sensitivity and dynamic range similar to conventional DAB chromogen, while enabling analysis of co-localized biomarkers. It is anticipated that this new paradigm will enable generation of a wide variety of new chromogens, useful for both research and clinical biomarker analysis that will benefit clinicians and patients.
Asunto(s)
Biomarcadores/análisis , Compuestos Cromogénicos/química , Colorantes/química , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , 3,3'-Diaminobencidina/química , Biomarcadores/química , Compuestos Cromogénicos/síntesis química , Colorantes/síntesis química , Humanos , Modelos Químicos , Estructura Molecular , Reproducibilidad de los Resultados , Tiramina/químicaRESUMEN
The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.
Asunto(s)
Encéfalo/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Animales , Imagenología Tridimensional , Ratones , Microscopía Electrónica de Rastreo/instrumentaciónRESUMEN
Fluid flow is observed when a volume of passivated Ag nanoparticles suspended in chloroform is mixed with a water/ethanol (v/v) mixture containing acidified 11-mercaptoundecanoic acid. Following mechanical agitation, Ag nanoparticles embedded in a film are driven from the organic-aqueous interface. A reddish-brown colored film, verified by transmission electron microscopy to contain uniformly dispersed Ag nanoparticles, is observed to spontaneously climb the interior surface of an ordinary, laboratory glass vial. This phenomenon is recorded by a digital video recorder, and a measurement of the distance traveled by the film front versus time is extracted. Surface (interfacial) tension gradients due to surfactant concentration, temperature, and electrostatic potential across immiscible fluids are known to drive interface motion; this well-known phenomenon is termed Marangoni flow or the Marangoni effect. Experimental results are presented that show the observed mass transfer is dependent on an acid surfactant concentration and on the volume fraction of water in the aqueous phase, consistent with fluid flow induced by interfacial tension gradients. In addition, an effective desorption rate constant for the Marangoni flow is measured in the range of approximately 0.01 to approximately 1 s(-1) from a fit to the relative film front distance traveled versus time data. The fit is based on a time-dependent expression for the surface (interface) excess for desorption kinetics. Such flow suggests that purposeful creation of interfacial tension gradients may aid in the transfer of 2- and 3-dimensional assemblies, made with nanostructures at the liquid-liquid interface, to solid surfaces.
Asunto(s)
Nanopartículas del Metal/química , Plata/química , Etanol/química , Ácidos Grasos/química , Microfluídica , Modelos Teóricos , Tamaño de la Partícula , Compuestos de Sulfhidrilo/química , Propiedades de Superficie , Tensoactivos/química , Agua/química , HumectabilidadRESUMEN
BACKGROUND: There is a paucity of articles in the surgical literature demonstrating transfer validity (transfer of training). The purpose of this study was to assess whether skills learned on the ArthroSim virtual-reality arthroscopic knee simulator transferred to greater skill levels in the operating room. METHODS: Postgraduate year-3 orthopaedic residents were randomized into simulator-trained and control groups at seven academic institutions. The experimental group trained on the simulator, performing a knee diagnostic arthroscopy procedure to a predetermined proficiency level based on the average proficiency of five community-based orthopaedic surgeons performing the same procedure on the simulator. The residents in the control group continued their institution-specific orthopaedic education and training. Both groups then performed a diagnostic knee arthroscopy procedure on a live patient. Video recordings of the arthroscopic surgery were analyzed by five pairs of expert arthroscopic surgeons blinded to the identity of the residents. A proprietary global rating scale and a procedural checklist, which included visualization and probing scales, were used for rating. RESULTS: Forty-eight (89%) of the fifty-four postgraduate year-3 residents from seven academic institutions completed the study. The simulator-trained group averaged eleven hours of training on the simulator to reach proficiency. The simulator-trained group performed significantly better when rated according to our procedural checklist (p = 0.031), including probing skills (p = 0.016) but not visualization skills (p = 0.34), compared with the control group. The procedural checklist weighted probing skills double the weight of visualization skills. The global rating scale failed to reach significance (p = 0.061) because of one extreme outlier. The duration of the procedure was not significant. This lack of a significant difference seemed to be related to the fact that residents in the control group were less thorough, which shortened their time to completion of the arthroscopic procedure. CONCLUSIONS: We have demonstrated transfer validity (transfer of training) that residents trained to proficiency on a high-fidelity realistic virtual-reality arthroscopic knee simulator showed a greater skill level in the operating room compared with the control group. CLINICAL RELEVANCE: We believe that the results of our study will stimulate residency program directors to incorporate surgical simulation into the core curriculum of their residency programs.
Asunto(s)
Artroscopía/educación , Internado y Residencia , Articulación de la Rodilla/cirugía , Procedimientos Ortopédicos/educación , Interfaz Usuario-Computador , Competencia Clínica , HumanosRESUMEN
Peptidyl prolyl cis/trans isomerase cyclophilin A (CypA) serves as a cellular receptor for the important immunosuppressant drug, cyclosporin A. In addition, CypA and its enzyme family have been found to play critical roles in a variety of biological processes, including protein trafficking, HIV and HCV infection/replication, and Ca(2+)-mediated intracellular signaling. For these reasons, cyclophilins have emerged as potential drug targets for several diseases. Therefore, it is extremely important to screen for novel small molecule cyclophilin inhibitors. Unfortunately, the biochemical assays reported so far are not adaptable to a high-throughput screening format. Here, we report a fluorescence polarization-based assay for human CypA that can be adapted to high-throughput screening for drug discovery. The technique is based on competition and uses a fluorescein-labeled cyclosporin A analog and purified human CypA to quantitatively measure the binding capacity of unlabeled inhibitors. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with CypA is highly advantageous for the accurate assessment of inhibitor binding.
Asunto(s)
Ciclofilina A/análisis , Polarización de Fluorescencia/métodos , Sitios de Unión , Unión Competitiva , Ciclofilina A/antagonistas & inhibidores , Ciclofilina A/metabolismo , Ciclosporina/química , Ciclosporina/metabolismo , Ciclosporinas/síntesis química , Ciclosporinas/química , Ciclosporinas/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fluoresceína , Humanos , Técnicas In Vitro , Cinética , Estructura Molecular , Proteínas Recombinantes/análisis , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismoRESUMEN
Chemiluminescent acridinium-9-carboxamide probes containing 1, 3, 9, and 27 phenylboronic acids were prepared and their chemiluminescent properties evaluated. The relative chemiluminescent signal from the probes varied from 4 to 0.83 x 10(19)counts/mol across the series, while the apparent affinity of the probes for the diabetes marker glycated hemoglobin increased from 211 to 0.43 microM. The dose-dependent modulation of the chemiluminescent intensity of the probes upon binding was used to demonstrate a homogeneous assay for glycated hemoglobin.
Asunto(s)
Acridinas/análisis , Acridinas/química , Hemoglobina Glucada/análisis , Mediciones Luminiscentes/métodos , Ácidos Borónicos/química , Relación Dosis-Respuesta a Droga , Estructura MolecularRESUMEN
An efficient method was developed for the preparation of polyanionic affinity agent (3), a key component in the measurement of glycated hemoglobin (GHb). Glycated hemoglobin is an important clinical marker for diagnosis of patients with diabetes and useful to monitor the management of disease. The affinity agent (3) was prepared based on coupling reaction between poly(acrylic acid) (1) and 3-aminophenylboronic acid (2) in water. The critical features of this polymeric affinity agent (3), such as size, boronic acid incorporation ratio and concentration, on the measurement of glycated hemoglobin were evaluated. It was found that the agent (3) prepared using poly(acrylic acid) (1) with 225 kDa molecular weight gave optimal GHb measurement. The performance test results demonstrated that the boronic acid incorporation ratio and concentration of affinity agent (3) play a critical role in the assay and determines the precision of glycated hemoglobin measurement.
Asunto(s)
Resinas Acrílicas/síntesis química , Ácidos Borónicos/síntesis química , Hemoglobina Glucada/análisis , Hemoglobina Glucada/metabolismo , Resinas Acrílicas/química , Resinas Acrílicas/metabolismo , Ácidos Borónicos/química , Ácidos Borónicos/metabolismo , Cromatografía de Afinidad , Estudios de Evaluación como Asunto , HumanosRESUMEN
An efficient synthesis of 4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]-N-(6-[[6-([6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl]amino)-6-oxohexyl]amino]-6-oxohexyl)cyclohexanecarboxamide (12), a heterobifunctional coupling agent, was developed, which is critical for chemoselective conjugation of proteins and enzymes. The synthesis involved seven steps starting from 6-{[(benzyloxy)carbonyl]amino}hexanoic acid (1), and multigram quantities of coupling agent (12) were prepared using this protocol in excellent overall yield and 99.6% purity by reversed phase HPLC. The new method is suitable for the synthesis of coupling agent 12, consistently with purity >99%, and is useful for the preparation of other analogous coupling agents.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/síntesis química , Pirroles/síntesis química , Pirrolidinas/síntesis química , Cromatografía Líquida de Alta Presión , Estructura Molecular , Pirroles/química , Pirrolidinas/químicaRESUMEN
Two cyanocobalamin-N-sulfonyl-acridinium-9-carboxamides with linkage through the N(10) or 9-position were prepared from B(12)-e-carboxylic acid. The noncovalent association of intrinsic factor with these ligands resulted in specific modulation of the associated chemiluminescence signal either by quenching or changing the emission profile. Either effect was sufficient to formulate a homogeneous assay to detect vitamin B(12) in buffer.