A Fluorescent Kinase Inhibitor that Exhibits Diagnostic Changes in Emission upon Binding

The development of a fluorescent LCK inhibitor that exhibits favourable solvatochromic properties upon binding the kinase is described. Fluorescent properties were realised through the inclusion of a prodan-derived fluorophore into the pharmacophore of an ATP-competitive kinase inhibitor. Fluorescence titration experiments demonstrate the solvatochromic properties of the inhibitor, in which dramatic increase in emission intensity and hypsochromic shift in emission maxima are clearly observed upon binding LCK. Microscopy experiments in cellular contexts together with flow cytometry show that the fluorescence intensity of the inhibitor correlates with the LCK concentration. Furthermore, multiphoton microscopy experiments demonstrate both the rapid cellular uptake of the inhibitor and that the two-photon cross section of the inhibitor is amenable for excitation at 700 nm

Posicionamiento visual con resolución subpixel de objetos marcados que se desplazan en un plano: conceptos básicos y aplicaciones

Vision is a convenient tool for position measurements. In this paper, we present several applications in which a reference pattern can be defined on the target for a priori knowledge of image features and further optimization by software. Selecting pseudoperiodic patterns leads to high resolution in absolute phase measurements. This method is adapted to position encoding of live cell culture boxes. Our goal is to capture each biological image along with its absolute highly accurate position regarding the culture box itself. Thus, it becomes straightforward to find again an already observed region of interest when a culture box is brought back to the microscope stage from the cell incubator where it was temporarily placed for cell culture. In order to evaluate the performance of this method, we tested it during a wound healing assay of human liver tumor-derived cells. In this case, the procedure enabled more accurate measurements of the wound healing rate than the usual method. It was also applied to the characterization of the in-plane vibration amplitude from a tapered probe of a shear force microscope. The amplitude was interpolated by a quartz tuning fork with an attached pseudo-periodic pattern. Nanometer vibration amplitude resolution is achieved by processing the pattern images. Such pictures were recorded by using a common 20x magnification lens.La visión es una herramienta conveniente para mediciones de posición. En este artículo, presentamos aplicaciones en las que un patrón de referencia puede ser adherido al objeto de interés. Ésto permite tener un conocimiento a priori de las características de la imagen y así poder optimizar el software. Como patrón de referencia se usan patrones pseudo-periódicos, los cuales permiten una alta resolución en las mediciones de fase absoluta. El método es adaptado para codificar la posición de soportes de cultivos celulares, con el fin de documentar cada imagen biológica registrada con su posición absoluta. Por lo tanto, resulta sencillo encontrar de nuevo una región de interés, observada previamente, cuando una caja de cultivo es traída de nuevo al microscopio luego de estar en una incubadora. Para evaluar el método, éste se utilizó durante un ensayo de “cicatrización de herida” de un cultivo celular derivado de tumores hepáticos. En este caso, el método permite obtener mediciones más precisas de la tasa de “cicatrización”, comparado a los resultados obtenidos con el método usual. El método propuesto también se aplica a la caracterización de la amplitud de vibración en el plano de una sonda de un microscopio de fuerza atómica. La amplitud fue interpolada por medio de un diapasón de cuarzo al cual se la adhirió un patrón pseudo-periódico. A partir del procesamiento de las imágenes del patrón, se logra obtener resolución nanométrica en la medida de la amplitud de la vibración. Estas imágenes fueron obtenidas con un microscopio óptico con magnificación 20x

Posicionamiento visual con resolución subpixel de objetos marcados que se desplazan en un plano: conceptos básicos y aplicaciones

Vision is a convenient tool for position measurements. In this paper, we present several applications in which a reference pattern can be defined on the target for a priori knowledge of image features and further optimization by software. Selecting pseudoperiodic patterns leads to high resolution in absolute phase measurements. This method is adapted to position encoding of live cell culture boxes. Our goal is to capture each biological image along with its absolute highly accurate position regarding the culture box itself. Thus, it becomes straightforward to find again an already observed region of interest when a culture box is brought back to the microscope stage from the cell incubator where it was temporarily placed for cell culture. In order to evaluate the performance of this method, we tested it during a wound healing assay of human liver tumor-derived cells. In this case, the procedure enabled more accurate measurements of the wound healing rate than the usual method. It was also applied to the characterization of the in-plane vibration amplitude from a tapered probe of a shear force microscope. The amplitude was interpolated by a quartz tuning fork with an attached pseudo-periodic pattern. Nanometer vibration amplitude resolution is achieved by processing the pattern images. Such pictures were recorded by using a common 20x magnification lens.La visión es una herramienta conveniente para mediciones de posición. En este artículo, presentamos aplicaciones en las que un patrón de referencia puede ser adherido al objeto de interés. Ésto permite tener un conocimiento a priori de las características de la imagen y así poder optimizar el software. Como patrón de referencia se usan patrones pseudo-periódicos, los cuales permiten una alta resolución en las mediciones de fase absoluta. El método es adaptado para codificar la posición de soportes de cultivos celulares, con el fin de documentar cada imagen biológica registrada con su posición absoluta. Por lo tanto, resulta sencillo encontrar de nuevo una región de interés, observada previamente, cuando una caja de cultivo es traída de nuevo al microscopio luego de estar en una incubadora. Para evaluar el método, éste se utilizó durante un ensayo de “cicatrización de herida” de un cultivo celular derivado de tumores hepáticos. En este caso, el método permite obtener mediciones más precisas de la tasa de “cicatrización”, comparado a los resultados obtenidos con el método usual. El método propuesto también se aplica a la caracterización de la amplitud de vibración en el plano de una sonda de un microscopio de fuerza atómica. La amplitud fue interpolada por medio de un diapasón de cuarzo al cual se la adhirió un patrón pseudo-periódico. A partir del procesamiento de las imágenes del patrón, se logra obtener resolución nanométrica en la medida de la amplitud de la vibración. Estas imágenes fueron obtenidas con un microscopio óptico con magnificación 20x

Microfabrication of position reference patterns onto glass microscope slides for high-accurate analysis of dynamic cellular events

Los portaobjetos de microscopio se utilizan ampliamente como sustratos base in situ para la realización de diversos sistemas o elementos microfabricados. Para estos fines, el proceso de microfabricación consiste en transferir un diseño predefinido sobre el sustrato correspondiente a una lámina de vidrio utilizada como portaobjetos de microscopio. Este proceso se conoce como “patterning”, que es una técnica que también se puede utilizar en la transferencia de diseños específicos que permite la recuperación de una región de interés (ROI) bajo el microscopio. En estos casos, aparecen dos desafíos principales: 1) Las perturbaciones en la transmisión de la luz deben permanecer mínimas para mantener la alta calidad de observación del objeto de interés bajo el microscopio. 2) El tamaño del patrón debe ser entonces suficientemente pequeño, pero, sin embargo, mayor que el límite de difracción para ser observable satisfactoriamente para propósitos de posicionamiento. En este artículo presentamos los procedimientos involucrados en la microfabricación de Patrones Pseudo-Periódicos (PPP) los cuales encriptan la posición absoluta de un área extendida. Esos patrones están embebidos en placas de Pétri para permitir la recuperación absoluta y de alta precisión de una ROI, al igual que su orientación. La microfabricación presentada se basa en una técnica conocida como “liftoff” que, tras el ajuste de parámetros, permite la obtención de PPP cumpliendo los dos requisitos anteriormente mencionados. Los resultados corresponden a la realización de PPP en portaobjetos de vidrio y compuesto por puntos laterales de 2μm hechos de aluminio con un grosor de 30nm.Glass microscopes slides are widely used as in situ base-substrates carrying diverse micro-fabricated systems or elements. For such purposes, the micro-fabrication process consists in transferring a pre-defined design onto the substrate made of a glass microscope slide. This is known as patterning, which is a technique that can also be used in transferring specific designs that allows region of interest (ROI) recovery under the microscope. In those cases, two main challenges appear: 1) Disturbances in light transmission should remain minimum to keep the high quality of observation of the object of interest under the microscope. 2) The pattern-size should then be small enough but, however, larger than the diffraction limit to be observable satisfactorily for positioning purposes. In this article, we present the procedures involved in the microfabrication of Pseudo-Periodic Patterns (PPP) encrypting the absolute position of an extended area. Those patterns are embedded in Pétri dishes in order to allow the highaccurate retrieval of absolute position and orientation. The presented microfabrication is based in a technique known as lift-off, which after parameter adjustment, allows the obtaining of PPP fulfilling the two previously mentioned requirements. The results report on PPP realized on glass microscope slides and composed by 2µm side dots made of aluminum with a thickness of 30nm

Position-referenced microscopy for live cell culture monitoring

Position-referenced microscopy (PRM) is based on smart sample holders that integrate a position reference pattern (PRP) in their depth, allowing the determination of the lateral coordinates with respect to the sample-holder itself. Regions of interest can thus be retrieved easily after culture dish transfers from a cell incubator to the microscope stage. Images recorded at different instants in time are superimposed in a common coordinate system with subpixel accuracy. This paper presents such smart Petri culture dishes and their use for live cell culture monitoring. The impact of the PRP on the light budget is discussed and performances are demonstrated. First results on the application of PRM to the observation of apoptotic body internalization are reported

Activation of JNK1 contributes to dystrophic muscle pathogenesis

AbstractDuchenne Muscular Dystrophy (DMD) originates from deleterious mutations in the dystrophin gene, with a complete loss of the protein product [1, 2]. Subsequently, the disease is manifested in severe striated muscle wasting and death in early adulthood [3]. Dystrophin provides a structural base for the assembly of an integral membrane protein complex [4]. As such, dystrophin deficiency leads to an altered mechanical integrity of the myofiber and a predisposition to contraction-induced damage [5–7]. However, the development of myofiber degeneration prior to an observed mechanical defect has been documented in various dystrophic models [8, 9]. Although activation of a detrimental signal transduction pathway has been suggested as a probable cause, a specific cellular cascade has yet to be defined. Here, it is shown that murine models of DMD displayed a muscle-specific activation of JNK1. Independent activation of JNK1 resulted in defects in myotube viability and integrity in vitro, similar to a dystrophic phenotype. In addition, direct muscle injection of an adenoviral construct containing the JNK1 inhibitory protein, JIP1, dramatically attenuated the progression of dystrophic myofiber destruction. Taken together, these results suggest that a JNK1-mediated signal cascade is a conserved feature of dystrophic muscle and contributes to the progression of the disease pathogenesis

Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non- palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC

Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists

Microfabrication of position reference patterns onto glass microscope slides for high-accurate analysis of dynamic cellular events

Glass microscopes slides are widely used as in situ base-substrates carrying diverse micro-fabricated systems or elements

Image-based analysis of living mammalian cells using label-free 3D refractive index maps reveals new organelle dynamics and dry mass flux.

Holo-tomographic microscopy (HTM) is a label-free microscopy method reporting the fine changes of a cell's refractive indices (RIs) in three dimensions at high spatial and temporal resolution. By combining HTM with epifluorescence, we demonstrate that mammalian cellular organelles such as lipid droplets (LDs) and mitochondria show specific RI 3D patterns. To go further, we developed a computer-vision strategy using FIJI, CellProfiler3 (CP3), and custom code that allows us to use the fine images obtained by HTM in quantitative approaches. We could observe the shape and dry mass dynamics of LDs, endocytic structures, and entire cells' division that have so far, to the best of our knowledge, been out of reach. We finally took advantage of the capacity of HTM to capture the motion of many organelles at the same time to report a multiorganelle spinning phenomenon and study its dynamic properties using pattern matching and homography analysis. This work demonstrates that HTM gives access to an uncharted field of biological dynamics and describes a unique set of simple computer-vision strategies that can be broadly used to quantify HTM images