Light-induced modulation of viscoelastic properties in azobenzene polymers.

Photo-induced isomerization of azobenzene molecules drives mass migrations in azopolymer samples. The resulting macroscopic directional photo-deformation of the material morphology has found many applications in literature, although the fundamental mechanisms behind this mass transfer are still under debate. Hence, it is of paramount importance to find quantitative observables that could drive the community toward a better understanding of this phenomenon. In this regard, azopolymer mechanical properties have been intensively studied, but the lack of a nanoscale technique capable of quantitative viscoelastic measurements has delayed the progress in the field. Here, we use bimodal atomic force microscopy (AFM) as a powerful technique for nanomechanical characterizations of azopolymers. With this multifrequency AFM approach, we map the azopolymer local elasticity and viscosity, with high resolution, after irradiation. We find that, while in the (previously) illuminated region, a general photo-softening is measured; locally, the Young modulus and the viscosity depend upon the inner structuring of the illuminating light spot. We then propose a possible interpretation based on a light-induced expansion plus a local alignment of the polymer chains (directional hole-burning effect), which explains the experimental observations. The possibility to access, in a reliable and quantitative way, both Young modulus and viscosity could trigger new theoretical-numerical investigations on the azopolymer mass migration dynamics since, as we show, both parameters can be considered measurable. Furthermore, our results provide a route for engineering the nanomechanical properties of azopolymers, which could find interesting applications in cell mechanobiology research.

Genomic and molecular evolutionary dynamics of transcriptional response regulator genes in bacterial species of the Harveyi clade of Vibrio.

Transcriptional response regulators (TRR) are the most abundant signal transducers in prokaryotic systems that mediate intracellular changes in response to environmental signals. They are involved in a wide range of biological processes that allow bacteria to persist in particular habitats. There is strong evidence that the bacterial habitat and their lifestyle influence the size of their TRR genetic repertoire. Therefore, it would be expected that the evolution of bacterial genomes could be linked to natural selection processes. To test this hypothesis, we explored the evolutionary dynamics of TRR genes of the widely studied Harveyi clade of the genus Vibrio at the molecular and genomic levels. Our results suggest that the TRR genetic repertoire of the species belonging to the Harveyi clade is a product of genomic reduction and expansion. The gene loss and gains that drive their genomic reduction and expansion could be attributed to natural selection and random genetic drift. It seems that natural selection acts to maintain the ancestral state of core TRR genes (shared by all species) by purifying processes and could be driving the loss of some accessory (found in certain species) genes through the diversification of sequences. The neutrality observed in gene gain could be attributed to spontaneous events as horizontal gene transfer driven by stochastic events as occurs in random genetic drift.

Bottom Effect in Atomic Force Microscopy Nanomechanics.

In this work, the influence of the rigid substrate on the determination of the sample Young's modulus, the so-called bottom-effect artifact, is demonstrated by an atomic force microscopy force-spectroscopy experiment. The nanomechanical properties of a one-component supported lipid membrane (SLM) exhibiting areas of two different thicknesses are studied: While a standard contact mechanics model (Sneddon) provides two different elastic moduli for these two morphologies, it is shown that Garcia's bottom-effect artifact correction yields a unique value, as expected for an intrinsic material property. Remarkably, it is demonstrated that the ratio between the contact radius (and not only the indentation) and the sample thickness is the key parameter addressing the relevance of the bottom-effect artifact. The experimental results are validated by finite element method simulations providing a solid support to Garcia's theory. The amphiphilic nature of the investigated material is representative of several kinds of lipids, suggesting that the results have far reaching implications for determining the correct Young's modulus of SLMs. The generality of Garcia's bottom-effect artifact correction allows its application to every kind of supported soft film.

Nanorheology of living cells measured by AFM-based force-distance curves.

Mechanobiology aims to establish functional relationships between the mechanical state of a living a cell and its physiology. The acquisition of force-distance curves with an AFM is by far the dominant method to characterize the nanomechanical properties of living cells. However, theoretical simulations have shown that the contact mechanics models used to determine the Young's modulus from a force-distance curve could be off by a factor 5 from its expected value. The semi-quantitative character arises from the lack of a theory that integrates the AFM data, a realistic viscoelastic model of a cell and its finite-thickness. Here, we develop a method to determine the mechanical response of a cell from a force-distance curve. The method incorporates bottom-effect corrections, a power-law rheology model and the deformation history of the cell. It transforms the experimental data into viscoelastic parameters of the cell as a function of the indentation frequency. The quantitative agreement obtained between the experiments performed on living fibroblast cells and the analytical theory supports the use of force-distance curves to measure the nanorheological properties of cells.

Subsurface Imaging of Cell Organelles by Force Microscopy.

The development of high-resolution, label-free, noninvasive, and subsurface microscopy methods of living cells remains a formidable problem. Force-microscopy-based stiffness measurements contribute to our understanding of single-cell nanomechanics. The elastic properties of the cell's outer structures, such as the plasma membrane and actin cytoskeleton, dominate stiffness measurements, which in turns prevents the imaging of intracellular structures. We propose that the above limitation could be overcome by combining 2D sections of the cell's viscoelastic properties. We show the simultaneous imaging of the outer cell's cytoskeleton and the organelles inside the nucleus. The elastic component of interaction force carries information on the cell's outer elements as the cortex and the actin cytoskeleton. The inelastic component is sensitive to the hydrodynamic drag of the inner structures such the nucleoli.

Determination of the viscoelastic properties of a single cell cultured on a rigid support by force microscopy.

Understanding the relationship between the mechanical properties of living cells and physiology is a central issue in mechanobiology. Mechanical properties are used as fingerprints of the pathological state of a single cell. The force exerted on a cell is influenced by the stiffness of the solid support needed to culture it. This effect is a consequence of the cell's boundary conditions. It causes a cell to appear with mechanical properties different from their real values. Here we develop a bottom effect viscoelastic theory to determine the viscoelastic response of a cell. The theory transforms a force-distance curve into the cell's Young's modulus, loss modulus, relaxation time or viscosity coefficient with independence of the stiffness of the rigid support. The theory predicts that, for a given indentation, the force exerted on the cell's periphery will be larger than on a perinuclear region. Results based on the use of semi-infinite contact mechanics models introduce large numerical errors in the determination of the mechanical properties. Finite element simulations confirm the theory and define its range of applicability.

Determination of the Elastic Moduli of a Single Cell Cultured on a Rigid Support by Force Microscopy.

The elastic response of a living cell is affected by its physiological state. This property provides mechanical fingerprints of a cell's dysfunctionality. The softness (kilopascal range) and thickness (2-15 µm) of mammalian cells imply that the force exerted by the probe might be affected by the stiffness of the solid support. This observation makes infinite sample thickness models unsuitable to describe quantitatively the forces and deformations on a cell. Here, we report a general theory to determine the true Young's moduli of a single cell from a force-indentation curve. Analytical expressions are deduced for common geometries such as flat punches, paraboloids, cones, needles, and nanowires. For a given cell and indentation, the influence of the solid support on the measurements is reduced by using sharp and high aspect ratio tips. The theory is validated by finite element simulations.

Time-resolved nanomechanics of a single cell under the depolymerization of the cytoskeleton.

Single cell stiffness measurements consider cells as passive and elastic materials which react instantaneously to an external force. This approximation is at odds with the complex structure of the cell which includes solid and liquid components. Here we develop a force microscopy method to measure the time and frequency dependencies of the elastic modulus, the viscosity coefficient, the loss modulus and the relaxation time of a single live cell. These parameters have different time and frequency dependencies. At low modulation frequencies (0.2-4 Hz), the elastic modulus remains unchanged; the loss modulus increases while the viscosity and the relaxation time decrease. We have followed the evolution of a fibroblast cell subjected to the depolymerization of its F-actin cytoskeleton. The elastic modulus, the loss modulus and the viscous coefficient decrease with the exposure time to the depolymerization drug while the relaxation time increases. The latter effect reflects that the changes in the elastic response happen at a higher rate than those affecting the viscous flow. The observed behavior is compatible with a cell mechanical response described by the poroelastic model.

Identification of N-(4-((1R,3S,5S)-3-Amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide (PIM447), a Potent and Selective Proviral Insertion Site of Moloney Murine Leukemia (PIM) 1, 2, and 3 Kinase Inhibitor in Clinical Trials for Hematological Malignancies.

Pan proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently begun to be tested in humans to assess whether pan PIM kinase inhibition may provide benefit to cancer patients. Herein, the synthesis, in vitro activity, in vivo activity in an acute myeloid leukemia xenograft model, and preclinical profile of the potent and selective pan PIM kinase inhibitor compound 8 (PIM447) are described. Starting from the reported aminopiperidyl pan PIM kinase inhibitor compound 3, a strategy to improve the microsomal stability was pursued resulting in the identification of potent aminocyclohexyl pan PIM inhibitors with high metabolic stability. From this aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several phase 1 trials of cancer patients with hematological malignancies.

Dynamic force microscopy simulator (dForce): A tool for planning and understanding tapping and bimodal AFM experiments.

We present a simulation environment, dForce, which can be used for a better understanding of dynamic force microscopy experiments. The simulator presents the cantilever-tip dynamics for two dynamic AFM methods, tapping mode AFM and bimodal AFM. It can be applied for a wide variety of experimental situations in air or liquid. The code provides all the variables and parameters relevant in those modes, for example, the instantaneous deflection and tip-surface force, velocity, virial, dissipated energy, sample deformation and peak force as a function of time or distance. The simulator includes a variety of interactions and contact mechanics models to describe AFM experiments including: van der Waals, Hertz, DMT, JKR, bottom effect cone correction, linear viscoelastic forces or the standard linear solid viscoelastic model. We have compared two numerical integration methods to select the one that offers optimal accuracy and speed. The graphical user interface has been designed to facilitate the navigation of non-experts in simulations. Finally, the accuracy of dForce has been tested against numerical simulations performed during the last 18 years.

Pan-PIM kinase inhibition provides a novel therapy for treating hematologic cancers.

PURPOSE: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies. EXPERIMENTAL DESIGN: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low Km for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context. RESULTS: LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model. CONCLUSIONS: We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies.

Pim2 is required for maintaining multiple myeloma cell growth through modulating TSC2 phosphorylation.

Multiple myeloma (MM) is the second most common hematologic malignancy. Despite recent treatment advances, it remains incurable. Here, we report that Pim2 kinase expression is highly elevated in MM cells and demonstrate that it is required for MM cell proliferation. Functional interference of Pim2 activity either by short hairpin RNAs or by a potent and selective small-molecule inhibitor leads to significant inhibition of MM cell proliferation. Pim inhibition results in a significant decrease of mammalian target of rapamycin C1 (mTOR-C1) activity, which is critical for cell proliferation. We identify TSC2, a negative regulator of mTOR-C1, as a novel Pim2 substrate and show that Pim2 directly phosphorylates TSC2 on Ser-1798 and relieves the suppression of TSC2 on mTOR-C1. These findings support Pim2 as a promising therapeutic target for MM and define a novel Pim2-TSC2-mTOR-C1 pathway that drives MM proliferation.

Structure Guided Optimization, in Vitro Activity, and in Vivo Activity of Pan-PIM Kinase Inhibitors.

Proviral insertion of Moloney virus (PIM) 1, 2, and 3 kinases are serine/threonine kinases that normally function in survival and proliferation of hematopoietic cells. As high expression of PIM1, 2, and 3 is frequently observed in many human malignancies, including multiple myeloma, non-Hodgkins lymphoma, and myeloid leukemias, there is interest in determining whether selective PIM inhibition can improve outcomes of these human cancers. Herein, we describe our efforts toward this goal. The structure guided optimization of a singleton high throughput screening hit in which the potency against all three PIM isoforms was increased >10,000-fold to yield compounds with pan PIM K is < 10 pM, nanomolar cellular potency, and in vivo activity in an acute myeloid leukemia Pim-dependent tumor model is described.

Discovery of novel 3,5-disubstituted indole derivatives as potent inhibitors of Pim-1, Pim-2, and Pim-3 protein kinases.

A series of novel 3,5-disubstituted indole derivatives as potent and selective inhibitors of all three members of the Pim kinase family is described. High throughput screen identified a pan-Pim kinase inhibitor with a promiscuous scaffold. Guided by structure-based drug design, SAR of the series afforded a highly selective indole chemotype that was further developed into a potent set of compounds against Pim-1, 2, and 3 (Pim-1 and Pim-3: IC(50)≤2nM and Pim-2: IC(50)≤100nM).

beta-Catenin regulates vascular endothelial growth factor expression in colon cancer.

To evaluate whether beta-catenin signaling has a role in the regulation of angiogenesis in colon cancer, a series of angiogenesis-related gene promoters was analyzed for beta-catenin/TCF binding sites. Strikingly, the gene promoter of human vascular endothelial growth factor (VEGF, or VEGF-A) contains seven consensus binding sites for beta-catenin/TCF. Analysis of laser capture microdissected human colon cancer tissue indicated a direct correlation between up-regulation of VEGF-A expression and adenomatous polyposis coli (APC) mutational status (activation of beta-catenin signaling) in primary tumors. In metastases, this correlation was not observed. Analysis by immunohistochemistry of intestinal polyps in mice heterozygous for the multiple intestinal neoplasia gene (Min/+) at 5 months revealed an increase and redistribution of VEGF-A in proximity to those cells expressing nuclear beta-catenin with a corresponding increase in vessel density. Transfection of normal colon epithelial cells with activated beta-catenin up-regulated levels of VEGF-A mRNA and protein by 250-300%. When colon cancer cells with elevated beta-catenin levels were treated with beta-catenin antisense oligodeoxynucleotides, VEGF-A expression was reduced by more than 50%. Taken together, our observations indicate a close link between beta-catenin signaling and the regulation of VEGF-A expression in colon cancer.