Overcoming clinical BCR-ABL1 compound mutant resistance with combined ponatinib and asciminib therapy.

BCR-ABL1 compound mutations can lead to resistance to ABL1 inhibitors in chronic myeloid leukemia (CML), which could be targeted by combining the ATP-site inhibitor ponatinib and the allosteric inhibitor asciminib. Here, we report the clinical validation of this approach in a CML patient, providing a basis for combination therapy to overcome such resistance.

Molecular-Scale Dynamics of Long Range Retrograde Brain-Derived Neurotrophic Factor Transport Shaped by Cellular Spatial Context.

Retrograde neurotrophin (NT) transport is a specialized form of signal transduction used to conduct information from axons to the cell bodies of central and peripheral nervous system neurons. It is activated upon NT-Trk receptor binding, NT-Trk internalization into signaling endosomes, and their motion along the axon toward the cell body. Brain-derived neurotrophic factor (BDNF) is an abundant NT that modulates key brain and spinal cord functions, and defects in BDNF trafficking are associated with neuronal death, neurodegenerative diseases and in nerve injury.

Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques.

Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present.

Multiplexed imaging reveals heterogeneity of PI3K/MAPK network signaling in breast lesions of known PIK3CA genotype.

Purpose: Activating genetic changes in the phosphatidylinositol-3-kinase (PI3K) signaling pathway are found in over half of invasive breast cancers (IBCs). Previously, we discovered numerous hotspot PIK3CA mutations in proliferative breast lesions. Here, we investigate the spatial nature of PI3K pathway signaling and its relationship with PI3K genotype in breast lesions.

Ultrasensitive proteomic quantitation of cellular signaling by digitized nanoparticle-protein counting.

Many important signaling and regulatory proteins are expressed at low abundance and are difficult to measure in single cells. We report a molecular imaging approach to quantitate protein levels by digitized, discrete counting of nanoparticle-tagged proteins. Digitized protein counting provides ultrasensitive molecular detection of proteins in single cells that surpasses conventional methods of quantitating total diffuse fluorescence, and offers a substantial improvement in protein quantitation.

Quantum dots for quantitative imaging: from single molecules to tissue.

Since their introduction to biological imaging, quantum dots (QDs) have progressed from a little known, but attractive, technology to one that has gained broad application in many areas of biology. The versatile properties of these fluorescent nanoparticles have allowed investigators to conduct biological studies with extended spatiotemporal capabilities that were previously not possible. In this review, we focus on QD applications that provide enhanced quantitative information concerning protein dynamics and localization, including single particle tracking and immunohistochemistry, and finish by examining the prospects of upcoming applications, such as correlative light and electron microscopy and super-resolution.

Heterogeneous intracellular trafficking dynamics of brain-derived neurotrophic factor complexes in the neuronal soma revealed by single quantum dot tracking.

Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells.

Tracing the endocytic pathways and trafficking kinetics of cell signaling receptors using single QD nanoparticles.

Cellular signaling is the fundamental process through which cells communicate with each other and respond to their environment. Regulation of this cellular signaling is crucial for healthy cellular function. Malfunctions in signaling are the cause for many diseases and disorders and therefore are under heavy investigation.

Kinetics of G-protein-coupled receptor endosomal trafficking pathways revealed by single quantum dots.

G-protein-coupled receptors (GPCRs) are the largest protein superfamily in the human genome; they comprise 30% of current drug targets and regulate diverse cellular signaling responses. The role of endosomal trafficking in GPCR signaling regulation is gaining substantial consideration. However, this process remains difficult to study due to the inability to distinguish among many individual receptors, simultaneously trafficking within multiple endosomal pathways.

Spatial structure and diffusive dynamics from single-particle trajectories using spline analysis.

Single-particle tracking of biomolecular probes has provided a wealth of information about intracellular trafficking and the dynamics of proteins and lipids in the cell membrane. Conventional mean-square displacement (MSD) analysis of single-particle trajectories often assumes that probes are moving in a uniform environment. However, the observed two-dimensional motion of probe particles is influenced by the local three-dimensional geometry of the cell membrane and intracellular structures, which are rarely flat at the submicron scale.

Nanotechnology research: applications in nutritional sciences.

The tantalizing potential of nanotechnology is to fabricate and combine nanoscale approaches and building blocks to make useful tools and, ultimately, interventions for medical science, including nutritional science, at the scale of approximately 1-100 nm. In the past few years, tools and techniques that facilitate studies and interventions in the nanoscale range have become widely available and have drawn widespread attention. Recently, investigators in the food and nutrition sciences have been applying the tools of nanotechnology in their research.

Quantum dot hybrid gel blotting: a technique for identifying quantum dot-protein/protein-protein interactions.

We describe an alternative to the molecular biology technique of polyacrylamide gel electrophoresis-based Western blotting and immunoprecipitation, which is an extensively used method for separating target proteins from complex cellular mixtures and for identification of protein expression and protein-protein interactions. This novel method, called quantum dot (QD) hybrid gel blotting, allows the purification and analysis of the action of QD bioconjugate-protein complexes in live cells. Moreover, these identified interactions can be correlated with spatial location in cells.

Single particle quantum dot imaging achieves ultrasensitive detection capabilities for Western immunoblot analysis.

Substantially improved detection methods are needed to detect fractionated protein samples present at trace concentrations in complex, heterogeneous tissue and biofluid samples. Here we describe a modification of traditional Western immunoblotting using a technique to count quantum-dot-tagged proteins on optically transparent PVDF membranes. Counts of quantum-dot-tagged proteins on immunoblots achieved optimal detection sensitivity of 0.

Quantitative analysis of multivesicular bodies (MVBs) in the hypoglossal nerve: evidence that neurotrophic factors do not use MVBs for retrograde axonal transport.

Multivesicular bodies (MVBs) are defined by multiple internal vesicles enclosed within an outer, limiting membrane. MVBs have previously been quantified in neuronal cell bodies and in dendrites, but their frequencies and significance in axons are controversial. Despite lack of conclusive evidence, it is widely believed that MVBs are the primary organelle that carries neurotrophic factors in axons.

Ligand-bound quantum dot probes for studying the molecular scale dynamics of receptor endocytic trafficking in live cells.

Endocytic receptor trafficking is a complex, dynamic process underlying fundamental cell function. An integrated understanding of endocytosis at the level of single or small numbers of ligand bound-receptor complexes inside live cells is currently hampered by technical limitations. Here, we develop and test ligand nerve growth factor-bound quantum dot (NGF-QD) bioconjugates for imaging discrete receptor endocytic events inside live NGF-responsive PC12 cells.

Distinct extracellular matrix microenvironments of progenitor and carotid endothelial cells.

Endothelial cells (ECs) produce and maintain the local extracellular matrix (ECM), a critical function that contributes to EC and blood vessel health. This function is also crucial to vascular tissue engineering, where endothelialization of vascular constructs require a cell source that readily produces and maintains ECM. In this study, baboon endothelial progenitor cell (EPC) deposition of ECM (laminin, collagen IV, and fibronectin) was characterized and compared to mature carotid ECs, evaluated in both elongated and cobblestone morphologies typically found in vivo.

Microcontact printing of quantum dot bioconjugate arrays for localized capture and detection of biomolecules.

The nanometer size scale of quantum dots (QDs) along with their unique luminescent properties offers great potential as photostable, color-metrically addressable nanoparticle platforms for high-throughput detection and identification of proteins. Here we apply microcontact printing for assembling quantum dot nanoparticle arrays with retained biomolecular capture functionality onto glass surfaces. This method allows the creation of addressable QD arrays on macroscopic glass surfaces.

Identification of quantum dot bioconjugates and cellular protein co-localization by hybrid gel blotting.

New approaches are needed to address the interaction of nanoparticles and cellular proteins at the molecular level. We present a modification of PAGE co-immunoprecipitation, QD-based PA-AGE electrophoresis blotting, and apply this to identify quantum dot (QD) bioconjugate-cellular protein association. This method provides the capability to isolate and evaluate the action of QD bioconjugate-protein complexes in intact cells and to correlate these identified interactions with their location in cells.

Quantum dots monitor TrkA receptor dynamics in the interior of neural PC12 cells.

Can quantum dots (QDs) serve as physiologically relevant receptor probes in the interior of cells? We directly visualize endocytosis, redistribution, and shuttling of QD bound-TrkA receptors to PC12 neural processes and far-reaching growth cone tips. Internalized QDs are contained in microtubule-associated vesicles and possess transport properties that reflect TrkA receptor dynamics. This opens up new possibilities for the development of QD platforms as molecular tools to image biochemical signaling and transport cargo in the cell interior.

Stoichiometry-dependent formation of quantum dot-antibody bioconjugates: a complementary atomic force microscopy and agarose gel electrophoresis study.

The unique advantages of quantum dot (QD) bioconjugates have motivated their application in biological assays. However, physical characterization of bioconjugated QDs after surface modification has often been overlooked. Here, biotinylated antibodies (biotin-IgG) were attached to commercial streptavidin-conjugated quantum dots (strep-QDs) at different stoichiometric ratios, and these QD bioconjugates were characterized with atomic force microscopy and discontinuous sodium dodecyl sulfate agarose gel electrophoresis (SDS-AGE).

Peptide-conjugated quantum dots activate neuronal receptors and initiate downstream signaling of neurite growth.

Quantum dots (QDs) could serve as fluorescent scaffolds for effecting specific physiological and pharmacological responses in cells. Here, we conjugate the peptide ligand betaNGF to QD surfaces, and confirm surface modification and single QD nanostructure using AFM. We show that betaNGF-QDs retain bioactivity, activate TrkA receptors, and initiate neuronal differentiation in PC12 cells.

Activation of membrane receptors by a neurotransmitter conjugate designed for surface attachment.

The derivatization of surfaces with bioactive molecules is a research area of growing importance for cell and tissue engineering. Tetherable molecules used in such applications must contain an anchoring moiety as well as the biofunctional group, typically along with a spacer to prevent steric clashes between the target molecule and the tethering surface. Post-synaptic membrane receptors at chemical synapses in neural tissue mediate signaling to the post-synaptic neuron and are activated by the binding of diffusible neurotransmitter molecules released by the pre-synaptic neuron.

Neurotransmitter analog tethered to a silicon platform for neuro-BioMEMS applications.

The design of chemically well-defined, machinable surfaces containing neuroactive molecules offers potential for fundamental neuroscience and clinical neural engineering applications. Here we report the assembly and characterization of silicon platforms containing a tethered form of muscimol. Muscimol, an analog of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), is a potent agonist at postsynaptic GABA(A) and GABA(C) receptors.

Assembly and characterization of biofunctional neurotransmitter-immobilized surfaces for interaction with postsynaptic membrane receptors.

Herein, we report progress toward the development of bioactive surfaces based on gamma-aminobutyric acid (GABA), a major neurotransmitter in the nervous system. Whereas immobilization techniques have focused largely on antibodies, enzymes, and receptors, to our knowledge, this is the first report of a prototype neurotransmitter-immobilized surface. Biosurfaces were assembled onto either mica or glass using passive adsorption of avidin and subsequent attachment of a derivatized form of GABA via a biotin-avidin affinity bond.