Image-based cell subpopulation identification through automated cell tracking, principal component analysis, and partitioning around medoids clustering.

In vitro cell culture model systems often employ monocultures, despite the fact that cells generally exist in a diverse, heterogeneous microenvironment in vivo. In response, heterogeneous cultures are increasingly being used to study how cell phenotypes interact. However, the ability to accurately identify and characterize distinct phenotypic subpopulations within heterogeneous systems remains a major challenge.

Identifying the mechanism for superdiffusivity in mouse fibroblast motility.

We seek to characterize the motility of mouse fibroblasts on 2D substrates. Utilizing automated tracking techniques, we find that cell trajectories are super-diffusive, where displacements scale faster than t1/2 in all directions. Two mechanisms have been proposed to explain such statistics in other cell types: run and tumble behavior with Lévy-distributed run times, and ensembles of cells with heterogeneous speed and rotational noise.

Value of Wellness Ratings and Countermovement Jumping Velocity to Monitor Performance.

This study examined the relationship between subjective ratings of overall wellness and neuromuscular performance throughout a 6-week intensive offseason strength and conditioning program. Thirty experienced NCAA Division II baseball players completed all phases of the program. A comprehensive wellness rating and 5 countermovement jumps (CMJ5) were measured and averaged for 4 phases of training.

Nuclear position relative to the Golgi body and nuclear orientation are differentially responsive indicators of cell polarized motility.

Cell motility is critical to biological processes from wound healing to cancer metastasis to embryonic development. The involvement of organelles in cell motility is well established, but the role of organelle positional reorganization in cell motility remains poorly understood. Here we present an automated image analysis technique for tracking the shape and motion of Golgi bodies and cell nuclei.

Shape memory activation can affect cell seeding of shape memory polymer scaffolds designed for tissue engineering and regenerative medicine.

The ability of a three-dimensional scaffold to support cell seeding prior to implantation is a critical criterion for many scaffold-based tissue engineering and regenerative medicine strategies. Shape memory polymer functionality may present important new opportunities and challenges in cell seeding, but the extent to which shape memory activation can positively or negatively affect cell seeding has yet to be reported. The goal of this study was to determine whether shape memory activation can affect cell seeding.

On-command on/off switching of progenitor cell and cancer cell polarized motility and aligned morphology via a cytocompatible shape memory polymer scaffold.

In vitro biomaterial models have enabled advances in understanding the role of extracellular matrix (ECM) architecture in the control of cell motility and polarity. Most models are, however, static and cannot mimic dynamic aspects of in vivo ECM remodeling and function. To address this limitation, we present an electrospun shape memory polymer scaffold that can change fiber alignment on command under cytocompatible conditions.

How Bacteria Respond to Material Stiffness during Attachment: A Role of Escherichia coli Flagellar Motility.

Material stiffness has been shown to have potent effects on bacterial attachment and biofilm formation, but the mechanism is still unknown. In this study, response to material stiffness by Escherichia coli during attachment was investigated with biofilm assays and cell tracking using the Automated Contour-base Tracking for in Vitro Environments (ACTIVE) computational algorithm. By comparing the movement of E.

Assembling Enzymatic Cascade Pathways inside Virus-Based Nanocages Using Dual-Tasking Nucleic Acid Tags.

The packaging of proteins into discrete compartments is an essential feature for cellular efficiency. Inspired by Nature, we harness virus-like assemblies as artificial nanocompartments for enzyme-catalyzed cascade reactions. Using the negative charges of nucleic acid tags, we develop a versatile strategy to promote an efficient noncovalent co-encapsulation of enzymes within a single protein cage of cowpea chlorotic mottle virus (CCMV) at neutral pH.

How Escherichia coli lands and forms cell clusters on a surface: a new role of surface topography.

Bacterial response to surface topography during biofilm formation was studied using 5 μm tall line patterns of poly (dimethylsiloxane) (PDMS). Escherichia coli cells attached on top of protruding line patterns were found to align more perpendicularly to the orientation of line patterns when the pattern narrowed. Consistently, cell cluster formation per unit area on 5 μm wide line patterns was reduced by 14-fold compared to flat PDMS.

Generation-dependent templated self-assembly of biohybrid protein nanoparticles around photosensitizer dendrimers.

In this article, we show the great potential of dendrimers for driving the self-assembly of biohybrid protein nanoparticles. Dendrimers are periodically branched macromolecules with a perfectly defined and monodisperse structure. Moreover, they allow the possibility to incorporate functional units at predetermined sites, either at their core, branches, or surface.

Automated, contour-based tracking and analysis of cell behaviour over long time scales in environments of varying complexity and cell density.

Understanding single and collective cell motility in model environments is foundational to many current research efforts in biology and bioengineering. To elucidate subtle differences in cell behaviour despite cell-to-cell variability, we introduce an algorithm for tracking large numbers of cells for long time periods and present a set of physics-based metrics that quantify differences in cell trajectories. Our algorithm, termed automated contour-based tracking for in vitro environments (ACTIVE), was designed for adherent cell populations subject to nuclear staining or transfection.

Self-assembly triggered by self-assembly: optically active, paramagnetic micelles encapsulated in protein cage nanoparticles.

In this contribution, optically active and paramagnetic micelles of the ligand 1,4,7,10-tetraaza-1-(1-carboxymethylundecane)-4,7,10-triacetic acid cyclododecane (DOTAC10) have been incorporated inside capsids of the cowpea chlorotic mottle virus (CCMV) protein through a hierarchical process of self-assembly triggered by self-assembly. The DOTAC10 ligand was used to complex Gd(III), in order to form paramagnetic micelles, as well as to encapsulate an amphiphilic Zn(II) phthalocyanine (ZnPc) dye that optically confirmed the encapsulation of the micelles. The incorporation of ZnPc molecules in the paramagnetic micelles led to high capsid loading of both Gd(III) and ZnPc, as the micelles were stabilized by the amphiphilic dye encapsulation.

Structural transitions and energy landscape for Cowpea Chlorotic Mottle Virus capsid mechanics from nanomanipulation in vitro and in silico.

Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Combined AFM experiments and computational modeling on subsecond timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus capsid show that the capsid's physical properties are dynamic and local characteristics of the structure, which change with the depth of indentation and depend on the magnitude and geometry of mechanical input. Under large deformations, the Cowpea Chlorotic Mottle Virus capsid transitions to the collapsed state without substantial local structural alterations.

Phototriggered cargo release from virus-like assemblies.

There has been tremendous progress towards the development of responsive polymers that are programmed to respond to an external stimulus such as light, pH and temperature. The unique combination of molecular packaging followed by slow, controlled release of molecular cargo is of particular importance for self-healing materials and the controlled release of drugs. While much focus and progress remains centred around synthetic carriers, viruses and virus-like particles can be considered ideal cargo carriers as they are intrinsically designed to package, protect and deliver nucleic acid cargo to host cells.

Supramolecularly oriented immobilization of proteins using cucurbit[8]uril.

A supramolecular strategy is used for oriented positioning of proteins on surfaces. A viologen-based guest molecule is attached to the surface, while a naphthol guest moiety is chemoselectively ligated to a yellow fluorescent protein. Cucurbit[8]uril (CB[8]) is used to link the proteins onto surfaces through specific charge-transfer interactions between naphthol and viologen inside the CB cavity.

Relative size selection of a conjugated polyelectrolyte in virus-like protein structures.

A conjugated polyelectrolyte poly[(2-methoxy-5-propyloxy sulfonate)-phenyl-ene vinylene] (MPS-PPV) drives the assembly of virus capsid proteins to form single virus-like particles (VLPs) and aggregates with more than two VLPs, with a relative selection of high molecular weight polymer in the latter.

Encapsulation of phthalocyanine supramolecular stacks into virus-like particles.

We report herein the encapsulation of a water-soluble phthalocyanine (Pc) into virus-like particles (VLPs) of two different sizes, depending on the conditions. At neutral pH, the cooperative encapsulation/templated assembly of the particles induces the formation of Pc stacks instead of Pc dimers, due to an increased confinement concentration. The Pc-containing VLPs may potentially be used as photosensitizer/vehicle systems for biomedical applications such as photodynamic therapy.

Virus-like particles templated by DNA micelles: a general method for loading virus nanocarriers.

DNA amphiphile particles template formation of virus capsids and enable their loading.

Catheter ablation within the sinus of Valsalva--a safe and effective approach for treatment of atrial and ventricular tachycardias.

Background: Ablation of the aortic sinus of Valsalva in adults for ectopic atrial tachycardia (EAT) and ventricular tachycardia (VT)/premature ventricular complexes (PVCs) has been reported in only a very few patients. Limited data exist concerning the safety of aortic ablation.

Objective: The purpose of this study was to confirm aortic wall and aortic valve integrity after ablation and to evaluate for potential cerebral embolism due to thrombus formation at aortic wall lesions.

A cell-based PDE4 assay in 1536-well plate format for high-throughput screening.

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease.

Interaction between the glutamate transporter GLT1b and the synaptic PDZ domain protein PICK1.

Synaptic plasticity is implemented by the interaction of glutamate receptors with PDZ domain proteins. Glutamate transporters provide the only known mechanism of clearance of glutamate from excitatory synapses, and GLT1 is the major glutamate transporter. We show here that GLT1 interacts with the PDZ domain protein PICK1, which plays a critical role in regulating the expression of glutamate receptors at excitatory synapses.

Improvements in live cell analysis of G protein coupled receptors using second generation BD calcium assay kits.

BD Calcium Assay Kits are designed for cell-based calcium mobilization high-throughput screening assays. The kits use a proprietary formulation including a non-fluorescent calcium indicator that becomes activated inside the cell and shows increased fluorescence upon calcium binding. The formulation includes a signal-enhancing reagent to maximize the signal over background in a homogeneous, no-wash assay format, based on a technology developed at BD.

Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones.

The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame.

Feasibility of genome-scale construction of promoter::reporter gene fusions for expression in Caenorhabditis elegans using a multisite gateway recombination system.

The understanding of gene function increasingly requires the characterization of DNA segments containing promoters and their associated regulatory sequences. We describe a novel approach for linking multiple DNA segments, here applied to the generation of promoter::reporter fusions. Promoters from Caenorhabditis elegans genes were cloned using the MultiSite Gateway cloning technology.