Benjamin L. Cox

3D Collagen Alignment Limits Protrusions to Enhance Breast Cancer Cell Persistence

Patients with mammographically dense breast tissue have a greatly increased risk of developing breast cancer. Dense breast tissue contains more stromal collagen, which contributes to increased matrix stiffness and alters normal cellular responses. Stromal collagen within and surrounding mammary tumors is frequently aligned and reoriented perpendicular to the tumor boundary. We have shown that aligned collagen predicts poor outcome in breast cancer patients, and postulate this is because it facilitates invasion by providing tracks on which cells migrate out of the tumor. However, the mechanisms by which alignment may promote migration are not understood. Here, we investigated the contribution of matrix stiffness and alignment to cell migration speed and persistence. Mechanical measurements of the stiffness of collagen matrices with varying density and alignment were compared with the results of a 3D microchannel alignment assay to quantify cell migration. We further interpreted the experimental results using a computational model of cell migration. We find that collagen alignment confers an increase in stiffness, but does not increase the speed of migrating cells. Instead, alignment enhances the efficiency of migration by increasing directional persistence and restricting protrusions along aligned fibers, resulting in a greater distance traveled. These results suggest that matrix topography, rather than stiffness, is the dominant feature by which an aligned matrix can enhance invasion through 3D collagen matrices.

Strategies from UW-Madison for rescuing biomedical research in the US

A cross-campus, cross-career stage and cross-disciplinary series of discussions at a large public university has produced a series of recommendations for addressing the problems confronting the biomedical research community in the US. DOI: http://dx.doi.org/10.7554/eLife.09305.001

Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination

The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 recA gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism in vivo. The growth deficiency is alleviated by expression of the functionally robust RecX protein from Neisseria gonorrhoeae. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism.

Multi-view second-harmonic generation imaging of mouse tail tendon via reflective micro-prisms

Here we experimentally show that second-harmonic generation (SHG) imaging is not sensitive to collagen fibers oriented parallel to the direction of laser propagation and, as a consequence, can potentially miss important structural information. As an alternative approach, we demonstrate the use of reflective micro-prisms to enable multi-view SHG imaging of mouse tail tendon by redirecting the focused excitation and collection of subsequent emission. Our approach data corroborates the theoretical treatment on vanishing and nonvanishing orientations, where fibers along the laser direction are largely transparent by SHG. In strong contrast, the two-photon excited fluorescence of dye-labeled collagen fibers is isotropic and is not subject to this constraint. We utilized Pearson correlation to quantify differences in fluorescent and backward detected SHG images of the tendon fiber structure, where the SHG and TPEF were highly statistically correlated (0.6-0.8) for perpendicular excitation but were uncorrelated for excitation parallel to the fiber axis. The results suggest that improved imaging of 3D collagen structure is possible with multi-view SHG microscopy.

An open source, 3D printed preclinical MRI phantom for repeated measures of contrast agents and reference standards

In medical imaging, clinicians, researchers and technicians have begun to use 3D printing to create specialized phantoms to replace commercial ones due to their customizable and iterative nature. Presented here is the design of a 3D printed open source, reusable magnetic resonance imaging (MRI) phantom, capable of flood-filling, with removable samples for measurements of contrast agent solutions and reference standards, and for use in evaluating acquisition techniques and image reconstruction performance. The phantom was designed using SolidWorks, a computer-aided design software package. The phantom consists of custom and off-the-shelf parts and incorporates an air hole and Luer Lock system to aid in flood filling, a marker for orientation of samples in the filled mode and bolt and tube holes for assembly. The cost of construction for all materials is under

Vacuum Seed Sowing Manifold: a novel device for high-throughput sowing of Arabidopsis seeds

90. All design files are open-source and available for download. To demonstrate utility, B0 field mapping was performed using a series of gadolinium concentrations in both the unfilled and flood-filled mode. An excellent linear agreement (R2>0.998) was observed between measured relaxation rates (R1/R2) and gadolinium concentration. The phantom provides a reliable setup to test data acquisition and reconstruction methods and verify physical alignment in alternative nuclei MRI techniques (e.g. carbon-13 and fluorine-19 MRI). A cost-effective, open-source MRI phantom design for repeated quantitative measurement of contrast agents and reference standards in preclinical research is presented. Specifically, the work is an example of how the emerging technology of 3D printing improves flexibility and access for custom phantom design.

3D second harmonic generation imaging tomography by multi-view excitation

The small size of Arabidopsis provides both opportunities and difficulties for laboratory research. Large numbers of plants can be grown in a relatively small area making it easy to observe and investigate interesting phenotypes. Conversely, their small size can also make it difficult to obtain large quantities of tissue for investigation using modern molecular techniques. Sowing large numbers of their seed can overcome this; however, their small seed size makes this difficult. Here we present the Vacuum Seed Sowing Manifold (VSSM), a simple device that can be printed using a 3D printer and provides a new high throughput method to sow large numbers of seeds at a range of densities.

Fabrication approaches for the creation of physical models from microscopy data

Biological tissues have complex 3D collagen fiber architecture that cannot be fully visualized by conventional second harmonic generation (SHG) microscopy due to electric dipole considerations. We have developed a multi-view SHG imaging platform that successfully visualizes all orientations of collagen fibers. This is achieved by rotating tissues relative to the excitation laser plane of incidence, where the complete fibrillar structure is then visualized following registration and reconstruction. We evaluated high frequency and Gaussian weighted fusion reconstruction algorithms, and found the former approach performs better in terms of the resulting resolution. The new approach is a first step toward SHG tomography.

SU‐E‐J‐187: Single‐Cell Dosimetric Simulation and Evaluation of Sb‐119 for Use as Auger Emitting Nuclide in Targeted Radiotherapy

BackgroundThree-dimensional (3D) printing has become a useful method of fabrication for many clinical applications. It is also a technique that is becoming increasingly accessible, as the price of the necessary tools and supplies decline. One emerging, and unreported, application for 3D printing is to aid in the visualization of 3D imaging data by creating physical models of select structures of interest.MethodsPresented here are three physical models that were fabricated from three different 3D microscopy datasets. Different methods of fabrication and imaging techniques were used in each case.ResultsEach model is presented in detail. This includes the imaging modality used to capture the raw data, the software used to create any computer models and the 3D printing tools used to create each model. Despite the differences in their creation, these examples follow a simple common workflow that is also detailed.ConclusionsFollowing these approaches, one can easily make 3D printed models from 3D microscopy datasets utilizing off the shelf commercially available software and hardware.

WE‐E‐108‐07: Design of Open‐Source Binary Micro MLC for Small Animal Radiotherapy: An OSMD Initiative

Purpose: Auger electrons have strong potential for targeted radiotherapy applications because of their relatively short ranged dose profile. 119Sb in particular shows promise as a radioisotope for this application because of its electron emissions with energies near 20 keV. A stochastic characterization of 119Sb and comparison to other auger emitting nuclides has been made to demonstrate this advantage. Methods: A Monte Carlo electron transport code has been written in Python to model the dose to a single cell for activity distributions within the cell membrane, cellular cytoplasm, and nuclear cytoplasm. A spherical geometry has been used, where energy is deposited in shells about the cell center. An extrapolated power fit has been used to model low‐energy electron stopping powers under a continual slowing down approximation. S‐values for dose to the nucleus were calculated from simulation results and were compared to previous analytic results (Thisgaard et. al., Med. Phys. 35, 2008). Finally, the results from this code were compared to other auger electron transport Monte Carlo codes for dosimetric verification. Results: For a cell and nucleus of radius 8μm and 6μm respectively, calculated 119Sb S‐values for activity distributions were 4.10E‐4 Gy/(s Bq) in the cell membrane, 5.93E‐4 Gy/(s Bq) in the cytoplasm, and 2.06E‐3 Gy/(s Bq) within the nucleus. These values are within 13% of S‐values calculated analytically. An explanation for this difference is the omission of CK electrons from this simulation. S‐values calculated for 123I compared with other auger electron transport codes typically agree within a 10% margin. Conclusion: Simulated 119Sb dose distributions have been shown to agree with analytic calculations. The ability of Auger electrons to localize dose to a single cell may lead to a significant dose reduction in healthy tissue. With the development of proper production and chelation techniques, 119Sb shows promise as a radiotherapeutic nuclide. This research was supported by the US Department of Energy: DE‐FG02‐12ER41882