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.

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.

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

Increasingly, in vitro culture of adherent cell types utilizes three-dimensional (3D) scaffolds or aggregate culture strategies to mimic tissue-like, microenvironmental conditions. In parallel, new flow cytometry-based technologies are emerging to accurately analyze the composition and function of these microtissues (i.e., large particles) in a non-invasive and high-throughput way. Lacking, however, is an accessible platform that can be used to effectively sort or purify large particles based on analysis parameters. Here we describe a microfluidic-based, electromechanical approach to sort large particles. Specifically, sheath-less asymmetric curving channels were employed to separate and hydrodynamically focus particles to be analyzed and subsequently sorted. This design was developed and characterized based on wall shear stress, tortuosity of the flow path, vorticity of the fluid in the channel, sorting efficiency and enrichment ratio. The large particle sorting device was capable of purifying fluorescently labelled embryoid bodies (EBs) from unlabelled EBs with an efficiency of 87.3% 6 13.5%, and enrichment ratio of 12.2 6 8.4 (n ¼ 8), while preserving cell viability, differentiation potential, and long-term function. V C 2012 American Institute of Physics. [http://dx