An optimized 3D inkjet printing process is demonstrated for structuring alginate into a tissue-like microvasculature capable of supporting physiological flow rates. Optimizing the reaction at the single-droplet level enables wet hydrogel droplets to be stacked, thus overcoming their natural tendancy to spread and coalesce. Live cells can be patterned using this process and it can be extended to a range of other hydrogels.

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53BP1, the vertebrate ortholog of the budding yeast Rad9 and fission yeast Crb2/Rhp9 checkpoint proteins, is recruited rapidly to sites of DNA double-strand breaks (DSBs). A tandem tudor domain in human 53BP1 that recognizes methylated residues in the histone core is necessary, but not sufficient, for efficient recruitment. By analysis of deletion mutants, we identify here additional elements in 53BP1 that facilitate recognition of DNA DSBs. The first element corresponds to an independently folding oligomerization domain. Replacement of this domain with heterologous tetramerization domains preserves the ability of 53BP1 to recognize DNA DSBs. A second element is only about 15 amino acids long and appears to be a C-terminal extension of the tudor domain, rather than an independently functioning domain. Recruitment of 53BP1 to sites of DNA DSBs is facilitated by histone H2AX phosphorylation and ubiquitination. However, none of the 53BP1 domains/ elements important for recruitment are known to bind phosphopeptides or ubiquitin, suggesting that histone phosphorylation and ubiquitination regulate 53BP1 recruitment to sites of DNA DSBs indirectly.Monitoring the presence of DNA double-strand breaks (DSBs) is critical for maintaining genomic stability. In eukaryotes, the DNA DSB checkpoint pathway senses the presence of DNA DSBs and activates effectors that induce cell cycle arrest, apoptosis, or senescence. Key components of this pathway in human cells are DNA DSB sensors such as 53BP1 and the Mre11-Rad50-NBS1 complex, the signal transducing kinase ATM, and effectors downstream of ATM such as the kinase Chk2 and the transcription factor p53 (1,16,25).53BP1 is one of the DNA damage response proteins that is recruited very efficiently to sites of DNA DSBs. Its recruitment can be visualized either by immunofluorescence of fixed cells or by monitoring live cells expressing 53BP1 fused to green fluorescent protein (GFP). In cells exposed to ionizing radiation (IR), the recruitment of 53BP1 to sites of DNA DSBs becomes evident by its localization to foci that are distributed throughout the nucleus; these foci are thought to correspond to sites of DNA DSBs (4,20,30,33,44). When DNA damage is induced in specific subnuclear compartments, for example, by UV laser light or by highly charged energetic particles, then 53BP1 localizes to the subnuclear compartments, where the DNA damage was induced (5, 8).The ability to easily monitor recruitment of 53BP1 to sites of DNA DSBs has allowed significant progress to be made regarding how this protein recognizes DNA damage. Mammalian 53BP1 and its orthologs Rad9 and Crb2/Rhp9, in budding and fission yeast, respectively, recognize DNA DSBs via a tandem tudor domain that binds to methylated histones (18, 31). Human 53BP1 recognizes either methylated K79 of histone H3 or methylated K20 of histone H4 (6,18,32,46), Rad9 recognizes exclusively methylated K79 of histone H3 (13, 43), and Crb2/Rhp9 recognizes exclusively methylated K20 of histone H4 (10, 31). Both K79 of histone H3 and K20 of hist...

We provide a common theoretical framework reuniting specific models for the Ca(2+)-alginate system and general reaction diffusion theory along with experimental validation on a microfluidic chip. As a starting point, we use a set of nonlinear, partial differential equations that are traditionally solved numerically: the Mikkelsen-Elgsaeter model. Applying the traveling-wave hypothesis as a major simplification, we obtain an analytical solution. The solution indicates that the fundamental properties of the alginate reaction front are governed by a single dimensionless parameter λ. For small λ values, a large depletion zone accompanies the reaction front. For large λ values, the alginate reacts before having the time to diffuse significantly. We show that the λ parameter is of general importance beyond the alginate model system, as it can be used to classify known solutions for second-order reaction diffusion schemes, along with the novel solution presented here. For experimental validation, we develop a microchip model system, in which the alginate gel formation can be carried out in a highly controlled, essentially 1D environment. The use of a filter barrier enables us to rapidly renew the CaCl(2) solution, while maintaining flow speeds lower than 1 μm/s for the alginate compartment. This allows one to impose an exactly known bulk CaCl(2) concentration and diffusion resistance. This experimental model system, taken together with the theoretical development, enables the determination of the entire set of physicochemical parameters governing the alginate reaction front in a single experiment.