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Depletion interactions are thought to significantly contribute to the organization of intracellular structures in the crowded cytosol. The strength of depletion interactions depends on physical parameters such as the depletant number density and the depletant size ratio. Cells are known to dynamically regulate these two parameters by varying the copy number of proteins of a wide distribution of sizes. However, mammalian cells are also known to keep the total protein mass density remarkably constant, to within 0.5% throughout the cell cycle. We thus ask how the strength of depletion interactions varies when the total depletant mass is held fixed, a.k.a. fixed-mass depletion. We answer this question via scaling arguments, as well as by studying depletion effects on networks of reconstituted semiflexible actin in silico and in vitro. We examine the maximum strength of the depletion interaction potential U∗ as a function of q, the size ratio between the depletant and the matter being depleted. We uncover a scaling relation U∗ ∼ qζ for two cases: fixed volume fraction φ and fixed mass density ρ. For fixed volume fraction, we report ζ < 0. For the fixed mass density case, we report ζ > 0, which suggests that the depletion interaction strength increases as the depletant size ratio is increased. To test this prediction, we prepared our filament networks at fixed mass concentrations with varying sizes of the depletant molecule poly(ethylene glycol) (PEG). We characterize the depletion interaction strength in our simulations via the mesh size. In experiments, we observe two distinct actin network morphologies, which we call weakly bundled and strongly bundled. We identify a mass concentration where different PEG depletant sizes lead to weakly bundled or strongly bundled morphologies. For these conditions, we find that the mesh size and intra-bundle spacing between filaments across the different morphologies do not show significant differences, while the dynamic light scattering relaxation time and storage modulus between the two states do show significant differences. Our results demonstrate the ability to tune actin network morphology and mechanics by controlling depletant size and give insights into depletion interaction mechanisms under the fixed-depletant-mass constraint relevant to living cells.

 

Human mesenchymal stromal cell (hMSC) manufacturing requires the production of large numbers of therapeutically potent cells. Licensing with soluble cytokines improves hMSC therapeutic potency by enhancing secretion of immunoactive factors but typically decreases proliferative ability. Soft hydrogels, however, have shown promise for boosting immunomodulatory potential, which may compensate for decreased proliferation. Here, hydrogels are cross‐linked with peptoids of different secondary structures to generate substrates of various bulk stiffnesses but fixed network connectivity. Secretions of interleukin 6, monocyte chemoattractive protein‐1, macrophage colony‐stimulating factor, and vascular endothelial growth factor are shown to depend on hydrogel stiffness in the presence of interferon gamma (IFN‐γ) supplementation, with soft substrates further improving secretion. The immunological function of these secreted cytokines is then investigated via coculture of hMSCs seeded on hydrogels with primary peripheral blood mononuclear cells (PBMCs) in the presence and absence of IFN‐γ. Cocultures with hMSCs seeded on softer hydrogels show decreased PBMC proliferation with IFN‐γ. To probe possible signaling pathways, immunofluorescent studies probe the nuclear factor kappa B pathway and demonstrate that IFN‐γ supplementation and softer hydrogel mechanics lead to higher activation of this pathway. Overall, these studies may allow for production of more efficacious therapeutic hMSCs in the presence of IFN‐γ.

 

Engineered living materials combine the advantages of biological and synthetic systems by leveraging genetic and metabolic programming to control material-wide properties. Here, we demonstrate that extracellular electron transfer (EET), a microbial respiration process, can serve as a tunable bridge between live cell metabolism and synthetic material properties. In this system, EET flux from Shewanella oneidensis to a copper catalyst controls hydrogel cross-linking via two distinct chemistries to form living synthetic polymer networks. We first demonstrate that synthetic biology-inspired design rules derived from fluorescence parameterization can be applied toward EET-based regulation of polymer network mechanics. We then program transcriptional Boolean logic gates to govern EET gene expression, which enables design of computational polymer networks that mechanically respond to combinations of molecular inputs. Finally, we control fibroblast morphology using EET as a bridge for programmed material properties. Our results demonstrate how rational genetic circuit design can emulate physiological behavior in engineered living materials. 

The utility of active proteases as biomarkers is often limited by overlapping substrate specificity. Here, this feature is leveraged to develop a quantitative pattern-recognition sensing system driven by the degradation patterns of peptide–peptoid hybrid substrates to classify proteases and estimate their concentration by multivariate data analysis. 

Due to their N -substitution, peptoids are generally regarded as resistant to biological degradation, such as enzymatic and hydrolytic mechanisms. This stability is an especially attractive feature for therapeutic development and is a selling point of many previous biological studies. However, another key mode of degradation remains to be fully explored, namely oxidative degradation mediated by reactive oxygen and nitrogen species (ROS/RNS). ROS and RNS are biologically relevant in numerous contexts where biomaterials may be present. Thus, improving understanding of peptoid oxidative susceptibility is crucial to exploit their full potential in the biomaterials field, where an oxidatively-labile but enzymatically stable molecule can offer attractive properties. Toward this end, we demonstrate a fundamental characterization of sequence-defined peptoid chains in the presence of chemically generated ROS, as compared to ROS-susceptible peptides such as proline and lysine oligomers. Lysine oligomers showed the fastest degradation rates to ROS and the enzyme trypsin. Peptoids degraded in metal catalyzed oxidation conditions at rates on par with poly(prolines), while maintaining resistance to enzymatic degradation. Furthermore, lysine-containing peptide–peptoid hybrid molecules showed tunability in both ROS-mediated and enzyme-mediated degradation, with rates intermediate to lysine and peptoid oligomers. When lysine-mimetic side-chains were incorporated into a peptoid backbone, the rate of degradation matched that of the lysine peptide oligomers, but remained resistant to enzymatic degradation. These results expand understanding of peptoid degradation to oxidative and enzymatic mechanisms, and demonstrate the potential for peptoid incorporation into materials where selectivity towards oxidative degradation is necessary, or directed enzymatic susceptibility is desired.