Hierarchical assemblies of proteins into fibrillar structures occur in both physiologic and pathologic extracellular spaces and often involve interactions between oppositely charged peptide domains. However, the interplay between tertiary structure dynamics and quaternary hierarchical structure formation remains unclear. In this work, we investigate supramolecular mimics of these systems by mixing one-dimensional assemblies of small alkylated peptides bearing opposite charge and varying in peptide sequence. We found that assemblies with weak cohesive interactions readily create fibrous superstructures of bundled filaments as molecules redistribute upon mixing. Low cohesion allows molecules to escape from the original assemblies and exchange dynamics help them reassemble into electrostatically stable bundles. However, we also found that kinetic barriers can be encountered in these systems and limit formation of the hierarchical structures at pH values where charge densities are high. Increasing intermolecular cohesion using longer peptide sequences that form stable β-sheets was found to suppress superstructure formation. Our findings suggest that low internal cohesion in protein systems could facilitate the conformational rearrangements required to create hierarchical structures.

An optimized fed-batch cultivation process for the production of the polyoma virus capsid protein VP1 in recombinant Escherichia coli BL21 bacteria is presented. The optimization procedure maximizing the amount of desired protein is based on a mathematical model. The model distinguishes an initial cell growth phase from a protein production phase initiated by inducer injection. A new approach to model the target protein formation rate was elaborated, where product formation is primarily dependent on the specific biomass growth rate. Lower growth rates led to higher specific protein concentrations. The model was identified from a series of fed-batch experiments designed for parameter identification purposes and possesses good prediction quality. Then the model was used to determine optimal open-loop control profiles by manipulating the substrate feed rates in both phases as well as the induction time. Feed-rate optimization has been solved using Pontryagin's maximum principle. The solution was validated experimentally. A significant improvement of the process performance index was achieved.

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Primary tumours can establish long‐range communication with distant organs to transform them into fertile soil for circulating tumour cells to implant and proliferate, a process called pre‐metastatic niche (PMN) formation. Tumour‐derived extracellular vesicles (EV) are potent mediators of PMN formation due to their diverse complement of pro‐malignant molecular cargo and their propensity to target specific cell types (Costa‐Silva et al., 2015; Hoshino et al., 2015; Peinado et al., 2012; Peinado et al., 2017). While significant progress has been made to understand the mechanisms by which pro‐metastatic EVs create tumour‐favouring microenvironments at pre‐metastatic organ sites, comparatively little attention has been paid to the factors intrinsic to recipient cells that may modify the extent to which pro‐metastatic EV signalling is received and transduced. Here, we investigated the role of recipient cell cholesterol homeostasis in prostate cancer (PCa) EV‐mediated signalling and metastasis. Using a bone metastatic model of enzalutamide‐resistant PCa, we first characterized an axis of EV‐mediated communication between PCa cells and bone marrow that is marked by in vitro and in vivo PCa EV uptake by bone marrow myeloid cells, activation of NF‐κB signalling, enhanced osteoclast differentiation, and reduced myeloid thrombospondin‐1 expression. We then employed a targeted, biomimetic approach to reduce myeloid cell cholesterol in vitro and in vivo prior to conditioning with PCa EVs. Reducing myeloid cell cholesterol prevented the uptake of PCa EVs by recipient myeloid cells, abolished NF‐κB activity and osteoclast differentiation, stabilized thrombospondin‐1 expression, and reduced metastatic burden by 77%. These results demonstrate that cholesterol homeostasis in bone marrow myeloid cells regulates pro‐metastatic EV signalling and metastasis by acting as a gatekeeper for EV signal transduction.

Synthetic high-density lipoprotein (HDL) mimics have emerged as promising therapeutic agents. However, approaches to date have been unable to reproduce key features of spherical HDLs, which are the most abundant human HDL species. Here, we report the synthesis and characterization of spherical HDL mimics using lipid-conjugated organic core scaffolds. The core design motif constrains and orients phospholipid geometry to facilitate the assembly of soft-core nanoparticles that are approximately 10 nm in diameter and resemble human HDLs in their size, shape, surface chemistry, composition, and protein secondary structure. These particles execute salient HDL functions, including efflux of cholesterol from macrophages, cholesterol delivery to hepatocytes, support lecithin:cholesterol acyltransferase activity, and suppress inflammation. These results represent a significant step toward a genuine functional mimic of human HDLs.