Design of cell-type-specific hyperstable IL-4 mimetics via modular de novo scaffolds.

The interleukin-4 (IL-4) cytokine plays a critical role in modulating immune homeostasis. Although there is great interest in harnessing this cytokine as a therapeutic in natural or engineered formats, the clinical potential of native IL-4 is limited by its instability and pleiotropic actions. Here, we design IL-4 cytokine mimetics (denoted Neo-4) based on a de novo engineered IL-2 mimetic scaffold and demonstrate that these cytokines can recapitulate physiological functions of IL-4 in cellular and animal models. In contrast with natural IL-4, Neo-4 is hyperstable and signals exclusively through the type I IL-4 receptor complex, providing previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors. Because of their hyperstability, our computationally designed mimetics can directly incorporate into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds. Neo-4 should be broadly useful for interrogating IL-4 biology, and the design workflow will inform targeted cytokine therapeutic development.

Proteome-Scale Screening to Identify High-Expression Signal Peptides with Minimal N-Terminus Biases via Yeast Display.

Signal peptides are critical for the efficient expression and routing of extracellular and secreted proteins. Most protein production and screening technologies rely upon a relatively small set of signal peptides. Despite their central role in biotechnology, there are limited studies comprehensively examining the interplay between signal peptides and expressed protein sequences. Here, we describe a high-throughput method to screen novel signal peptides that maintain a high degree of surface expression across a range of protein scaffolds with highly variable N-termini. We find that the canonical signal peptide used in yeast surface display, derived from Aga2p, fails to achieve high surface expression for 42.5% of constructs containing diverse N-termini. To circumvent this, we have identified two novel signal peptides derived from endogenous yeast proteins, SRL1 and KISH, which are highly tolerant to diverse N-terminal sequences. This pipeline can be used to expand our understanding of signal peptide function, identify improved signal peptides for protein expression, and refine the computational tools used for signal peptide prediction.

THE DESIGN AND USE OF A MINIMALLY-INVASIVE, EXPANDABLE RETRACTOR FOR DEEP-SEATED BRAIN LESIONS.

Access to deep-seated brain lesions (e.g., tumors, aneurysms, hematomas, and other malformations) is challenging due to the potential for retraction-induced injury. Traditionally, neurosurgeons use dissection and blade retractors to push apart tissue to visualize and operate on target lesions. These blades apply focal pressure onto the brain, resulting in ischemia, edema, and parenchymal trauma, leading to complications in up to 29% of cases. Tubular retractors were introduced to distribute forces radially and have led to improved safety and clinical outcomes. However, reports indicate that tubular retractors still led to complications in up to 9.1% of cases. Other concerns include significant pressure in the direction of insertion and the displacement of anatomic landmarks leading to inaccurate stereotaxis. We present a novel, minimally-invasive brain retractor that utilizes an expandable soft balloon to further reduce retraction-induced injury and increase stereotactic accuracy with a minimal port of entry. The device consists of a balloon catheter system, a clear sheath, and integration with neuronavigation stylets. This approach can reduce the rate of iatrogenic injury and improve clinical outcomes for brain lesion operations. Furthermore, we illustrate the efficacy of this device in use compared to those of conventional tubular and blade retractors in a pig cadaver.

Development of a bioprinting approach for automated manufacturing of multi-cell type biocomposite TRACER strips using contact capillary-wicking.

In many types of solid cancer, interactions between tumour cells and their surrounding microenvironment significantly impact disease progression, and patient prognosis. Tissue engineered models permit investigations of cellular behaviour and interactions in the context of this diseased microenvironment. Previously our group developed the tissue roll for analysis of cellular environment and response (TRACER) platform. To improve the manufacturing robustness of the TRACER platform and to enhance its use for studies involving multiple cell types, we have developed a bioprinting process that deposits cell-laden collagen hydrogel into a thin cellulose scaffolding sheet though a contact-wicking printing process. Printed scaffolds can then be assembled into layered 3D cultures where the location of cells in 3D is dependent on their printed position in the 2D sheet. After a desired culture time 3D TRACERs can be disassembled to easily assess printed cell re-location and phenotype within the heterogeneous microenvironments of the 3D tissue. In our bioprinting manufacturing process, cells are printed into scaffolding sheets, using a modified 3D bioprinter to extrude cells encapsulated in unmodified collagen hydrogel through a polydimethylsiloxane (PDMS) printer extrusion nozzle. This nozzle design reproducibly generated bioink deposition profiles in the scaffold without causing significant cellular damage or compromising scaffold integrity. We assessed print pattern quality and reproducibility and demonstrated printing of co-culture strips containing tumour cells and fibroblasts in separate compartments (i.e. separate TRACER layers). This printing approach will potentially enable adoption of the TRACER platform to the broader community to better understand multi-cell type interactions in 3D tumours and tissues.