We previously developed chicken interleukin-1β (IL-1β) mutants as single-dose adjuvants that induce protective immunity when co-administered with an avian vaccine. However, livestock such as pigs may require a vaccine adjuvant delivery system that provides long-lasting protection to reduce the need for successive booster doses. Therefore, we developed chitosan-coated alginate microparticles as a carrier for bovine serum albumin (BSA) or porcine IL-1β (pIL-1β) and assessed their physical, chemical, and biological properties. Electrospraying of the BSA-loaded alginate microparticles (BSA/ALG MPs) resulted in an encapsulation efficiency of 50%, and those MPs were then coated with chitosan (BSA/ALG/CHI MPs). Optical and scanning electron microscopy, zeta potential analysis, and Fourier transform infrared spectroscopy were used to characterize these MPs. The BSA encapsulation parameters were applied to ALG/CHI MPs loaded with pIL-1β, which were not cytotoxic to porcine fibroblasts but had enhanced bio-activity over unencapsulated pIL-1β. The chitosan layer of the BSA/ALG/CHI MPs prevented burst release and facilitated sustained release of pIL-1β for at least 28 days. In conclusion, BSA/ALG/CHI MPs prepared as a carrier for pIL-1β may be used as an adjuvant for the formulation of pig vaccines.

Glutamine synthetase (GS) catalyzes the condensation of ammonia and glutamate, along with ATP, to form glutamine. Despite extensive studies on GSs from eukaryotes and prokaryotes, the roles of the N-terminus and other structural features in catalysis remain unclear. Here we report the decameric structure of Drosophila melanogaster GS 2 (DmGS2). The N-terminal short helices, α1 and α2, constitute a meander region, and form hydrogen bonds with residues 3–5 in the N-terminal loop, which are not present in the GSs of other species. Deletion of α1 or α1-α2 inactivates DmGS2. Notably, the Arg4 in each monomer of one pentamer forms hydrogen bonds with Glu7, and Asp8 in the adjacent monomer of the other pentamer. Replacement of Arg4 with Asp (R4D) abolishes activity. Analytical ultracentrifugation revealed that Arg4 is crucial for oligomerization. Circular dichroism spectra revealed that R4D may alter the secondary structure. We mutated key residues to identify the substrate-binding site. As Glu140 binds glutamate and Glu311 binds ammonia, mutants E140A and E311A have little activity. Conversely, mutant P214A (P contributes to ATP binding) has higher activity than wild-type DmGS2. These findings expand the understanding of the structural and functional features of the N-terminal meander region of DmGS2 and the residues important for catalytic efficiency.

Colorectal cancer (CRC) is currently the third most common cancer in the world. Due to the development of treatment resistance, the efficacy of current chemotherapeutic agents against CRC has reached a plateau. Drug activity depends on the entire physiological response; therefore, drug‐dose parameters cannot be designed efficiently by using conventional prediction‐based methodologies. In this work, the AI‐PRS (artificial intelligence‐based phenotypic response surface) platform is successfully applied to find optimal drug‐dose combinations in vitro from a pool of ten approved drugs. The AI‐PRS platform optimizes effective drug‐dose combinations without reference to molecular pathways or drug interaction data. With the aid of AI‐PRS platform, efficient one, two, three, and four drug‐dose combinations from in vitro studies are found. Of hundreds of combinations, regorafenib (R)/gemcitabine (G)/cetuximab (C)/5‐fluorouracil (U) drug‐dose combination exhibits the best activity on four CRC cell lines, two circulating tumor cells (CTCs), and one patient derived xenografts (PDX) cell lines. The three‐drug combination of R/G/U shows the highest toxicity (70%) in the PDX cell line. Four‐drug combination of R/G/C/U displays the best toxicity (80%) in in vitro cultured CTCs. The findings from the present derived cells reveal the prospective validation of the AI‐PRS platform, which may help identify customized and highly efficient drug‐dose combinations for future CRC treatment.