not-yet-known not-yet-known not-yet-known unknown Glycosylation is a critical quality attribute in biopharmaceuticals that influences crucial properties, such as immunogenicity and stability. Current methods for modeling glycosylation typically rely on imprecise or limited data on nucleotide sugar donor dynamics. These methods use in vitro transporter kinetics or flux balance analysis, which overlook the key aspects of metabolic regulation. We devised an integrative workflow for absolute subcellular NSD quantification in both cytoplasm and secretory organelles. Using subcellular fractionation, exhaustive sample extraction, and liquid chromatography triple-quadrupole tandem mass spectrometry, we accurately measured NSD concentrations ranging 1.6 amol/cell to 3 fmol/cell. Our NSD concentration profiles aligned closely with the glycan distributions on antibodies, particularly after nutrient pulsing to stimulate NSD production. This method enables empirical observation of compartment-specific NSD dynamics. Thus, this study provides novel insights indicating that N-glycosylation, which governs NSD supply, is primarily regulated within the Golgi apparatus. This method offers a novel tool to obtain sophisticated data for the more efficient optimization of glycosylation processes in production cell lines.

Traditional beta-cyclodextrin (beta-CD) in biomedical applications faces challenges due to its inherent physical and biochemical limitations. One of the most effective strategies to enhance the properties of beta-CD for drug delivery is the synthesis of supramolecular polycyclodextrins. In this study, we designed a novel beta-CD nanocage-like structure for drug delivery, incorporating imine and disulfide bonds through Schiff base reactions. Aldehyde group-functionalized beta-CD units were used to construct the main backbone of the nanocage, forming dual-dynamic covalent bonds. The chemical structure of the beta-CD nanocage was confirmed using ¹H nuclear magnetic resonance (¹H NMR) and fourier transform infrared spectroscopy (FTIR). Additionally, atomic force microscopy (AFM) and dynamic light scattering (DLS) revealed that varying amounts of beta-CD crosslinked with cystamine resulted in nanocages approximately 200 nm in size. In vitro drug release experiments demonstrated that doxorubicin (DOX)-loaded beta-CD nanocages exhibited accelerated DOX release in acidic and reductive environments compared to normal physiological conditions, owing to the pH-sensitive imine bond and the glutathione (GSH)-cleavable disulfide bond. The DOX-loaded beta-CD nanocages showed exceptional tumor-killing effects, particularly in acid/reduction-enhanced tumor cells. Both cellular fluorescence imaging and flow cytometry confirmed the potential of the beta-CD nanocages for acid/reduction-specific drug release. Consequently, this precision medicine model using imine/disulfide-linked beta-CD nanocage structures as acidity/reduction-sensitive drug carriers promises to improve oncotherapy through more targeted drug delivery and release, supporting individualized treatment approaches.

Optimisation of isoamylase production in Escherichia coli under different nutrient limitation conditions

The Alzhai Grottoes are challenged by a saline-alkali environment that causes the murals to crumble and peel. Traditional restoration methods are not effective in this environment and can lead to secondary damage. This study employs microbial-induced calcium carbonate precipitation (MICP) technology, using specific bacteria to induce mineral deposition under saline-alkali conditions, forming a protective mineralized layer. The study isolated and screened a mineralized strain, Bacillus oceanicum, which was compared to the traditional mineralizing bacterium Pasteurella. Laboratory experiments revealed that Bacillus oceanicum exhibited higher solidification strength, uniform solidification, and better salt-alkali tolerance. The cohesion of specimens treated with Bacillus oceanicus increased by 83.18%, and the internal friction angle increased by 29.06% compared to Bacillus pasteurii-treated specimens. The study demonstrated that Bacillus oceanicum effectively treated the saline-alkali diseases in the Alzhai Caves’ murals, reducing the height of phthalate, the volume of crisp powder, and the thickness of salt crust in MICP-treated samples by 29.1%, 45.0%, and 36.4%, respectively, compared to untreated specimens. This research provides new insights and methods for the protection and restoration of cultural relics in special environments, focusing on the green microbial technology for disease control and preventive protection of murals.

In this work, double-layer heterogeneous calcium alginate scaffolds were designed for alveolar bone defects where the outer layer featured high hardness and slow degradation, and the inner layer characterized by large pores and rapid degradation. The morphology of the calcium alginate scaffold was akin to that of bone defects, and its direct implantation reduced the operation time. A higher concentration of calcium alginate resulted in smaller pores and slower degradation. Calcium alginate can promote the formation of mineralized nodules and the expression of genes related to mineralization without inducing cytotoxic effects. It also promoted the expression of cellular inflammatory factors, potentially through the TLR4 pathway. In vivo studies confirmed that calcium alginate did not promote the aggregation of inflammatory cells nor the expression of inflammatory factors. In conclusion, the scaffold's characteristics of high surface hardness and slow degradation were beneficial for surface osteogenesis and maintaining the defect's shape and osteogenic space. Conversely, rapid internal degradation favors the formation of bone tissue.

The mutual interactions of ER resident proteins in the ER maintain its functions, prompting the protein folding, modification, and transportation. Here, a new method, named YST-PPI (YESS-based Split fast TEV protease System for Protein-protein Interaction) was developed, targeting the characterization of protein interactions in ER. YST-PPI method integrated the YESS system, split-TEV technology and endoplasmic reticulum retention signal peptide (ERS) to provide an effective strategy for studying ER in situ PPIs in a fast and quantitative manner. The interactions among 15 ER resident proteins of S. cerevisiae were explored using the YST-PPI system, and their interaction network map was constructed, in which more than 74 interacting resident protein pairs were identified. Our studies also showed that Lhs1p plays a critical role in regulating the interactions of most of the ER resident proteins, expect the Sil1p, indicating its potential role in controlling the ER molecular chaperones. Moreover, the mutual interaction revealed by our studies further confirmed that the ER resident proteins perform their functions in a synergetic way and multimer complex might be formed during the process.

Laccases have a wide range of uses in the textile industry for their ability to decolorize dyes, modify the surface of fabrics and bleach textiles. Screening a laccase with high thermal stability and alkali tolerance suitable for textile applications is challenging. In this study, we identified a novel alkaline laccase, LacCT, from Caldalkalibacillus thermarum, and expressed it in Escherichia coli. It showed an optimal temperature of 65°C and a pH range of high stability from 6.0 to 10.0, with an optimum pH of 7.5. Through rational design, the thermal stability of the best variant, G190P/Q254Y/G336M/D510F (LacCT-11), was significantly improved, exhibiting a half-life of 63.2 minutes at 60°C, 1.8 times longer than the wild type. This research presents a new laccase with significant potential for decolorizing textile wastewater and enhancing the ramie degumming process.