Biotechnology and Bioengineering最新文献

Bioprocesses for stem cell-based therapeutics are resource- and time-intensive, hindering the generation of sufficient data for machine learning-informed development. We describe a framework combining data augmentation, multivariate regression, and feature importance analysis to investigate the relationship between metabolites and critical quality attributes (CQAs) of cultured stem cells. A first-principles model (FP), a hybrid model using neural ordinary differential equations (ODEs) with stoichiometric constraints (HD), and a purely statistical neural ODE model (NODEAM) were considered. Probing these models through data augmentation, we generated synthetic data and amplified the biological information encoded in each modality, directly linking architectural choices to their ability to capture relevant dynamics. For all models, the validation error remained relatively constant, and the convergence of the feature importance tensor followed a power law with the number of augmented runs. Temporal feature importance and functional data analysis of variance revealed key time windows during which glucose and lactate showed strong correlation with CQAs. The HD model provided the best fidelity and accuracy, underscoring the value of combining mechanistic and statistical modeling for improved interpretability at lower complexity. Overall, this framework can yield insights into the physiology of cultivated cells and can be adapted for various culture-based biomanufacturing systems.

Ultraviolet-B (UVB) radiation exacerbates oxidative stress, accelerating collagen degradation and skin aging. While collagen and its hydrolysates offer therapeutic potential, medical apparatus and instruments call for high quality and traceability. Here we investigated the synergistic effects of collagen and collagen peptides derived from Shad (Alosa sapidissima) scales against UVB-induced skin damage. Collagen was extracted and peptides were obtained through enzymatic hydrolysis. Structural characterization confirmed collagen's triple helix and peptide fragments. In vitro assays demonstrated concentration-dependent antioxidant activity: collagen peptides (5%–10%) and collagen (0.5%–1%) exhibited potent DPPH/ABTS⁺ radical scavenging, with their combination outperforming individual components. In UVB-irradiated mice, topical application of the collagen-peptide mixture significantly reduced epidermal thickening, suppressed ROS production, and enhanced SOD activity. Histological analysis revealed mitigated wrinkle formation and improved dermal blood flow. The mixture also formed stable hydrogels, enhancing bioavailability. These findings highlight the dual mechanism of collagen (structural support) and peptides (antioxidant/moisturizing effects), offering a promising strategy for combating photoaging. This study provides foundational insights for developing marine collagen-based dermatological therapies.

Nicotinamide adenine dinucleotide (NAD⁺) is an essential redox cofactor widely used in industrial biocatalysis and therapeutic applications. Conventional chemical synthesis of NAD⁺ is hazardous and inefficient, while enzymatic approaches, particularly dual-enzyme cascades involving nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), suffer from low catalytic efficiency due to spatially separated active sites and poor intermediate channeling. Here, we report a direct, one-step NAD⁺ biosynthetic route using Haemophilus influenzae NadR, a naturally bifunctional enzyme that naturally integrates NRK and NMNAT activities into a single polypeptide chain. To address the rate-limiting NR phosphorylation step, we developed a loop engineering–docking combinatorial simulation (LEDCS) strategy to rationally redesign the NRK domain. The resulting V241S/L387Q mutant displayed a fourfold increase in enzymatic activity and a 2.4-fold improvement in catalytic efficiency relative to the wild type. Structural and interaction analyses revealed that the P-loop plays a critical role in coordinating NR and ATP binding, while enhanced hydrophilicity in the substrate-binding pocket improved substrate affinity. This study highlights the catalytic advantage of intramolecular substrate channeling and presents a generalizable strategy for enhancing the performance of multifunctional biocatalysts.

The developability of antibodies is a critical concern in antibody discovery, encompassing issues such as self-interaction, aggregation, and thermal stability. The use of computational and structure-based tools has greatly improved the evaluation and prioritization of initial antibody sequences. With the increasing demand for subcutaneous administration of small-volume, high-concentration antibody formulations, there is a need for more accurate prediction tools based on protein structures. Our study introduces AB-Panda, a tool based on AlphaFold2-predicted antibody structures and three innovative structure-related metrics. AB-Panda utilizes unit-area hydrophobic value (UHV), unit-area positive charge (UPC), and unit-area negative charge (UNC) to automatically identify hydrophobic and charged patches within the complementarity determining regions (CDRs) of antibodies. Through the analysis of the 919 clinical stage therapeutic (CST) antibodies, we have established recommended ranges of UHV, UPC, and UNC as reference standards for antibody developability. AB-Panda offers clear visualizations of surface hydrophobic and charge distribution, facilitating the identification of problematic amino acids and providing suggestions for further sequence engineering. Additionally, AB-Panda has been integrated into a web application, available at https://www.antibodydev.com, by combining UHV, UPC, UNC, and other established computational metrics for the early screening and optimization of antibody sequences.

Bacillus subtilis is widely recognized as a microbial chassis for industrial protein production due to its robust secretory capacity. In B. subtilis, most proteins are exported from the cytoplasm via classical secretion pathways, including the Sec and Tat systems, as well as ABC transporters. However, efficient secretion of heterologous proteins using signal peptides remains a major challenge, largely due to bottlenecks within these pathways. In this study, we demonstrate that the pyruvate dehydrogenase E3 subunit (PdhD) from B. subtilis (BsPdhD) exhibits significantly higher secretion efficiency compared to conventional signal peptides. Through systematic truncation analysis of BsPdhD's N-terminal FAD/NAD-binding domain and C-terminal dimerization domain, coupled with biophysical characterization of its oligomeric state, we uncover a unique dimerization-dependent secretion mechanism. Notably, disruption of BsPdhD dimerization via removal of its C-terminal dimerization domain completely abolished secretion, whereas the N-terminal domain was primarily responsible for protein expression. To our knowledge, this study provides the first mechanistic evidence that BsPdhD-mediated secretion strictly depends on C-terminal dimerization. These findings not only reveal a previously unrecognized mechanism of protein trafficking but also establish BsPdhD as a promising tool for engineering high-efficiency secretory systems for industrial protein production.

Traditional wound treatment involves protecting the wound with dressing and administering antibiotics to prevent tissue infection due to bacteria. However, these methods are inadequate due to the side effects of antibiotics on healthy cells and microbial resistance to antibiotics. Therefore, new strategies involving the application of natural resources such as essential oils as antimicrobial agents in combination with biomaterials as wound dressings have been tested in the treatment of wounds. Furthermore, oxygen (O₂)-releasing biomaterials have attracted great interest due to the important role of O₂ in wound healing processes. However, the co-application of O₂ and essential oil as antimicrobial and cell-promoting agents has not been studied. In this context, we report a novel biomaterial capable of co-delivering O₂ and natural antimicrobial tea tree oil (TTO) for 15 and 5 days, respectively. The biomaterial consists of an alginate scaffold (Alg-PMOF-O) containing O₂-carrying nanomaterial, laponite and TTO. In vitro bacterial experiments have shown that O₂ release from Alg-PMOF-O is an additional parameter acting as an antibacterial agent to inhibit bacterial growth but is not sufficient alone to inhibit bacteria. 5 µL of TTO in Alg-PMOF-O is necessary to suppress both E. coli and S. aureus over a 1-day incubation period. The effect of TTO and O₂ alone or in combination on cell viability is examined using WST-1 and PrestoBlue assays. According to the WST-1 and PrestoBlue tests, the combined application of TTO and O₂ does not show any toxic effect on fibroblast cells under normoxic conditions during the 5-day incubation period. Under hypoxic conditions, the WST-1 test shows no toxic effect after only 1 day of incubation, while the PrestoBlue test shows no toxicity under hypoxia during both 1 and 5 days of incubation. On the other hand, the combined application of TTO and O₂ indicates toxic effects on cancer Malme-3M cells during both normoxic and hypoxic conditions over 1 and 5 days of incubation. This effect is confirmed by both the WST-1 and PrestoBlue tests. The overall results demonstrate that Alg-PMOF-O exhibits antibacterial activity while having a lower toxic effect on fibroblasts under hypoxic conditions, and therefore has potential for use as wound dressing.