Biotechnology Journal最新文献

Computational fluid dynamics (CFD) offers a powerful tool in characterizing the complex biophysical environment inducing by dynamic storage conditions, providing insights often beyond the reach of conventional experimental approaches. As our understanding of platelet (PLT) biology has advanced, increased attention has been directed toward mechanical stresses, attributing shear forces encountered during collection, processing, and storage to an acceleration decline in PLT concentrate (PC) quality. CFD simulations using the volume of fluid model were used to simulate PC storage under varying agitation frequencies. Key parameters assessed include fluid velocity, wall shear stress (WSS), and gas–liquid mass transfer. Agitation increased fluid velocity and WSS while preserving the temporal symmetry characteristic of sinusoidal motion. Enhanced oxygen transfer was observed in open-top containers; however, when accounting for the gas permeability of storage materials, oxygen availability was ultimately constrained by container permeability rather than fluid motion. These results highlight the dual role of agitation: promoting oxygen transfer while simultaneously introducing mechanical stress that may contribute to PLT storage lesions. Importantly, since oxygen supply is limited by container permeability, reducing agitation could minimize shear-induced PLT damage without compromising oxygenation. Future optimization strategies may involve modifying storage container geometry or permeability to further improve oxygen delivery during storage.

Phospholipid-based nanocarriers are an adaptable and chemically tunable class of drug delivery systems that self-assemble into bilayered vesicles due to the amphiphilic nature of phospholipids. Phospholipid-based nanocarriers can encapsulate both hydrophilic and hydrophobic drugs through non-covalent interactions and modulation of lipid phase behavior. This review explores the molecular and supramolecular principles governing the formation, stability, and function of key phospholipid-derived nanocarriers, including liposomes, transferosomes, ethosomes, invasomes, phytosomes, pharmacosomes, and virosomes. The structural attributes—such as bilayer packing, surface charge, curvature elasticity, and membrane permeability—are critically evaluated for the impact of those structural parameters on optimizing drug loading, drug release, and bioavailability. For example, variations in bilayer packing affect the encapsulation efficiency and drug release profiles, while surface charge modulates cellular uptake and colloidal stability. Curvature elasticity plays a pivotal role in membrane fusion and drug release, and membrane permeability determines the rate at which drugs diffuse from the nanocarrier. Emerging multilamellar systems such as vesosomes and spongosomes are also discussed for their potential in site-specific, controlled drug release. Common fabrication techniques (e.g., thin-film hydration, ethanol injection, freeze–thaw, and microfluidics) and characterization methods (e.g., DLS, DSC, FTIR, and cryo-TEM) are reviewed. The translational landscape is assessed through clinically approved liposomal drugs, patent trends, and ongoing trials involving stimuli-responsive systems. Challenges related to colloidal stability, tumor penetration, immune interactions, and large-scale manufacturing are addressed. This review provides a chemistry-centered framework to guide the rational design and clinical development of phospholipid nanocarriers, particularly in cancer therapeutics.

In this study, lincomycin A (Lin-A), a lincosamide antibiotic primarily synthesized by Streptomyces lincolnensis, was selected as a model to demonstrate a combined genetic regulation and process engineering strategy for production improvement. The GntR-family transcriptional regulator SLCG_2790 was identified as a negative modulator of Lin-A biosynthesis. Disruption of SLCG_2790 in the industrial strain S. lincolnensis L-427 led to enhanced Lin-A accumulation, accompanied by increased transcriptional levels of mycothiol and ergothioneine biosynthesis genes. Metabolic flux analysis revealed an 83.6% elevation in pentose phosphate pathway activity compared to the parental strain. Despite the observed reduction in mycelial growth resulting from SLCG_2790 deletion, fermentation performance was significantly improved through medium optimization using Plackett–Burman design, steepest ascent, and response surface methodology. The optimized conditions yielded a 38.1% increase in Lin-A production in shake-flask culture, along with an accelerated onset of physiological activity. In a 5-L bioreactor, the engineered strain achieved a maximum Lin-A titer of 3640.6 mg/L, representing a 101.4% improvement over the parental strain cultured in the original medium. These findings underscore the potential of transcriptional regulation coupled with rational process optimization to overcome metabolic constraints and enhance antibiotic production in actinomycetes.

To address the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 in Bacillus subtilis, such as low transformation efficiency and strong dependence on specific PAM sequences, this study developed a novel genome-editing tool based on AsCas12f1 nuclease derived from Acidibacillus sulfuroxidans. Using the CRISPR-AsCas12f1 system, we successfully achieved gene knockout and targeted insertion in B. subtilis with a knockout efficiency of up to 100%. We further demonstrated that the length of the donor DNA homology arms and the choice of PAM motifs significantly influenced the editing efficiency. To expand the applicability of this system, gene interference and activation experiments were performed using green fluorescent protein (GFP) as a reporter. The system achieved more than 90% gene knockdown efficiency and effectively activated the reported gene transcription, with a maximum activation fold of 3.20. In conclusion, the CRISPR-AsCas12f1 system established in this study provides an efficient and reliable genome editing tool for the functional gene research and industrial applications of B. subtilis.

Solid tumors are characterized by a metabolically dysregulated tumor microenvironment (TME) enriched with reactive oxygen species (ROS), which suppresses immune cell function. Natural killer (NK) cells are promising effectors in cancer immunotherapy due to their intrinsic cytotoxicity without prior antigen sensitization. However, oxidative stress impairs NK cell cytotoxicity by reducing degranulation, interferon-γ production, and survival. Therefore, maintaining NK cell redox balance is a crucial obstacle to achieving optimal therapeutic results in ROS-rich TMEs. Here, we proposed a straightforward, non-genetic ex vivo membrane modification approach to reinforce the redox balance of NK cells via ROS scavenging. Using tris(2-carboxyethyl)phosphine (TCEP), a mild reducing agent, we selectively introduced free thiol groups onto the exterior surface of NK cell plasma membranes. The engineered surface-thiol-riched NK cells (STR-NK) demonstrated (1) increased membrane thiols, (2) efficiently eliminated extracellular ROS, (3) attenuated intracellular ROS accumulation, and (4) preserved cytotoxicity-associated gene expression under oxidative stress. Importantly, STR-NK cells maintained potent cytotoxicity against diverse solid tumor cells despite the presence of ROS. Overall, this uncomplicated and scalable surface redox modulation approach enhances NK cell anticancer activity under oxidative stress, offering a promising strategy to improve NK cell-based cancer immunotherapies in ROS-enriched solid TMEs.

Chinese Hamster Ovary (CHO) cells are the predominant host for the production of biotherapeutics; however, there remains considerable potential to further enhance their cellular productivity. Adaptive laboratory evolution (ALE) combined with omics-based analysis has emerged as a promising approach toward generating host cell lines with desirable characteristics. In this study, CHOK1 cells were gradually adapted to tunicamycin (TM), an endoplasmic reticulum (ER) stressor, resulting in an 8-fold increase in resistance compared to the non-adapted cells with a marked enlargement of the ER. Notably, the per-cell secretion rate of total protein was 3- to 4-fold higher in the TM-adapted cells. Transcriptomic analysis revealed upregulation of several genes in the protein processing pathway, such as Dpagt1, the TM target gene, and ER stress response genes. The protein transport, secretion and ubiquitination pathways were also altered, potentially contributing to the increased protein secretion. Furthermore, genes participating in signaling cascades of PI3K-AKT, MAPK, and Ras pathways were differentially expressed, thereby aiding in its survival and proliferation. Whole genome sequencing confirmed the amplification of a large genome segment of chromosome 4, which included several genes upregulated at the mRNA level, including Dpagt1. Thus, the survival and increased protein secretion of TM-adapted cells can be attributed to a combination of transcriptional level changes and amplification of a large genome segment. Further, transient expression of a recombinant protein, SEAP, in TM-adapted cells showed an improvement in specific productivity of ∼1.4 fold as compared to the non-adapted cells, underscoring the importance of ALE as a cell engineering strategy.

Cardiovascular diseases are a leading global cause of mortality, with cardiac patches (CPs) emerging as a novel surgical treatment. This study used a combined decellularization method with sodium dodecyl sulfate (SDS), Triton X-100, and DNase to process porcine myocardial tissue (PMT), yielding decellularized extracellular matrix (dECM). Quantitative analysis revealed that the dECM contained collagen, DNA, and glycosaminoglycans (GAGs) at concentrations of 2.63 ± 0.37 µg/mg, 4.27 ± 0.79 ng/mg, and 15.94 ± 0.60 µg/mg. Additionally, a conductive hydrogel patch (PDA/CSCA/PAM) with a uniform porous structure and excellent mechanical properties was developed. Its adhesive strengths on glass, stainless steel, and PMT were 50.71 ± 2.88 kPa, 34.19 ± 3.63 kPa, and 54.71 ± 3.24 kPa, respectively. Bacterial inhibition rates reached 103.26 ± 3.52% (Day 1) and 99.26 ± 5.35% (Day 3), indicating significant antimicrobial efficacy. The patch met myocardial tissue engineering standards for swelling ratio, hydrophilicity (contact angle <90°), hemolysis rate (<5%), and conductivity (10⁻⁴ S cm⁻¹⁾, with biosafety certified by ISO 10993-5. These results highlight its mechanical compatibility, antimicrobial activity, and biocompatibility, offering a multifunctional solution for myocardial infarction repair with high clinical translation potential.