Artificial blood vessel transplantation is considered the preferred surgical therapy for treating blocked blood vessels. Artificial blood vessels less than 6 mm frequently fail in vivo due to restenosis and thrombosis, significantly reducing the lifespan of the grafts. It is therefore crucial to develop antithrombotic materials for artificial blood vessels. This study presented a hydrogel with nitric oxide (NO) release, reactive oxygen species (ROS) scavenging, and antithrombotic properties, designed for eventual application in 3D printing artificial blood vessels. The hydrogel was primarily composed of double bond-modified recombinant collagen and hyaluronic acid (HA), along with caffeic acid arginine amide grafted onto HA, mimicking the protein/polysaccharide dual-network structure of the extracellular matrix. After modification, this hydrogel exhibited strong light-curing capabilities and shear-thinning qualities which were highly desirable for bioprinting. The hydrogel was capable of dynamically triggering and sustaining the release of NO, thereby effectively eliminating excess ROS at sites of inflammation. NO produced by the hydrogel enhanced the migration and proliferation of human umbilical vein endothelial cells while significantly inhibiting the proliferation of vascular smooth muscle cells. In terms of angiogenesis, the hydrogel demonstrated a significant ability to promote neovascularization. Furthermore, experimental results showed that platelet adhesion was virtually undetectable on the material surface, and protein adhesion was inhibited, thus minimizing the risk of thrombosis. Overall, this hydrogel bio-ink shows great potential for the 3D printing of small-diameter vascular scaffolds, offering a novel solution to address the issues of thrombosis and restenosis in artificial blood vessels.
Adeno-associated virus (AAV) is a promising delivery system for gene therapy. However, current manufacturing of AAV suffers from very low yields compared to other biotherapeutics. The AAV dose per patient ranges between 1011and 1015 viral genomes (vg), requiring an average of 10 to 30 L production/dose. As a consequence, production costs are prohibitive for most indications. Our recent studies revealed that only 10% of the HEK293 cells that have received the AAV encoding DNA produce assembled AAV capsids. This observation prompts the question: Why would cells that have been successfully transfected, be unable to produce AAV. To answer this question, we undertook a detailed study to characterize the two sub-populations from the same transfection, the cells that were making assembled capsids and those that were not. We found that the two populations had distinct cell cycle profiles, with a block in cell cycle progression characterizing the producer population. RNA-seq analysis of the two populations reveals differences in the molecular pathways impacted and provides a basis for making changes to improve productivity.
Epithelial tissues actively deform their surrounding extracellular matrix mechanically. Traction forces represent an intrinsic mechanism by which cells actively sense and adapt to their extracellular environment, which has been increasingly recognized to play a crucial role in cancer progression, metastasis, and treatment failure. However, current traction force research has predominantly concentrated at the single-cell level, overlooking the multicellular spatio-temporal dynamics and collective effects inherent in cancer as an integrated multi-cellular system. Herein, the collective-level traction forces of cancer spheroids were mapped using traction force microscopy. Our results revealed an inherent spatial distribution pattern of cancer spheroid traction force at the spheroid–substrate contact plane, with peaks concentrated along the periphery of the contact interface. Besides, the cancer spheroid traction force was regulated by the spheroid size when the spheroid did not undergo dispersion, which was positively correlated with the spheroid dispersion ability. Moreover, there existed an inherent temporal correlation between the spheroid traction force and dispersion. The onset of cancer spheroid dispersion was accompanied with a marked suppression of the traction force dynamics. Furthermore, the traction force of cancer spheroids was validated to hold potential as a biomechanics-related phenotypic readout for anticancer drug testing.
Mycelial biocomposites are sustainable alternatives to nonbiodegradable materials in building and packaging. Efficient manufacturing requires accurate, non-destructive quantification of growth over time, yet existing methods are often destructive or imprecise. This study develops and evaluates several non-destructive quantification methods for wood-flour biocomposites by using images to define mycelial density levels, low (no visible growth, removable surface hyphal coverage < 7.8%/cm²), medium (light growth, 7.8%–26.7%/cm²), and high (dense coverage, > 26.7%/cm²), and tracking changes in each level over time. The first method, the manual creation of masks for each growth level, provided rapid but coarse classification, estimating 64.1% high growth after 16 days with 3.5% user variability. An algorithmic masking approach improved detail detection, increasing estimated high-growth coverage to 81.7% but also variability to 9.3%. A fully automated deep-learning model proved fastest and most consistent, yielding 77.8% high-growth coverage and eliminating intra-user variability (0%). The deep-learning method was then applied to assess the effects of substrate supplements, revealing distinct growth patterns for each—differences not captured by traditional methods. These results demonstrate the effectiveness of automated quantification for mycelial biocomposites, enabling reproducible, high-resolution, non-destructive monitoring of growth and providing a foundation for more precise engineering and wider adoption of these materials.
Clostridioides difficile infection presents an escalating clinical challenge due to the proliferation of hypervirulent and antibiotic-resistant strains. The primary symptoms of disease, namely colitis and diarrhea, are induced by the release of two toxins: TcdA and TcdB. Targeting these toxins with peptide inhibitors provides an attractive therapeutic strategy that can be used alone or synergistically with standard antibiotic treatments to alleviate severe symptoms and reduce the risk of resistance development. In this study, we present the rational discovery and optimization of potent TcdB peptide inhibitors. The lead sequences effectively inhibit TcdB glucosyltransferase activity, the crucial enzymatic process leading to disease symptoms, by directly competing with the toxin's molecular targets, Rho proteins. Detailed enzymatic studies also elucidate distinct Michaelis constants, KM, for each substrate, UDP-glucose and Rho-proteins, for multiple TcdB GTD subtypes. The selected peptides demonstrated broad efficacy against the three most common TcdB subtypes, which are used in over 90% of clinical isolates. Additionally, the peptides delayed TcdB-induced loss of barrier integrity and decreased apoptosis in a primary human colon epithelial monolayer model. This study highlights a novel therapeutic avenue with significant potential to enhance the treatment and management of C. difficile infections.
Carcinine, a valuable imidazole dipeptide with antioxidant and therapeutic properties, faces biosynthesis challenges due to enzyme aggregation and substrate inhibition. In this study, an integrated strategy combining linker peptide engineering and immobilization was applied to address these challenges and enhance carcinine production. Rational design of linker peptides (D5, L2, L3) in the sfp-Ebony fusion protein enabled its highest soluble expression in WSL2E strain, achieving 93.1% conversion efficiency—3.5-fold higher catalytic efficiency than WSGE strain. Response surface methodology optimized sodium alginate-polyvinyl alcohol (SA-PVA) immobilization parameters (5% PVA, 3% SA, 2.3% CaCl₂), yielding excellent immobilized WSL2E@SA-PVA cells with 95.93% activity recovery. Structural characterization by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) confirmed the formation of a porous SA-PVA matrix that protected cells from harsh conditions. The immobilized biocatalyst exhibited superior operational stability (retaining > 80% activity after 7 cycles) and storage stability (maintaining 44.89% activity after 14 days at 4°C). Fed-batch scale-up (50 mL) achieved a record carcinine titer of 71.13 mM, mitigating the inhibitory effect of high substrate concentrations through phased substrate feeding. This study provides a scalable biocatalytic platform for industrial carcinine production, effectively addressing key bottlenecks in biocatalyst stability and substrate tolerance.
Opportunistic colonization and recurring infection by Pseudomonas aeruginosa are substantial risks to lung functionality for people with underlying respiratory diseases such as cystic fibrosis and chronic obstructive pulmonary disease. The complex metabolic and phenotypic adaptations P. aeruginosa exhibits in response to its environmental conditions make relevant in vitro models of pathogenic populations crucial for identifying and evaluating effective antimicrobial targets. However, an extracellular component that is rarely integrated into these experimental platforms is a spatially extensive, semisolid gel medium representative of biological respiratory mucus layers that P. aeruginosa propagates through via active motility. In this investigation, we examine the applicability of swim plate assays, a qualitative methodology for measuring flagellar swimming motility, as an in vitro platform to study the spatiotemporal development of P. aeruginosa strain PA14. The propagation behavior of PA14 was tracked through time-lapse microscopy and studied under different agar gel compositions incorporating methylcellulose as well as native MUC5AC mucin. To aid quantitative characterization of PA14 population expansion, we paired this experimental workflow with a continuum model that would fit density profile fluctuations to changes in PA14 swimming motility and growth kinetics. We observed higher extracellular concentration and production of the phenazine pyocyanin when PA14 populations were grown in swim plate assays, supporting the emergence of heterogeneous growth environments within the microbial population. PA14 swim plates exhibited a significantly lower spreading velocity in gels containing 0.30% w/v MUC5AC, which model-to-experiment fitting results determined to be driven by reductions in PA14 swimming motility. Continuum model parameters additionally portrayed PA14 expansion in mucin gels, having cell growth outcompeting cell motility, which aligned with experimental assay observations of macrocolonies rapidly developing to high biomass density states. In contrast, PA14 did not show spreading velocity differences in gels containing 0.30% methylcellulose, and fitted parameters did not identify major growth and motility differences when compared to agar-only gels. Combined with the resource accessibility of this experimental platform, the swim plate assay as an in vitro model is well suited to investigations of pathogenic community dynamics in gel conditions over more extensive spatial and time scales.
Microglia are critical regulators of brain homeostasis and immune responses in the central nervous system (CNS). However, existing human-based models fail to reproduce the early and complex microglia-neural cell interactions. The differentiation of human induced pluripotent stem cells (hiPSCs) into specialized cell types offers promising avenues for understanding human development and disease modeling. Herein, a methodology for the differentiation of hiPSC-derived erythromyeloid progenitors (iEMPs) and their 3D co-culture with hiPSC-derived neurospheres were explored, utilizing the Ambr 250 Modular stirred-tank bioreactor (STB) system. The aim of this study was to build a complex co-culture model between iEMP and neurospheres in a scalable and controlled environment. Our results demonstrate that the STB effectively supports the co-culture process, with iEMP integration into the neurospheres, exhibiting cell density, aggregate morphology, and concentration similar to the neurosphere cultures. The co-culture environment induced the upregulation of transcription factors critical for microglial lineage commitment. iEMP-neurospheres displayed a unique secretory profile, releasing proteins involved in extracellular matrix remodeling and neuronal differentiation, essential for microenvironment remodeling. In conclusion, this study underscores the role of iEMPs in CNS development and presents a robust platform for preclinical research.
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