Biotechnology and Bioengineering最新文献

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, K_M, 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.

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.

Hydrogels are commonly used to immobilize mammalian cells, offering mechanical support in 3D cultures and acting as barriers for immunoprotection in transplantation, such as islet encapsulation for diabetes therapy. Cell immobilization restricts bulk fluid motion, resulting in diffusion-limited molecular transport and nutrient concentration gradients, particularly for oxygen consumed by immobilized cells. Oxygen mass transport models are essential for designing immobilization strategies but often rely on assumed diffusion coefficients due to a lack of experimental data. We propose a cost-effective, accessible system for experimentally measuring oxygen diffusion coefficients in cell-laden hydrogels, tested on alginate-immobilized pancreatic beta cells (MIN6). Compared to water, oxygen diffusivity was significantly lower in alginate gels and inversely correlated with the dynamic loss modulus. Diffusivity also decreased with increasing alginate concentration from 2% to 5%. Cell viability depended heavily on gel concentration and cell density, as predicted by Thiele modulus and effectiveness factor values calculated from the measured diffusion coefficients. This platform, combining a simple experimental setup with dimensionless numbers, offers a practical way to predict maximal diffusion distances in cell immobilization strategies. The proposed approach can support rational design of cell encapsulation, immobilized cell culture, and tissue engineering strategies.

The cover image is based on the article Succinic Acid Production by Engineered Mannheimia succiniciproducens and Its Use in Chemoenzymatic Poly(Butylene Succinate) Synthesis by Ji Yeon Kim et al., https://doi.org/10.1002/bit.70072.

Removing host cell proteins (HCPs) during biotherapeutic manufacturing is essential to ensure patient safety and supply continuity. Yet, this goal remains exceptionally challenging for some HCP species. For example, phospholipase B-like-2 (PLBL2) from Chinese hamster ovary (CHO) cells plagues process engineers by evading typical purification strategies. Separation challenges for CHO PLBL2 and other HCPs are due in large part to a dearth of detailed biochemical characterization for HCPs, such as their proteoforms or variants. Herein, we present as a case study the elucidation of proteoforms of PLBL2, which was endogenously expressed in CHO alongside pembrolizumab (a monoclonal IgG4 antibody) and inadvertently co-purified following a typical downstream process. Using site-specifically modified polyclonal anti-CHO PLBL2 antibodies immobilized onto a solid support, CHO PLBL2 was captured from pembrolizumab, enriched, and recovered, enabling deep characterization by mass spectrometry and other techniques. These analyses revealed a detailed understanding of CHO PLBL2's proteoforms, including charge variants, N-linked and O-linked glycosylation, phosphorylation, and cleavage to afford multiple molecular weight isoforms. To the best of our knowledge, this is the first published, deep characterization of a challenging CHO HCP. Armed with a greater understanding of the impurities that plague purification processes, we can design new, targeted strategies to ensure their removal and safeguard both patient safety and biotherapeutic supplies. While our work centered on CHO PLBL2, our antibody immobilization, affinity enrichment, and characterization approach should be broadly applicable to enable the examination of innumerable challenging HCPs from modalities across the increasingly diverse bioprocessing landscape.

Human embryonic kidney cells HEK293 are widely used in biopharmaceutical manufacturing, with a recent surge particularly in recombinant adeno-associated virus production. Despite their industrial relevance, comprehensive data on their genomic background and stability remains limited. Here, we systematically analyze the genetic landscape of various HEK293 cell lines in response to cultivation conditions, clonal selection, genetic manipulation and over time in culture. Adherent HEK293 were adapted to suspension growth using different serum-free media. Whole genome sequences from these cell lines were analyzed together with previously published data from additional variants in common use. All data sets were aligned against the human reference genome, enabling the assessment of genome stability by evaluation of variants and revealing a conserved genetic core across all lines, regardless of cultivation history or phenotypic divergence. Evaluation of the functional implications of conserved core mutations identified an enrichment in genes related to cellular structure, morphology and cellular connectivity. The distribution of structural variants and single nucleotide polymorphisms indicated a gradual accumulation of mutations over time in culture rather than abrupt shifts in response to environmental changes. Notably, the integrated adenoviral genes remained highly conserved with respect to copy number, integration site and sequence integrity. These findings provide insight into the genomic evolution of HEK293 cells and offer a foundation for further multi-omics studies aimed at optimizing HEK293 cells for applications in biopharmaceutical production.