Journal of Biological Engineering最新文献

Cell fate decisions are regulated by intricate signaling networks, with Extracellular signal-Regulated Kinase (ERK) being a central node in the control of cell proliferation and differentiation. ERK typically cooperates with a network of other regulators, making it necessary to study multiple signaling pathways simultaneously at the single-cell level. Many existing fluorescent biosensors for ERK and other pathways have significant spectral overlap, limiting their utility for multiplexing. To address this limitation, we developed two novel red-FRET ERK biosensors, REKAR67 and REKAR76, which operate in the 670-720 nm range using the fluorescent proteins miRFP670nano3 and miRFP720. REKAR67 and REKAR76 differ in fluorophore position, which impacts biosensor characteristics; REKAR67 displayed a higher dynamic range but greater signal variance than REKAR76. In both polyclonal and clonal populations, REKAR67- or REKAR76-expressing cells displayed similar Signal-to-Noise ratios (SNR). Overall, the red-FRET ERK biosensors were highly consistent with existing CFP/YFP biosensors in reporting ERK activity. Both REKAR biosensors expand the available tools for measuring single-cell ERK activity by being spectrally compatible with other CFP/YFP FRET and cpGFP -based biosensors, allowing for multiplexed imaging.

Background: Cellular transport systems are key determinants of intracellular molecule concentrations and can be utilized to modulate the responsiveness of transcription factor (TF)-based biosensors. Efflux pumps facilitate the export of small molecules; therefore, their activity can directly compromise the accuracy of biosensor-based assays. We engineered a cellular export machinery to enhance the responsiveness and reduce crosstalk of a DmpR-based biosensor, which employs the phenol-responsive TF DmpR to detect intracellular phenolic ligands but is affected by ligand diffusion between cells. To overcome this limitation, efflux pump genes were knocked out to minimize ligand diffusion and improve signal fidelity.

Results: Among the efflux pumps tested, deletion of mdtA was found to promote effective intracellular accumulation of phenolic compounds. This strategy not only increased biosensor sensitivity by up to 19-fold but also reduced false positives during enzyme screening by suppressing intercellular diffusion of enzymatic products. In the mock library experiment, the proportion of false positives relative to the total positive cells was 74% in the wild-type strain, whereas it was only 5% in the ΔmdtA strain. To demonstrate the applicability of this approach to an enzyme screening platform, we targeted penicillin G acylase (PGA), an enzyme useful for producing semi-synthetic antibiotics. Knockout of mdtA effectively reduced false positives during flow cytometry-based high-throughput screening. Using this biosensor platform, several PGA variants with improved catalytic activity were successfully identified from a random mutagenesis library.

Conclusions: Overall, host engineering to adjust cellular conditions and ligand concentrations provides a versatile approach to enhance the sensitivity, precision, and efficiency of single-cell-based enzyme screening platforms.

Background: Chinese hamster ovary (CHO) cells are the predominant cell line used for biotherapeutic production. To reduce the cost of therapeutic recombinant proteins produced in CHO cells, efforts have been made over decades to improve overall yield through media and process optimization, as well as genetic engineering aimed at enhancing cell proliferation or productivity. Within an intricate cellular framework, such as the CHO cell system, it is indisputable that numerous genes with seemingly disparate functions may be involved in the process of expressing recombinant proteins.

Results: Thrombomodulin (TM), encoded by the Thbd gene, is primarily known for its roles in coagulation, innate immunity, inflammation, and tumor cell proliferation. Our research was the first to reveal that the presence of thrombomodulin is highly correlated with recombinant protein production in CHO cells producing an Fc-fusion protein. Knocking out Thbd resulted in approximately an 82% reduction in recombinant protein yield by the end of fed-batch culture, indicating that TM is essential for efficient production. Further investigation revealed that this loss was due to a dramatic reduction in mRNA levels of the recombinant protein. Re-expression of TM in the Thbd-knockout cell line restored mRNA levels, confirming TM's role in maintaining transcription. Phosphorylation levels of PKC, MEK, and ERK were elevated in the knockout cells compared to untreated wild-type cells, whereas phosphorylation of mTOR and AKT was decreased. Additionally, overexpression of Thbd led to moderate increases in c-Myc and Bcl2 expression, which appeared to slow the decline in cell viability during cultivation. Functional analyses of different TM domains revealed that both the N-terminal lectin-like domain and the C-terminal cytoplasmic tail have greater impacts on recombinant protein production than the other regions.

Conclusions: This study demonstrates the essential role of thrombomodulin in recombinant Fc-fusion protein production in Chinese hamster ovary cells, reveals novel biological functions of thrombomodulin, and expands our understanding of the complex cellular machinery underlying recombinant protein expression in CHO cells.

Multiple myeloma (MM) is a hematologic malignancy characterized by uncontrolled expansion of malignant plasma cells within the bone marrow microenvironment. While suspension cultures of MM cell lines and murine models have been the cornerstone of research with MM pathogenesis, these conventional systems fail to recapitulate critical aspects of the human tumor microenvironment. Specifically, current models inadequately address key biological questions including mechanisms of drug resistance acquisition, immune evasion strategies, and the role of cellular crosstalk in disease progression. These limitations stem from the absence of physiologically relevant extracellular matrix architecture, lack of primary human stromal and immune cell populations, and inability to model the bone marrow niche with functional vasculature. Three-dimensional (3D) culture platforms have emerged to address these deficiencies by incorporating structural complexity and cellular heterogeneity. However, many existing 3D models remain insufficient for comprehensive MM modeling, as they typically lack integrated human-derived stromal compartments, functional immune surveillance mechanisms, and physiological vascular networks that collectively regulate MM pathobiology. Advanced humanized in vitro models-particularly those incorporating patient-derived cells within immunocompetent microenvironments-are needed to bridge the translational gap between preclinical findings and clinical outcomes. We analyze the evolution from conventional in suspension cultures to current organotypic systems while examining their applications in mechanistic studies and capabilities in therapeutic screening. Lastly, we outline the emerging challenges in model development and propose future research directions, with particular emphasis on establishing fully humanized, immunocompetent platforms that authentically reproduce the bone marrow ecosystem for predictive drug testing.

Preterm premature rupture of membranes (PPROM) remains a leading cause of spontaneous preterm birth and neonatal morbidity, yet current clinical management strategies are limited. The fetal membranes, composed of the amnion and chorion, possess limited regenerative capacity once ruptured. Recent advances in biomaterials and tissue engineering have introduced promising therapeutic platforms capable of sealing membrane defects and promoting biological healing. These approaches offer significant advantages over traditional methods by providing dynamic, customizable, and biologically integrated solutions that better mimic the native extracellular matrix (ECM) and enhance tissue regeneration. This review summarizes biomaterial-based strategies, including collagen-based and natural ECM-derived materials, growth factor and drug delivery platforms, bioadhesives, membrane patch systems, and 3D bioprinting and in situ biofabrication. These materials are increasingly engineered to mimic native extracellular matrix properties, support cell migration, modulate local inflammation, and conform to the dynamic intrauterine environment. Preclinical studies in small and large animal models have demonstrated the feasibility of these systems in achieving defect closure, reducing inflammation, and prolonging gestation. Despite encouraging results, challenges remain related to biocompatibility, degradation kinetics, intrauterine delivery, and regulatory approval. This review underscores the potential of biomaterial strategies to transform the management of PPROM and outlines future directions for translating these technologies into clinical practice.

Active transport of chemical species across the cell membrane represents a critical biological and biotechnological function, allowing the cell to selectively import compounds of nutritional value whilst exporting potentially toxic compounds. Major facilitator superfamily (MFS) transporters represent a ubiquitous class able to uptake and export an array of different chemical species. When designing biosynthetic pathways within microbial hosts, for production or remediation, transport is often critical to the efficiency of the resulting engineered strain. However, transport is a commonly neglected node for characterisation and engineering given difficulties in producing, purifying and assaying membrane transport proteins outside of their native environment. Here, using syntenic analysis and genetically encoded biosensors a library of 11 TphK and 10 PcaK homologs were screened for their ability to uptake the aromatic acids, protocatechuic acid and terephthalic acid. The structure activity relationships of the corresponding PcaK and TphK transporter-biosensor constructs, were then assessed with a library of aromatic acid effectors. Finally, the feasibility of protein engineering was assessed, by the creation of chimeric MFS transporter-biosensor constructs, revealing a degree of effector recognition plasticity and the modularity of core transmembrane domains. This study provides a library of validated TphK and PcaK homologs and demonstrates the value of employing genetically encoded biosensors in the characterisation and engineering of this important transport function.

Background: Tantalum (Ta) and its derivatives are inert, possess mechanical qualities such as corrosion resistance, and are biocompatible. They also offer structural support during surgical correction, such as bone grafts during surgery, in instances of dental or skeletal disabilities. However, various sizes of Ta particles could be expelled from the implant's surface due to mechanical stress and load-induced wear caused by micromotion between loose implant surfaces during usage. Therefore, the study examined the effects of nano (25 nm) and micro-sized Ta particles (10 μm and 40-50 μm) on osteoblasts and osteoclasts.

Results: Osteoblasts efficiently phagocytosed 25 nm sized Ta particles compared to micro-sized particles and triggered significant biological effects. Only 25 nm Ta particles suppressed ALP activity, downregulated osteogenic regulators and markers, and inhibited collagen synthesis and mineralization. Moreover, 25 nm sized Ta particles induced inflammatory responses in osteoblasts by increasing Cox-2 expression and activating the NFkB signaling pathway. Nano-sized Ta particles induced intracellular ROS generation in osteoblasts and osteoclasts. Compared to micro-sized Ta particles, 25 nm sized Ta particles stimulated osteoclast formation, but ROS scavenging by N-acetyl cysteine (NAC) inhibited Ta particle-mediated osteoclastogenesis. Likewise, ALP activity of osteoblasts was partially restored after NAC treatment. 25 nm Ta particles suppressed Axin-2 reporter activity and protein levels of pGSK3β and β-catenin stability, implicating suppressed WNT signaling in treated osteoblasts. Expression levels of several families of antagonists like DKK, sFRP, and SOST of the WNT signaling pathway were found elevated several-fold in 25 nm-sized Ta particles treated osteoblasts, explaining suppressed WNT signaling pathway in exposed osteoblasts.

Conclusion: Ta supports osseointegration and biocompatibility, but micromotion-induced nanoscale wear particles may disrupt osteoprogenitor function and enhance osteoclast activity, risking implant loosening. Thus, vigilant post-operative monitoring for nano-sized Ta particles is advisable and critical in detecting early osteolysis and ensuring implant longevity.