Journal of Biological Engineering最新文献

This study addresses the challenges in the early diagnosis of deep infiltrating endometriosis (DIE) by exploring the potential role of the deubiquitinating enzyme USP14. By analyzing the GSE141549 dataset from the Gene Expression Omnibus (GEO) database, using bioinformatics methods and three machine learning algorithms (LASSO, Random Forest, and Support Vector Machine), the key feature gene USP14 was identified. The results indicated that USP14 is significantly upregulated in DIE and exhibits good predictive value (AUC = 0.786). Further analysis revealed the important role of USP14 in muscle function, cellular growth factor response, and maintenance of chromosome structure, and its close association with various immune cell functions. Immunohistochemical staining confirmed the high expression of USP14 in DIE tissues. This study provides a new molecular target for the early diagnosis of DIE, which holds significant clinical implications and potential application value.

Background: Yeast Saccharomyces cerevisiae is widely recognised as a versatile chassis for constructing microbial cell factories. However, producing chemicals from toxic, highly concentrated, or cell-impermeable substrates, or chemicals dependent on enzymatic reactions incompatible with the yeast's intracellular environment, remains challenging. One such chemical is 2-O-(α-D-glucopyranosyl)-sn-glycerol (glucosyl glycerol, αGG), a natural osmolyte used in the cosmetics and healthcare industries. This compound can be synthesised in a one-enzyme reaction from sucrose and glycerol by Leuconostoc mesenteroides sucrose phosphorylase (SucP), an enzyme which, in a low-water, glycerol-rich, phosphate-free environment, transfers the glucosyl moiety from sucrose to glycerol.

Results: In this study, we engineered a yeast microbial cell factory for αGG production. For this purpose, we first focused on the abundant yeast GPI-anchored cell wall protein Ccw12 and used our insights to develop a miniature Ccw12-tag, which adds only 1.1 kDa to the enzyme of interest while enabling its covalent attachment to the cell wall. Next, we Ccw12-tagged SucP and expressed it in an invertase-negative strain of yeast S. cerevisiae from the PHO5 promoter, i.e., promoter strongly induced under phosphate-free conditions. Such SucP isoform, covalently C-terminally anchored to the outer cell surface, produced extracellularly 37.3 g l^- 1 (146 mM) of αGG in five days, while the underlying chassis metabolised reaction by-products, thereby simplifying downstream processing.

Conclusions: The here-described S. cerevisiae strain, displaying C-terminally anchored sucrose phosphorylase on its cell surface, is the first eukaryotic microbial cell factory capable of a one-step αGG production from the readily available substrates sucrose and glycerol.

Background: The successful production of industrially relevant natural products hinges on two key factors: the cultivation of robust microbial chassis capable of synthesizing the desired compounds, and the availability of reliable genetic tools for expressing target genes. The development of versatile and portable genetic tools offers a streamlined pathway to efficiently produce a variety of compounds in well-established chassis organisms. The σ⁷⁰lac and tet expression systems - adaptations of the widely used lac and tet regulatory systems developed in our laboratory - have shown effective regulation and robust expression of recombinant proteins in various Gram-negative bacteria. Understanding the strengths and limitations of these regulatory systems in controlling recombinant protein production is essential for progress in this area.

Results: To assess their capacity for combinatorial control, both the σ⁷⁰lac and tet expression systems were combined into a single plasmid and assessed for their performance in producing fluorescent reporters as well as the terpenoids lycopene and β-carotene. We thoroughly characterized the induction range, potential for synergistic effects, and metabolic costs of our dual σ⁷⁰lac and tet expression system in the well-established microorganisms Escherichia coli, Pseudomonas putida, and Vibrio natriegens using combinations of fluorescent reporters. The dynamic range and basal transcriptional control of the σ⁷⁰ expression systems were further improved through the incorporation of translational control mechanisms via toehold switches. This improvement was assessed using the highly sensitive luciferase reporter system. The improvement in control afforded by the integration of the toehold switches enabled the accumulation of a biosynthetic intermediate (lycopene) in the β-carotene synthesis pathway.

Conclusion: This study presents the development and remaining challenges of a set of versatile genetic tools that are portable across well-established gammaproteobacterial chassis and capable of controlling the expression of multigene biosynthetic pathways. The enhanced σ⁷⁰ expression systems, combined with toehold switches, facilitate the biosynthesis and study of enzymes, recombinant proteins, and natural products, thus providing a valuable resource for producing a variety of compounds in microbial cell factories.

Mitochondrial abnormalities underscore a variety of neurologic injuries and diseases and are well-studied in adult populations. Clinical studies identify critical roles of mitochondria in a wide range of developmental brain injuries, but models that capture mitochondrial abnormalities in systems representative of the neonatal brain environment are lacking. Here, we develop an organotypic whole-hemisphere (OWH) brain slice model of mitochondrial dysfunction in the neonatal brain. We extended the utility of complex I inhibitor rotenone (ROT), canonically used in models of adult neurodegenerative diseases, to inflict mitochondrial damage in OWH slices from term-equivalent rats. We quantified whole-slice health over 6 days of exposure for a range of doses represented in ROT literature. We identified 50 nM ROT as a suitable exposure level for OWH slices to inflict injury without compromising viability. At the selected exposure level, we confirmed exposure- and time-dependent mitochondrial responses showing differences in mitochondrial fluorescence and nuclear localization using MitoTracker imaging in live OWH slices and dysregulated mitochondrial markers via RT-qPCR screening. We leveraged the regional structures present in OWH slices to quantify cell density and cell death in the cortex and the midbrain regions, observing higher susceptibilities to damage in the midbrain as a function of exposure and culture time. We supplemented these findings with analysis of microglia and mature neurons showing time-, region-, and exposure-dependent differences in microglial responses. We demonstrated changes in tissue microstructure as a function of region, culture time, and exposure level using live-video epifluorescence microscopy of extracellularly diffusing nanoparticle probes in live OWH slices. Our results highlight severity-, time-, and region-dependent responses and establish a complimentary model system of mitochondrial abnormalities for high-throughput or live-tissue experimental needs.

Peptide drugs have seen rapid advancement in biopharmaceutical development, with over 80 candidates approved globally. Despite their therapeutic potential, the clinical translation of peptide drugs is hampered by challenges in production yields and stability. Engineered bacterial therapeutics is a unique approach being explored to overcome these issues by using bacteria to produce and deliver therapeutic compounds at the body site of use. A key advantage of this technology is the possibility to control drug delivery within the body in real time using genetic switches. However, the performance of such genetic switches suffers when used to control drugs that require post-translational modifications or are toxic to the host. In this study, these challenges were experienced when attempting to establish a thermal switch for the production of a ribosomally synthesized and post-translationally modified peptide antibiotic, darobactin, in probiotic E. coli. These challenges were overcome by developing a thermo-amplifier circuit that combined the thermal switch with a T7 RNA Polymerase. Due to the orthogonality of the Polymerase, this strategy overcame limitations imposed by the host transcriptional machinery. This circuit enabled production of pathogen-inhibitory levels of darobactin at 40 °C while maintaining leakiness below the detection limit at 37 °C. Furthermore, the thermo-amplifier circuit sustained gene expression beyond the thermal induction duration such that with only 2 h of induction, the bacteria were able to produce pathogen-inhibitory levels of darobactin. This performance was maintained even in physiologically relevant simulated conditions of the intestines that include bile salts and low nutrient levels.

The NK-92MI cell line has displayed significant promise in clinical trials for cancer treatment. However, challenges persist in obtaining sufficient cell quantities and achieving optimal cytotoxicity. The proliferation of natural killer (NK) cells involves the formation of cell aggregates, but excessively large aggregates can impede nutrient and waste transport, leading to reduced cell survival rates. In this study, a custom bioreactor was designed to mimic pseudostatic culture conditions by integrating brief mechanical rotation during a 6-h static culture period. This method aimed to achieve an optimal aggregate size while improving cell viability. The findings revealed a 144-fold expansion of 3D NK-92MI cell aggregates, reaching an ideal size of 80-150 µm, significantly increasing both cell proliferation and survival rates. After 14 days of culture, the NK-92MI cells maintained their phenotype during the subsequent phase of cell activation. Moreover, these cells presented elevated levels of IFN-γ expression after IL-18 activation, resulting in enhanced NK cell-mediated cytotoxicity against K562 cells. This innovative strategy, which uses a closed suspension-based culture system, presents a promising approach for improving cell expansion and activation techniques in immunocellular therapy.

Adenosine triphosphate (ATP) assays have a faster turnaround time and higher sensitivity than traditional cultivation methods for microbial monitoring. Challenges implementing ATP testing include incompatibility with chlorine quenching agents and hold time sensitivity, which are not well-studied. Chlorinated distribution system samples were collected from two Canadian utilities, Metro Vancouver (n = 40 samples) and Halifax Water (n = 283). No significant correlations were observed between heterotrophic cell count (HPC) and cellular ATP, suggesting these do not correlate well in waters with low biological activity (median HPC < 2 CFU/mL). However, interpretation of HPC and cATP results (based on the HPC guideline of 100 CFU/mL and cATP of 10 pg/mL) yielded the same conclusion for 95% of samples, suggesting a potential decision-making framework to replace HPC with cATP. Moreover, cATP correlates better with free chlorine (p < 0.04) compared with HPC for one of the studied systems. Importantly, adding chlorine quench (10% sodium thiosulfate) did not produce significantly different cATP results, nor did analyzing at various hold times of 4-, 6-, and 24-h. This study supports the integration of ATP testing into existing sampling procedures for water utilities, as a sensitive, fast, and reliable monitoring method.

Background: De-novo-designed synthetic transcriptional regulators have great potential as the genetic parts for constructing complex multilayered gene circuits. The design flexibility afforded by advanced nucleic acid sequence design tools vastly expands the repertoire of regulatory elements for circuit design. In principle, the design space of synthetic regulators should allow for the construction of regulatory circuits of arbitrary complexity; still, the orthogonality and robustness of such components have not been fully elucidated, thereby limiting the depth and width of synthetic circuits.

Results: In this work, we systematically explored the design strategy of synthetic transcriptional regulators, termed switchable transcription terminators. Specifically, by redesigning key sequence domains, we created a high-performance switchable transcription terminator with a maximum fold change of 283.11 upon activation by its cognate input RNA. Further, an automated design algorithm was developed for these elements to improve orthogonality for a complex multi-layered circuit construction. The resulting orthogonal switchable transcription terminators could be used to construct a three-layer cascade circuit and a two-input three-layer OR gate.

Conclusions: We demonstrated a practical strategy for designing standardized regulatory elements and assembling modular gene circuits, ultimately laying the foundation for the streamlined construction of complex synthetic gene circuits.

The production of value-added bio-compounds from rejuvenated sources and their recruitment for healthcare services are paramount objectives in the agenda of white biotechnology. Hereupon, the current study focused on economic production of single cell oils (SCOs) from oleaginous fungi Alternaria sp. (A-OS) and Drechslera sp. (D-OS) using cheese whey waste stream, followed by their evaluation as antibiofilm and anticancer agents, for the first time. As a sole substrate for growth, the whey aided in lipid accumulation by 3.22 and 4.33 g/L, which representing 45.3 and 48.2% lipid content in Drechslera sp. (D-OS) and Alternaria sp. (A-OS), respectively. Meanwhile, a higher unsaturation degree was detected in A-OS by 62.18% comparing to 53.15% of D-OS, with advantageous presence of omega-6 poly unsaturated fatty acid by 22.67% and 15.04% for A-OS and D-OD, respectively, as revealed by GC-MS and FTIR characterization analysis. Interestingly, an eminent and significant (P ≤ 0.05) antibiofilm potency was observed in a dose-dependent modality upon employing both SCOs as antibiofilm agents. Whereas, 100 µg/mL of A-OS recorded superior inhibition of P. aeruginosa, S. aureus and C. albicans biofilms development by 84.10 ± 0.445, 90.37 ± 0.065 and 94.96 ± 0.21%, respectively. Whereas, D-OS (100 µg/mL) thwarted the biofilms of P. aeruginosa, S. aureus and C. albicans by 47.41 ± 2.83, 62.63 ± 5.82 and 78.67 ± 0.23%, correspondingly. Besides, the metabolic performance of cells within biofilm matrix, protein, carbohydrate contents and hydrophobicity of examined biofilms were also curtailed in a significant correlation with biofilm biomass (r ≥ 0.9). Further, as anticancer agents, D-OS recorded higher potency against A549 and CaCo-2 cell lines with IC50 values of 2.55 and 3.425% and SI values of 10.1 and 7.5, respectively. However, A-OS recorded 8.275% and 2.88 for IC50 and SI of Caco-2 cells, respectively. Additionally, A-OS activated caspase 3 by 64.23 ± 1.18% and 53.77 ± 0.995% more than D-OS (52.09 ± 0.222% and 49.72 ± 0.952%) in A549 and Caco-2 cells, respectively. Furthermore, the enzymes, which associated with cancer invasion, metastasis, and angiogenesis (i.e., MMP2 and MMP9) were strongly inhibited by A-OS with 18.58% and 8.295%, respectively as IC50 values; while D-OS results recorded 23.61% and 13.16%, respectively, which could be ascribed to the higher ω-6/ω-3 contents of A-OS. The promising results of the current study opens up the vision to employ SCOs as anti-infective nutraceuticals and in complementary/alternative therapy and prophylactic programs as well.

Type 2 diabetes mellitus (T2DM) is a major public health concern with significant cardiovascular complications (CVD). Despite extensive epidemiological data, the molecular mechanisms relating hyperglycemia to CVD remain incompletely understood. We here investigated the impact of chronic hyperglycemia on human aortic smooth muscle cells (HASMCs) cultured under varying glucose conditions in vitro, mimicking normal (5 mmol/L), pre-diabetic (10 mmol/L), and diabetic (20 mmol/L) conditions, respectively. Normal HASMC cultures served as baseline controls, and patient-derived T2DM-SMCs served as disease controls. Results showed significant increases in cellular proliferation, area, perimeter, and F-actin expression with increasing glucose concentration (p < 0.01), albeit not exceeding the levels in T2DM cells. Atomic force microscopy analysis revealed significant decreases in Young's moduli, membrane tether forces, membrane tension, and surface adhesion in SMCs at higher glucose levels (p < 0.001), with T2DM-SMCs being the lowest among all the cases (p < 0.001). T2DM-SMCs exhibited elevated levels of selected pro-inflammatory markers (e.g., ILs-6, 8, 23; MCP-1; M-CSF; MMPs-1, 2, 3) compared to glucose-treated SMCs (p < 0.01). Conversely, growth factors (e.g., VEGF-A, PDGF-AA, TGF-β1) were higher in SMCs exposed to high glucose levels but lower in T2DM-SMCs (p < 0.01). Pathway enrichment analysis showed significant increases in the expression of inflammatory cytokine-associated pathways, especially involving IL-10, IL-4 and IL-13 signaling in genes that are up-regulated by elevated glucose levels. Differentially regulated gene analysis showed that compared to SMCs receiving normal glucose, 513 genes were upregulated and 590 genes were downregulated in T2DM-SMCs; fewer genes were differentially expressed in SMCs receiving higher glucose levels. Finally, the altered levels in genes involved in ECM organization, elastic fiber synthesis and formation, laminin interactions, and ECM proteoglycans were identified. Growing literature suggests that phenotypic switching in SMCs lead to arterial wall remodeling (e.g., change in stiffness, calcific deposits formation), with direct implications in the onset of CVD complications. Our results suggest that chronic hyperglycemia is one such factor that leads to morphological, biomechanical, and functional alterations in vascular SMCs, potentially contributing to the pathogenesis of T2DM-associated arterial remodeling. The observed differences in gene expression patterns between in vitro hyperglycemic models and patient-derived T2DM-SMCs highlight the complexity of T2DM pathophysiology and underline the need for further studies.