Journal of Biological Engineering最新文献

Background: The clustered regulatory interspaced short palindromic repeats (CRISPR)-Cas13a system has strong potential for highly sensitive detection of exogenous sequences. The detection of KRAS^G12 point mutations with low allele frequencies may prove powerful for the formal diagnosis of pancreatic ductal adenocarcinoma (PDAC).

Results: We implemented preamplification of KRAS alleles (wild-type and mutant) to reveal the presence of mutant KRAS with CRISPR-Cas13a. The discrimination of KRAS^G12D from KRAS^WT was poor for the generic KRAS preamplification templates and depended on the crRNA design, the secondary structure of the target templates, and the nature of the mismatches between the guide and the templates. To improve the specificity, we used an allele-specific PCR preamplification method called CASPER (Cas13a Allele-Specific PCR Enzyme Recognition). CASPER enabled specific and sensitive detection of KRAS^G12D with low DNA input. CASPER detected KRAS mutations in DNA extracted from patients' pancreatic ultrasound-guided fine-needle aspiration fluid.

Conclusion: CASPER is easy to implement and is a versatile and reliable method that is virtually adaptable to any point mutation.

RNA recognition motifs (RRMs) are widespread RNA-binding protein domains in eukaryotes, which represent promising synthetic biology tools due to their compact structure and efficient activity. Yet, their use in prokaryotes is limited and their functionality poorly characterized. Recently, we repurposed a mammalian Musashi protein containing two RRMs as a translation regulator in Escherichia coli. Here, employing high-throughput RNA sequencing, we explored the impact of Musashi expression on the transcriptomic and translatomic profiles of E. coli, revealing certain metabolic interference, induction of post-transcriptional regulatory processes, and spurious protein-RNA interactions. Engineered Musashi protein mutants displayed compromised regulatory activity, emphasizing the importance of both RRMs for specific and sensitive RNA binding. We found that a mutation known to impede allosteric regulation led to similar translation control activity. Evolutionary experiments disclosed a loss of function of the synthetic circuit in about 40 generations, with the gene coding for the Musashi protein showing a stability comparable to other heterologous genes. Overall, this work expands our understanding of RRMs for post-transcriptional regulation in prokaryotes and highlight their potential for biotechnological and biomedical applications.

Epithelial tissues respond strongly to the mechanical stress caused by collective cell migration and are able to regulate it, which is important for biological processes such as morphogenesis, wound healing, and suppression of the spread of cancer. Compressive, tensional, and shear stress components are produced in cells when epithelial monolayers on substrate matrices are actively or passively wetted or de-wetted. Increased compressive stress on cells leads to enhanced cell-cell interactions by increasing the frequency of change the cell-cell distances, triggering various signalling pathways within the cells. This can ultimately lead either to cell jamming or to the extrusion of live cells. Despite extensive research in this field, it remains unclear how cells decide whether to jam, or to extrude a cell or cells, and how cells can reduce the compressive mechanical stress. Live cell extrusion from the overcrowded regions of the monolayers is associated with the presence of topological defects of cell alignment, induced by an interplay between the cell compressive and shear stress components. These topological defects stimulate cell re-alignment, as a part of the cells' tendency to re-establish an ordered trend of cell migration, by intensifying the glancing interactions in overcrowded regions. In addition to individual cell extrusion, collective cell extrusion has also been documented during monolayer active de-wetting, depending on the cell type, matrix stiffness, and boundary conditions. Cell jamming has been discussed in the context of the cells' contact inhibition of locomotion caused by cell head-on interactions. Since cell-cell interactions play a crucial role in cell rearrangement in an overcrowded environment, this review is focused on physical aspects of these interactions in order to stimulate further biological research in the field.

Effective enzyme stabilization through immobilization is essential for the functional usage of enzymatic reactions. We propose a new method for synthesizing elastic hydroxyapatite microgel (E-HAp-M) materials and immobilizing lipase using this mesoporous mineral via the ship-in-a-bottle-neck strategy. The physicochemical parameters of E-HAp-M were thoroughly studied, revealing that E-HAp-M provides efficient space for enzyme immobilization. As a model enzyme, lipase (LP) was entrapped and then cross-linked enzyme structure, preventing leaching from mesopores, resulting in highly active and stable LP/E-HAp-M composites. By comparing LP activity under different temperature and pH conditions, it was observed that the cross-linked LP exhibited improved thermal stability and pH resistance compared to the free enzyme. In addition, they demonstrated a 156% increase in catalytic activity compared with free LP in hydrolysis reactions at room temperature. The immobilized LP maintained 45% of its initial activity after 10 cycles of recycling and remained stable for over 160 days. This report presents the first demonstration of a stabilized cross-linked LP in E-HAp-M, suggesting its potential application in enzyme-catalyzed processes within biocatalysis technology.

Introduction: Microphysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation.

Methods: Human valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts.

Results: Hypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h.

Conclusion: In hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS.

Considerable attention has been paid to exploring the biotechnological applications of several Monascus sp. for pigment production. In this study, our focus is on enhancing the bioprocessing of red pigment (RP) derived from the endophytic fungus Monascus ruber SRZ112. To achieve this, we developed a stable mutant strain with improved productivity through gamma irradiation. This mutant was then employed in the immobilization technique using various entrapment carriers. Subsequently, we optimized the culture medium for maximal RP production using the Response Surface Methodology. Finally, these immobilized cultures were successfully utilized for RP production using a semi-continuous mode of fermentation. After eight cycles of fermentation, the highest RP yield by immobilized mycelia reached 309.17 CV mL^-1, a significant increase compared to the original titer. Importantly, this study marks the first report on the successful production of Monascus RP in a semi-continuous mode using gamma rays' mutant strain, offering prospects for commercial production.