Biotechnology Journal最新文献

Global climate deterioration intensifies the demand for exploiting efficient CO₂ utilization approaches. Converting CO₂ to biorefinery feedstock affords an alternative strategy for third-generation biorefineries. However, upcycling CO₂ into complex chiral carbohydrates remains a major challenge. Previous attempts at sugar synthesis from CO₂ either produce mixtures with poor stereoselectivity or require ATP as a cofactor. Here, by redesigning glycolaldehyde synthase, the authors constructed a synthetic pathway for biorefinery feedstock D-xylulose from CO₂ that does not require ATP as a cofactor. The artificial D-xylulose pathway only requires a three-step enzyme cascade reaction to achieve the stereoselective synthesis of D-xylulose at a concentration of 1.2 g L⁻¹. Our research opens up an alternative route toward future production of chemicals and fuels from CO₂.

The 3D multicellular tumor spheroid (MTS) model exhibits enhanced fidelity in replicating the tumor microenvironment and demonstrates exceptional resistance to clinical drugs compared to the 2D monolayer model. In this study, we used multiomics (transcriptome, proteomics, and metabolomics) tools to explore the molecular mechanisms and metabolic differences of the two culture models. Analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways revealed that the differentially expressed genes between the two culture models were mainly enriched in cellular components and biological processes associated with extracellular matrix, extracellular structural organization, and mitochondrial function. An integrated analysis of three omics data revealed 11 possible drug resistance targets. Among these targets, seven genes, AKR1B1, ALDOC, GFPT2, GYS1, LAMB2, PFKFB4, and SLC2A1, exhibited significant upregulation. Conversely, four genes, COA7, DLD, IFNGR1, and QRSL1, were significantly downregulated. Clinical prognostic analysis using the TCGA survival database indicated that high-expression groups of SLC2A1, ALDOC, and PFKFB4 exhibited a significant negative correlation with patient survival. We further validated their involvement in chemotherapy drug resistance, indicating their potential significance in improving prognosis and chemotherapy outcomes. These results provide valuable insights into potential therapeutic targets that can potentially enhance treatment efficacy and patient outcomes.

Laccases are extensively used in the textile industry due to their ability to decolorize dyes, modify fabric surfaces, and bleach textiles. Identifying a laccase with both high thermal stability and alkali tolerance suitable for textile applications presents a significant challenge. A novel alkaline laccase, LacCT, was discovered from Caldalkalibacillus thermarum and successfully expressed it in Escherichia coli. LacCT displayed optimal activity at 65°C and maintained high stability across a pH range of 6.0–10.0, with an optimal pH of 7.5. Through rational design, the thermal stability of the best variant, G190P/Q254Y/G336M/D510F (LacCT-11), was significantly enhanced, resulting in a half-life of 63.2 min at 60°C – 1.8 times longer than that of the wild type. This research introduces a promising new laccase with considerable potential for decolorizing textile wastewater and improving the ramie degumming process.

Interzone/cavitation are key steps in early stage joint formation that have not been successfully developed in vitro. Further, current models of endochondral ossification, an important step in early bone formation, lack key morphology morphological structures such as microcavities found during development in vivo. This is possibly due to the lack of appropriate strategies for incorporating chemical and mechanical stimuli that are thought to be involved in joint development. We designed a bioreactor system and investigated the synergic effect of chemical stimuli (chondrogenesis-inducing [CIM] and hypertrophy-inducing medium [HIM]) and mechanical stimuli (flexion) on the growth of human mesenchymal stem cells (hMSCs) based linear aggregates under different conditions over 4 weeks of perfusion culture. Computational studies were used to evaluate tissue stress qualitatively. After harvesting, both Safranin-O and hematoxylin & eosin (H&E) staining histology demonstrated microcavity structures and void structures in the region of higher stresses for tissue aggregates cultured only in HIM under flexion. In comparison to either HIM treatment or flexion only, increased glycosaminoglycan (GAG) content in the extracellular matrix (ECM) at this region indicates the morphological change resembles the early stage of joint cavitation; while decreased type II collagen (Col II), and increased type X collagen (Col X) and vascular endothelial growth factor (VEGF) with a clear boundary in the staining section indicates it resembles the early stage of ossification. Further, cell alignment analysis indicated that cells were mostly oriented toward the direction of flexion in high-stress region only in HIM under flexion, resembling cell morphology in both joint cavitation and hypertrophic cartilage in growth plate. Collectively, our results suggest that flexion and HIM inhibit chondrogenesis and promote hypertrophy and development of microcavities that resemble the early stage of joint cavitation and endochondral ossification. We believe the tissue model described in this work can be used to develop in vitro models of joint tissue for applications such as pathophysiology and drug discovery.

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.

The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.

The differentiation of bone marrow mesenchymal stem cells (BMSCs) toward osteogenesis can be induced by low-intensity pulsed ultrasound (LIPUS). However, the molecular mechanisms responsible for LIPUS stimulation are unclear. The possible molecular mechanisms by which LIPUS promotes osteogenic differentiation of BMSCs were investigated in this study. The quantification of alkaline phosphatase (ALP) activity, Alizarin Red S staining, ALP staining, and the establishment of a calvarial defect model were used to evaluate osteogenic effects. Immunofluorescence was performed to observe the expression of microfilaments and transient receptor potential melastatin 7 (TRPM7). The levels of F-actin/G-actin and osteogenesis-related proteins under LIPUS alone or LIPUS combined with cytoskeleton interfering drugs (Cytochalasin D [CytoD] or Jasplakinolide [JA]) were assayed by western blot. Quantitative real-time reverse transcription polymerase chain reaction was utilized to measure the expression of Trpm7 mRNA. Moreover, adenoviral Trpm7 knockdown was verified using western blot. The results demonstrated that LIPUS promoted bone formation in vivo. Under osteogenic induction in vitro, the osteogenesis of BMSCs induced by LIPUS was accompanied by the depolymerization and rearrangement of microfilaments and increased levels of TRPM7. By perturbing intracellular actin dynamics, CytoD enhanced the pro-osteogenicity of LIPUS and increased TRPM7 level, while JA inhibited the pro-osteogenicity of LIPUS and reduced TRPM7 level. Additionally, the knockdown of Trpm7 suppressed the osteogenic promotion of BMSCs induced by LIPUS. The transient depolymerization and rearrangement of the cytoskeleton microfilaments mediated by LIPUS can affect TRPM7 expression and subsequently promote the osteogenesis of BMSCs. This study provides further direction for exploring the molecular mechanism of LIPUS, as a mechanical stress, in facilitating the osteogenic differentiation of BMSCs.

The development of 3D organoids has provided a valuable tool for studying human tissue and organ development in vitro. Cerebral organoids, in particular, offer a unique platform for investigating neural diseases. However, current methods for generating cerebral organoids suffer from limitations such as labor-intensive protocols and high heterogeneity among organoids. To address these challenges, we present a microfluidic device designed to automate and streamline the formation and differentiation of cerebral organoids. The device utilizes microwells with two different shapes to promote the formation of a single aggregate per well and incorporates continuous medium flow for optimal nutrient exchange. In silico simulations supported the effectiveness of the microfluidic chip in replicating cellular microenvironments. Our results demonstrate that the microfluidic chip enables uniform growth of cerebral organoids, significantly reducing the hands-on time required for maintenance. Importantly, the performance of the microfluidic system is comparable to the standard 96-well plate format even when using half the amount of culture medium, and the resulting organoids exhibit substantially developed neuroepithelial buds and cortical structures. This study highlights the potential of custom-designed microfluidic technology in improving the efficiency of cerebral organoid culture.