This study evaluated the performance of biofilm reactors inoculated with azo dye degrading Shewanella for the decolorization of Reactive Black 5 (RB5), using three different carrier materials, namely almond shell biochar, moving bed biofilm reactor (MBBR), and polypropylene carrier (PPC). The reactors were fed with low-nutrient artificial wastewater containing RB5 for a long term, and all three carriers showed good RB5 decolorization performance, with varying efficiencies. LC-MS analysis revealed significant differences in the degradation pathways of RB5 among the carriers, indicating the role of carrier materials and microbial communities. The MBBR carrier exhibited good stability due to its rough surface and microbial aggregates. Sequencing results revealed significant differences in the microbial community structure among the carriers. Shewanella was the dominant functional bacteria in the MBBR and PPC carriers, while highly efficient degrading microbial communities were observed on the biochar carrier. Overall, the physicochemical properties of the carrier materials had a significant impact on the microbial community in the reactor, which affected the degradation efficiency of RB5. These findings provide valuable insights into the optimization of biofilm reactors for dye-containing wastewater treatment.

Tracheal resection and end-to-end anastomosis has been the standard clinical approach for the treatment of most airway diseases, especially invading the lower trachea or carina. However, when long-length (exceeding 2 cm in children or 5 cm in adults) tracheal circular resection is performed, tracheal replacement therapy is often required. In this study, we aimed to utilize autologous tracheal epithelia and bone marrow mesenchymal stem cells (BMSCs) as the seeding cells, utilize polycaprolactone (PCL) coated with Silk Fibroin Methacryloyl (SilMA) as the scaffold to carry the cells and Kartogenin (KGN). Firstly, SilMA with the concentration of 10%, 15% and 20% was made, and the experiment of swelling and degradation was performed. With the increase of the concentration, the swelling ratio decreased, the degradation progress slowed down. Upon the result of CCK-8 test and HE staining of 3D co-culture, the 20% SilMA was selected. Next, SilMA and the cells attached to SilMA were characterized by scanning electron microscopy (SEM). Furthermore, in vitro cytotoxicity test shows that 20% SilMA has good cytocompatibility. The hybrid scaffold was then made by PCL coated with 20% SilMA. The mechanical test shows this hybrid scaffold has better biomechanical properties. In vivo tracheal defect repair assays were done to evaluate the effect of the hybrid substitution. H&E staining, immunohistochemical (IHC) and immunofluorescence (IF) staining showed that this hybrid substitution ensured the viability, proliferation and migration of epithelium. This study is expected to provide new strategies for the fields of tracheal replacement therapy needing mechanical properties and epithelization.

Chlamydomonas reinhardtii has been successfully engineered to produce compounds of interest following transgene integration and heterologous protein expression. The advantages of this model include the availability of validated tools for bioengineering, its photosynthetic ability and its potential use as biofuel. Despite this, breakthroughs have been hindered by its ability to silence transgene expression through epigenetic changes. Histone deacetylases (HDAC) are main players in gene expression. We hypothesized that transgene silencing can be reverted with chemical treatments using HDAC inhibitors. To analyze this, we transformed C. reinhardtii, integrating into its genome the mVenus reporter gene under the HSP70-rbcs2 promoter. From 384 transformed clones, 88 (22.9 %) displayed mVenus positive (mVenus+) cells upon flow-cytometry analysis. Five clones with different fluorescence intensities were selected. The number of integrated copies was measured by qPCR. Transgene expression levels were followed over the growth cycle and upon SAHA treatment, using a microplate reader, flow cytometry, RT-qPCR, and western blot analysis. First, we observed that expression varies with the cell cycle, reaching a maximum level just before the stationary phase in all clones. Second, we uncovered that supplementation with HDAC inhibitors of the hydroxamate family, such as vorinostat (suberoylanilide-hydroxamic-acid, SAHA) at the initiation of culture increases the frequency (% of mVenus+ cells) and the level of transgene expression per cell over the whole growth cycle, through histone deacetylase inhibition. Thus, we propose a new tool to successfully trigger the expression of heterologous proteins in the green algae C. reinhardtii, overcoming its main handicap as an expression platform.

Schizochytrium sp. is a heterotrophic microorganism capable of accumulating polyunsaturated fatty acids, and has achieved industrial production of docosahexaenoic acid (DHA). It also has the potential for eicosapentaenoic acid (EPA) production. In this study, it was found that the cell growth, lipid synthesis and fatty acid composition of Schizochytrium sp. were significantly affected by the level of cobalamin in the medium, especially with regards to the content of EPA in the fatty acids. The content of EPA in the fatty acids increased 17.91 times, reaching 12.0%, but cell growth and lipid synthesis were significantly inhibited under cobalamin deficiency. The response mechanism for this phenomenon was revealed through combined lipidomic and transcriptomic analysis. Although cell growth was inhibited under cobalamin deficiency, the genes encoding key enzymes in central carbon metabolism were still up-regulated to provide precursors (Acetyl-CoA) and reducing power (NADPH) for the synthesis and accumulation of fatty acids. Moreover, the main lipid subclasses observed during cobalamin deficiency were glycerolipids (including glycerophospholipids), with EPA primarily distributed in them. The genes involved in the biosynthesis of these lipid subclasses were significantly up-regulated, such as the key enzymes in the Kennedy pathway for the synthesis of triglycerides. Thus, this study provided insights into the specific response of Schizochytrium sp. to cobalamin deficiency and identified a subset of new genes that can be engineered for modification.

DNA ligases catalyze bond formation in the backbone of nucleic acids via the formation of a phosphodiester bond between adjacent 5’ phosphates and 3’ hydroxyl groups on one strand of the duplex. While DNA ligases preferentially ligate single breaks in double stranded DNA (dsDNA), they are capable of ligating a multitude of other nucleic acids substrates like blunt-ended dsDNA, TA overhangs, short overhangs and various DNA-RNA hybrids. Here we report a novel DNA ligase from Chronobacter phage CR 9 (AZ R2D Ligase) with an unexpected DNA-to-RNA ligation activity. The R2D ligase shows excellent efficiency when ligating DNA to either end of RNA molecules using a DNA template. Furthermore, we show that DNA can be ligated simultaneously to both the 5’ and 3’ ends of microRNA-like molecules in a single reaction mixture. Abortive adenylated side product formation is suppressed at lower ATP concentrations and the ligase reaction reaches near completion when ligating RNA to DNA or DNA to RNA. The ligation of a DNA strand to the 5’-PO4 end of RNA is unique among the commercially available ligases and may facilitate novel workflows in microRNA analysis, RNA sequencing and in the preparation of chimeric guide DNA-RNA for gene editing applications.

The Chinese hamster ovary (CHO) cell is a fibroblast-like cell that produces proteins with post-translational modifications similar to human glycosylation. It is widely used in the production of recombinant therapeutic proteins and monoclonal antibodies. Culturing CHO cells typically requires the addition of a certain proportion of fetal bovine serum (FBS) to maintain cell proliferation and passaging. However, serum is characterized by its complex composition, batch-to-batch variability, high cost, and potential risk of exogenous contaminants such as mycoplasma and viruses, which impact the purity and safety of the synthesized proteins. Therefore, search for serum alternatives and development of serum-free media for CHO-based protein biomanufacturing are of great significance. This review systematically summarizes the application advantages of CHO cells and strategies for high-density expression. It highlights the developmental trends of serum substitutes from human platelet lysates to animal-free extracts and microbial-derived substances and elucidates the mechanisms by which these substitutes enhance CHO cell culture performance and recombinant protein production, aiming to provide theoretical guidance for exploring novel serum alternatives and developing serum-free media for CHO cells.

Squalene (C30H50) is an acyclic triterpenoid compound renowned for its myriad physiological functions, such as anticancer and antioxidative properties, rendering it invaluable in both the food and pharmaceutical sectors. Owing to the constraints on natural resources, microbial fermentation has emerged as a prominent trend. Schizochytrium sp. known to harbor the intact Mevalonate (MVA) pathway, possesses the inherent capability to biosynthesize squalene. However, there is a dearth of reported key genes in both the MVA pathway and the squalene synthesis pathway, along with the associated promoter elements for their modification. This study commenced by cloning and characterizing 13 endogenous promoters derived from transcriptome sequencing data. Subsequently, five promoters exhibiting varying expression intensities were chosen from the aforementioned pool to facilitate the overexpression of the squalene synthase gene SQS, pivotal in the MVA pathway. Ultimately, a transformed strain designated as SQS-3626, exhibiting squalene production 2.8 times greater than that of the wild-type strain, was identified. Lastly, the optimization of nitrogen source concentrations and trace element contents in the fermentation medium was conducted. Following 120 hours of fed-batch fermentation, the accumulated final squalene yield in the transformed strain SQS-3626 reached 2.2 g/L.

The mutual interactions of ER resident proteins in the ER maintain its functions, prompting the protein folding, modification, and transportation. Here, a new method, named YST-PPI (YESS-based Split fast TEV protease System for Protein-protein Interaction) was developed, targeting the characterization of protein interactions in ER. YST-PPI method integrated the YESS system, split-TEV technology and endoplasmic reticulum retention signal peptide (ERS) to provide an effective strategy for studying ER in situ PPIs in a fast and quantitative manner. The interactions among 15 ER resident proteins of S. cerevisiae were explored using the YST-PPI system, and their interaction network map was constructed, in which more than 74 interacting resident protein pairs were identified. Our studies also showed that Lhs1p plays a critical role in regulating the interactions of most of the ER resident proteins, expect the Sil1p, indicating its potential role in controlling the ER molecular chaperones. Moreover, the mutual interaction revealed by our studies further confirmed that the ER resident proteins perform their functions in a synergetic way and multimer complex might be formed during the process.

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 rAAV production by targeting the 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 success. 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. Our study also established a comprehensive analytical workflow to detect and evaluate the gene knockouts generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.

Laccases have a wide range of uses in the textile industry for their ability to decolorize dyes, modify the surface of fabrics and bleach textiles. Screening a laccase with high thermal stability and alkali tolerance suitable for textile applications is challenging. In this study, we identified a novel alkaline laccase, LacCT, from Caldalkalibacillus thermarum, and expressed it in Escherichia coli. It showed an optimal temperature of 65°C and a pH range of high stability from 6.0 to 10.0, with an optimum pH of 7.5. Through rational design, the thermal stability of the best variant, G190P/Q254Y/G336M/D510F (LacCT-11), was significantly improved, exhibiting a half-life of 63.2 minutes at 60°C, 1.8 times longer than the wild type. This research presents a new laccase with significant potential for decolorizing textile wastewater and enhancing the ramie degumming process.

The performance of industrial strains has gradually improved with the rapid development of synthetic biotechnology. The production efficiency of traditional batch and fed-batch culture is limited and product quality varies since both are dynamic processes, whereas multi-stage continuous culture can maximise the production efficiency of specific fermentation processes and achieve consistent product quality. However, each single-stage fermentation under multi-stage continuous fermentation requires accurate steady-state control, and a model with adequate accuracy is required for designing and controlling a multi-stage continuous fermentation process. At present, there are few reports on kinetic models for the control of multi-stage continuous fermentation. In this work, we constructed a hybrid model for Saccharomyces cerevisiae multi-stage continuous culture, taking both oxygen limitation and Crabtree effect. The accuracy of the model was ~ 80%, advantages and limitations of the model are discussed and potential improvement strategy is proposed.

Amino acids, including asparagine, aspartate, glutamine, and glutamate, play important roles in the purine and pyrimidine biosynthesis as well as serve as anaplerotic sources fueling the tricarboxylic acid (TCA) cycle for mitochondrial energy generation in mammalian cells. Despite extensive studies on glutamine and glutamate in CHO cell cultures, the roles of asparagine and aspartate, especially in feed media, remain underexplored. In this study, we utilized the CHO genome scale model to first deeply characterize the intracellular metabolic states of CHO cells cultured in different combinations of basal and feed media to understand the traits of asparagine/aspartate-dependent and glutamate-dependent feeds. Subsequently, we identified the critical role of asparagine and aspartate in the feed media as anaplerotic sources and conduct in silico simulations to ascertain their optimal ratios to improve cell culture performance. Finally, based on the model simulations, we reformulated the feed media by tailoring the concentrations of asparagine and aspartate. Our experimental data reveal a CHO cell preference for asparagine compared with aspartate, and thus maintaining an optimal ratio of these amino acids is a key factor for achieving optimal CHO cell culture performance in biopharmaceutical production.

After the COVID-19 pandemic, several companies and organisations are actively developing, scaling up and optimising mRNA manufacturing processes to produce their vaccines and therapeutics. Herein, we evaluated tangential flow filtration (TFF) for high-recovery, and high-purity separation of mRNA from unreacted NTPs in the IVT reaction mixture. For the first time, the membrane fouling behaviour caused by mRNA and separation of nucleoside triphosphates (NTPs) was mathematically modelled. The mRNA membrane fouling model is necessary for optimising mRNA separation processes, designing a suitable strategy for membrane clean-up, estimating the end of production life and reducing the process cost. Recovery greater than 70% mRNA without degradation was obtained by implementing a capacity load of ~19 g/m2, <2.5 psi transmembrane pressure (TMP) and feed flux of 300 LMH. This approach enables the purification of multiple RNA drug substance sequences for the prevention and treatment of a wide range of diseases.

Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the CRISPR-Cas9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to non-model plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.

Background: The functions of GOLPH3, STIP1, and the STAT3 signaling pathway in the invasion and migration of CC cells were examined in this study. Method: High-speed centrifugation was used to collect the exosomes. The expression of GOLPH3, STIP1, and epithelial-mesenchymal transition (EMT)-related proteins in CC tissues, cells, and exosomes were analyzed using Western blotting (WB) experiments. The abilities of CC cell invasion and migration were evaluated by the Transwell assay. The binding relationship between GOLPH3 and STIP1 was validated through Co-immunoprecipitation (Co-IP), and their sublocalization in CC cells was determined by immunofluorescence detection under laser confocal microscopy. Immunohistochemistry (IHC) experiments detected the expression levels of each protein in the transplanted tumor mass. Animal experiments confirmed the impact of the GOLPH3/STIP1/STAT3 regulatory axis on the CC growth. Results: In CC tissues and cells, GOLPH3 was highly expressed, and silencing GOLPH3 not only greatly reduced CC cell invasion and migration but also prevented EMT. Furthermore, GOLPH3 and STIP1 interacted in CC cells, and the GOLPH3-STIP1 complex affected the capacity for cell invasion and migration by triggering the STAT3 signaling pathway. Noteworthily, GOLPH3, and STIP1 could also be detected in CC cell exosomes, and the exosomes carried the GOLPH3-ST1P1 complex to act on CC cells to activate intracellular STAT3 signaling, ultimately affecting the cancer cell migration and invasion. The above molecular regulatory mechanisms have also been validated in mice. Conclusion: The GOLPH3-STIP1 complex acted on surrounding CC cells through exosomes and activated the STAT3 signaling pathway to stimulate CC cell invasion and migration

Ultrasound (US) can easily penetrate media with excellent spatial precision corresponding to its wavelength. Naturally, US plays a pivotal role in the echolocation abilities of certain mammals such as bats and dolphins. In addition, medical US generated by transducers interact with tissues via delivering ultrasonic energy in the modes of heat generation, exertion of acoustic radiation force (ARF), and acoustic cavitation. Based on the principle of echolocation, various assistive devices for visual impairment people have been developed. High-Intensity Focused Ultrasound (HIFU) are developed for targeted ablation and tissue destruction. Besides thermal ablation, histotripsy with US is designed to damage tissue purely via mechanical effect without thermal coagulation. Low-Intensity Focused Ultrasound (LIFU) has been proven to be an effective stimulation method for neuromodulation. Furthermore, US has been reported to transiently increase permeability of biological membranes, enabling acoustic transfection and blood-brain barrier open. All of these advances in US are changing the clinic. This review mainly introduces the advances in these aspects, focusing on the physical and biological principles, challenges and future direction.