not-yet-known not-yet-known not-yet-known unknown Glycosylation is a critical quality attribute in biopharmaceuticals that influences crucial properties, such as immunogenicity and stability. Current methods for modeling glycosylation typically rely on imprecise or limited data on nucleotide sugar donor dynamics. These methods use in vitro transporter kinetics or flux balance analysis, which overlook the key aspects of metabolic regulation. We devised an integrative workflow for absolute subcellular NSD quantification in both cytoplasm and secretory organelles. Using subcellular fractionation, exhaustive sample extraction, and liquid chromatography triple-quadrupole tandem mass spectrometry, we accurately measured NSD concentrations ranging 1.6 amol/cell to 3 fmol/cell. Our NSD concentration profiles aligned closely with the glycan distributions on antibodies, particularly after nutrient pulsing to stimulate NSD production. This method enables empirical observation of compartment-specific NSD dynamics. Thus, this study provides novel insights indicating that N-glycosylation, which governs NSD supply, is primarily regulated within the Golgi apparatus. This method offers a novel tool to obtain sophisticated data for the more efficient optimization of glycosylation processes in production cell lines.

In this work, double-layer heterogeneous calcium alginate scaffolds were designed for alveolar bone defects where the outer layer featured high hardness and slow degradation, and the inner layer characterized by large pores and rapid degradation. The morphology of the calcium alginate scaffold was akin to that of bone defects, and its direct implantation reduced the operation time. A higher concentration of calcium alginate resulted in smaller pores and slower degradation. Calcium alginate can promote the formation of mineralized nodules and the expression of genes related to mineralization without inducing cytotoxic effects. It also promoted the expression of cellular inflammatory factors, potentially through the TLR4 pathway. In vivo studies confirmed that calcium alginate did not promote the aggregation of inflammatory cells nor the expression of inflammatory factors. In conclusion, the scaffold's characteristics of high surface hardness and slow degradation were beneficial for surface osteogenesis and maintaining the defect's shape and osteogenic space. Conversely, rapid internal degradation favors the formation of bone tissue.

Studying neurological disorders in vitro is still challenging due to the human brain’s complexity and the difficulty of obtaining primary neural cells. However, tissue engineering and tridimensional (3D) cell culture have become increasingly important tools for disease modeling. By providing an extracellular matrix (ECM) substrate that closely resembles physiological conditions, 3D cell culture offers several advantages over standard monolayer cell culture, including enhanced cell-cell and cell-matrix interactions. This results in a microenvironment that more accurately reflects in vivo biology. In this study, we performed an in-depth analysis of the proteome and matrisome of 3D tissue-engineered dermis, made from human primary dermal fibroblasts cultivated in 3D and embedded in a self-produced ECM. Interestingly, in silico analysis revealed that neurogenesis and associated functions were predicted to be strongly activated in this tissue-engineered 3D model. Indeed, we showed that ECM proteins involved in neuronal development and maintenance, typically produced by cerebral cells, were also expressed by dermal fibroblasts. Of particular interest, the 3D co-cultivation of dermal fibroblasts with iPSC-derived motor neurons readily enabled long-lasting culture periods without costly media supplementation with exogenous additives. Patient-derived dermal fibroblasts, cultivated in 3D, could therefore become valuable models for the study of neurological diseases. This approach offers a cost-effective and a less invasive alternative to brain biopsies for modeling complex neurological disorders in vitro.

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

Chinese hamster ovary (CHO) cells are widely used to produce recombinant proteins, including monoclonal antibodies (mAbs), through various process modes. Traditionally, fed batch (FB) processes have been the standard. However, the shift towards high-density perfusion processes is driven by increased productivity, flexible facility footprints, and lower costs. Ensuring the clearance of process-related impurities, such as host cell proteins (HCPs), is crucial in biologics manufacturing. While purification processes remove most impurities, integrated strategies are being developed to enhance biologics purity and address high-risk HCPs. Current understanding of HCP expression dynamics in cell culture is limited. This study utilized data-independent acquisition (DIA) proteomics to compare the proteomic profiles of cell culture supernatants from 14 FB clones and 3 perfusion clones, all expressing the same mAb from the same host cell line. Results showed that perfusion processes enhance cell growth and productivity, exhibiting distinct proteomic profiles compared to FB processes. Perfusion processes also maintain a more stable HCP profile across clones, especially for 46 problematic HCPs monitored. Cluster analysis of FB proteomics revealed distinct abundance patterns and correlations with process parameters. Differential abundance analysis identified significant protein differences between the two processes. Compared to FB, the perfusion process may provide a less stressful cellular environment. This is the first extensive study characterizing HCPs expressed by different clones under different process modes. Further research could lead to strategies for preventing or managing problematic HCPs in biologics manufacturing.

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.

javascript:void(0) Antioxidants are known for their health beneficial effects by reducing oxidative damage, increasing free radical scavenging rate, blocking and terminating oxidation reactions. They have been widely used as supplements in food, cosmetics, nutraceuticals and medicines. Emerging researches increasingly indicate that the development of diverse research methodologies and the exploration of new microorganisms for heterogeneous synthesis of antioxidants play an indispensable role in socioeconomic development. In this study, we engineered Saccharomyces boulardii for probiotic supplementation of antioxidants such as L-ergothioneine (EGT). We first constructed double knockout of ura3 and trp1 gene in S. boulardii, to facilitate plasmid-based expressions. To further enable effective genome editing of S. boulardii, we implemented the PiggyBac system to transpose the heterologous gene expression cassettes into the chromosomes of S. boulardii. By using enhanced green fluorescent protein (EGFP) as the reporter gene, we achieved random chromosomal integration of EGFP expression cassette. EGT-producing strains were subsequently obtained via the PiggyBac transposon system. One isolated S. boulardii mutant produced EGT at 17.50 mg/L after 120 h cultivation. In summary, we have applied the PiggyBac transposon system to S. boulardii for the first time for genetic engineering. The engineered probiotic yeast S. boulardii has been endowed with new antioxidant properties and produces EGT. It has potential applications in developing novel therapeutics and dietary supplements for the prevention and treatment of gastrointestinal disorders.

Mesenchymal stem cells (MSCs) derived exosomes, as a cell-free therapy to replace MSCs, have higher safety and great potential in regenerative medicine. The isolation of exosomes is a challenge that complicates their application. The commonly used ultracentrifugation and tangential flow filtration methods are inconvenient due to the need for expensive instruments and ultrafilter membranes. The cost of commercial kits limits their application in handling large samples. PEG precipitation is convenient and cost-effective. However, there is a need for optimized and standardized isolation methods based on PEG precipitation in different cell types or liquids to ensure consistent quality and yield. In this work, we optimized the PEG precipitation method for exosomes isolation and compared its effectiveness to two commonly used methods: ultracentrifugation (UC) and commercial exosome isolation kits (ExoQuick). The recovery rate of the optimized PEG method ( about 61.74%) was comparable to that of the commercial ExoQuick kit (about 62.19%), which was significantly higher than UC (about 45.80%). Exosome cargo analysis validated no significant differences in miRNA and protein profiles associated with the proliferation and migration of exosomes isolated by UC and PEG precipitation, which was confirmed by gap closure and CCK8 assays. These findings suggest that PEG-based exosomes isolation could be a highly efficient and high-quality method and may facilitate the development of exosome-based therapies for regenerative medicine.

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.

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.

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.

A new method has been developed to improve the detection of norovirus (NoV) in complex fecal samples using nanomaterials-based immunoassays. The method involves using multifunctional trimetallic nanoparticles, known as Ag@Fe3O4@Au NPs. These nanoparticles consist of a core of silver (Ag) and a shell of iron oxide (Fe3O4) decorated with isolated gold (Au) nanoparticles. The nanoparticles have enhanced catalytic activity, making them an ideal nanocatalyst for reducing substrate molecules. The developed Ag@Fe3O4@Au NPs-based immunoassay achieved a limit of detection (LOD) of 1.9 pg/mL for norovirus-like particles (NoV-LP) and 6.97 fecal NoV copy number/mL. Achieving a LOD under 10 copy numbers is important for detailed protein analysis. The study also found that heat treatment of fecal suspension at 65°C was necessary to prevent the degradation of the target analyte for sensitive NoV detection. This work successfully combined multifunctional nanocatalysts for advanced immunoassays, which could contribute to developing nano-biosensing platforms.

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 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.

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.

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.

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.