Biocatalysis in high-concentration organic solvents has been applied to produce various industrial products with many advantages. However, using enzymes in organic solvents often suffers from inactivation or decreased catalytic activity and stability. So, improving the tolerance of enzymes in organic solvents is essential. Herein, the method of regional random mutation combined with combinatorial mutation was used to improve the resistance of transaminase from Aspergillus terreus (AtATA) in organic solvents, and the best mutant T23I/T200K/P260S (M3) was acquired. In different concentrations of dimethyl sulfoxide (DMSO), the catalytic efficiency toward 1-acetylnaphthalene and the stability were higher than the wild-type (WT) of AtATA. M3 also showed enhanced stability against six organic solvents with different oil-water partition coefficients (log P values). The results of decreased Root Mean Square Fluctuation (RMSF) values via 20-ns molecular dynamics simulations under different concentration DMSO revealed that mutant M3 had lower flexibility, acquiring a more stable protein structure and contributing to its organic solvents stability than WT. Intra- and intermolecular interaction analysis indicated that the increased hydrogen bonds and hydrophobic interactions within monomers or at the interface of two monomers also strengthened the stability of the overall structure against organic solvents. Furthermore, M3 was applied to convert 1-acetylnaphthalene for synthesizing (R)-(+)-1(1-naphthyl)-ethylamine ((R)-NEA), which was and an intermediate of Cinacalcet Hydrochloride. Moreover, 3~10 mM 1-acetylnaphthalene can be converted to (R)-NEA with 94.2~38.9% yield and a strict R-stereoselectivity within 10 h under 25% DMSO, which was higher than WT and expected to be a potential biocatalyst for industrial application.
Periosteum has shown potential as an effective barrier membrane for guided bone regeneration (GBR). However, if recognized as a “foreign body”, insertion of a barrier membrane in GBR treatment will inevitably alter the local immune microenvironment and subsequently influence bone regeneration. The aim of this investigation was to fabricate decellularized periosteum (DP) and investigate its immunomodulatory properties in GBR. DP was successfully fabricated from periosteum from the mini-pig cranium. In vitro experiments indicated that the DP scaffold modulated macrophage polarization toward a pro-regenerative M2 phenotype, which in turn facilitated migration and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. A rat GBR model with a cranial critical-size defect was established, and our in vivo experiment confirmed the beneficial effects of DP on the local immune microenvironment and bone regeneration. Collectively, the findings of this study indicate that the prepared DP possesses immunomodulatory properties and represents a promising barrier membrane for GBR procedures.
In the past decade, recombinant adeno-associated virus (rAAV) has gained increased attention as a prominent gene therapy technology to treat monogenetic disease. One of the challenges in rAAV production is the enrichment of full-rAAV particles containing the gene of interested (GOI) payload. Herein, we demonstrated that by adjusting the mobile phase properties of anion-exchange chromatography (AEX), Empty and Full separation of rAAV was improved in monolith based preparative AEX chromatography. When compared to the baseline method using NaCl, the presence of tetraethylammonium acetate (TEA-Ac) in the AEX mobile phase resulted in enhanced resolution (from 0.75 to 1.23) between Empty and Full peaks by salt linear gradient elution, as well as increased the percentage of full-rAAV particles from 20% to 36% and GOI genome recovery (from 59% to 62%). Furthermore, a dual wash + step elution AEX method was developed to harness TEA-Ac contribution on Empty and Full separation in the first wash (wash1) step while removing TEA-Ac in the second wash (wash2) step to ensure product safety. The resulting optimized AEX purification method could be easily adapted in scaled-up manufacturing and could also be applied to purification processes involving other AAV serotypes facing similar Empty and Full rAAV separation challenges.
The use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has risen considerably in recent years. Consequently, the amount of AAV vectors required during the validation and clinical trials has also increased. AAV serotype 6 (AAV6) is well-documented for its efficiency in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG). Overall, this means that large-scale production of AAV6 is needed. Suspension cell-based platforms are currently limited to low-cell-density productions, hindering the potential of this production process to increase yields. Here, we investigate the improvement of the production of AAV6 at higher cell densities. The production was performed by transient transfection of HEK293SF cells. When the plasmid DNA is provided on a cell basis, the production can be carried out at medium cell density without effects on cell-specific titer or particle functionality, resulting in titers above 1010 VG/mL. Medium supplementation alleviated the cell density effect, in terms of VG/cell, at high-cell-density productions. On the other hand, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations. The medium-cell-density production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing.
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.
Adipose-derived stem cells (ADSCs) have important applications in basic research, especially in fat transplantation. Some studies have found that three-dimensional (3D) spheroids formed by mesenchymal stem cells have enhanced therapeutic potential. However, the fundamental basics of this effect are still being discussed. In this study, ADSCs were harvested from subcutaneous adipose tissues and 3D spheroids were formed by the automatic aggregation of ADSCs in a non-adhesive 6-well plate. Oxygen glucose deprivation (OGD) was used to simulate the transplantation microenvironment. We found that 3D culture of ADSCs triggered cell autophagy. After inhibiting autophagy by Chloroquine, the rates of apoptosis were increased. When the 3D ADSC-spheroids were re-planked, the number of senescent ADSCs decreased, and the proliferation ability was promoted. In addition, there were more cytokines secreted by 3D ADSC-spheroids including VEGF, IGF-1 and TGF-β. After adding the conditioned medium with human umbilical vein endothelial cells (HUVECs), 3D ADSC-spheroids were more likely to promote migration, and tube formation, stimulating the formation of new blood vessels. Fat grafting experiments in nude mice also showed that 3D ADSC-spheroids enhanced survival and neovascularization of fat grafts. These results suggested that 3D spheroids culturing of ADSCs can increase the therapeutic potential in fat transplantation.
Ultrasound-guided protein delivery is promising for site-specific control of cellular functions in the deep interior of the body in a noninvasive manner. Herein, we propose a method for cytosolic protein delivery based on ultrasound-guided intracellular vaporization of perfluorocarbon nano-droplets. The nano-droplets were conjugated with cargo proteins through a bio-reductively cleavable linker and introduced into living cells via antibody-mediated binding to a cell-surface receptor, which gets internalized through endocytosis. After the cells were exposed to ultrasound for endosomal escape of proteins, the ultrasound-responsive cytosolic release of a cargo enzyme was confirmed by visualizing the hydrolysis of the fluorogenic substrate using confocal microscopy. Moreover, a significant decrease in cell viability was achieved via the release of a cytotoxic protein in response to ultrasound treatment. The results of this study provide the proof of a principle that protein-conjugated nano-droplets can be used as carriers in ultrasound-guided cytosolic delivery of proteins.
In recent years, health care providers have seen an increase in the number of patients with difficult-to-treat wounds and burns. The bio polymer-based wound dressing shields the injured part and aids in the recapture of epithelial and dermal tissues throughout the process of healing. The total count of a person with chronic lesions has been expanding whole due to developing society, over weight and cardiovascular illness. The development of ideal wound dressing material with excellent characteristics like antimicrobial activity, biocompatibility, free radical scavenging capacity, non-adherent property, the hydrophilicity of alginate, cellulose, chitosan, collagen has an increasing demand for the treatment of chronic wounds. Nevertheless, owing to the above mention property, natural polymers are being used for several key functions of biomedicine like narcotic distribution systems, tissue manufacturing, bandages etc. accordingly, the significance of these bio-based polymers interfered with healing functions that lead to inform and inspire youth and scientist researchers worldwide to grab with these far-reaching areas of medicine and biology. The review highlights the physiochemical property of natural polymer, biological evaluation of various materials as wound dress, along with their synthesis and mechanical properties, clinical status, challenges and future perspectives.
Cas13 are the only CRISPR/Cas systems found so far, which target RNA strand while preserving chromosomal integrity. Cas13b or Cas13d cleaves RNA by the crRNA guidance. However, the effect of the characteristics of the spacer sequences, such as the length and sequence preference, on the activity of Cas13b and Cas13d remains unclear. Our study shows that neither Cas13b nor Cas13d has a particular preference for the sequence composition of gRNA, including the sequence of crRNA and its flanking sites on target RNA. However, the crRNA, complementary to the middle part of the target RNA, seems to show higher cleavage efficiency for both Cas13b and Cas13d. As for the length of crRNAs, the most appropriate crRNA length for Cas13b is 22-25 nt and crRNA as short as 15 nt is still functional. Whereas, Cas13d requires longer crRNA, and 22-30 nt crRNA can achieve good effect. Both Cas13b and Cas13d show the ability to process precursor crRNAs. Our study suggests that Cas13b may have a stronger precursor processing ability than Cas13d. There are few in vivo studies on the application of Cas13b or Cas13d in mammals. With the methods of transgenic mice and hydrodynamic injection via tail vein, our study showed that both of them had high knock-down efficiency against target RNA in vivo. These results indicate that Cas13b and Cas13d have great potential for in vivo RNA operation and disease treatment without damaging genomic DNA.
Self-sufficient cytochromes P450 of the sub-family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions towards a wide range of organic compounds without the need of a separate reductase partner. However, these P450s are often unstable in solution and their activity is limited to short reaction time. As the isolated heme domain of CYP116B5 has been shown to work as a peroxygenase with H2O2 without the need for expensive NAD(P)H, in this work protein engineering was used to generate a chimeric enzyme (CYP116B5-SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) that is able to produce H2O2 with a controlled and continuous release in time. The full-length form enzyme (CYP116B5-fl) is expressed and characterized for the first time, allowing a detailed comparison to both the isolated heme domain (CYP116B5-hd) and CYP116B5-SOX. The catalytic activity of the three forms of the enzyme was studied using p-nitrophenol as substrate, and adding NADPH (CYP116B5-fl), H2O2 (CYP116B5-hd) and sarcosine (CYP116B5-SOX) as direct or indirect source of electrons. CYP116B5-SOX outperforms CYP116B5-fl by 10 folds and CYP116B5-hd by 3 folds, in terms of p-nitrocatechol produced per mg of enzyme per minute. CYP116B5-SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class.
D-Allulose has many health-benefiting properties, physiological functions, and sustainable applications in food, pharmaceutical, and nutrition industries. The aldol reaction based route is a very promising alternative to Izumoring strategy in D-allulose production. Remarkable studies have been reported in this field, but still suffer from by-product formation and costly purified enzyme involvement. In the present study, we explored the glycerol assimilation, alditol oxidase, alcohol dehydrogenase, aldolase, and dephosphorylation pathways, and modularly designed, assembled, and optimized the D-allulose synthetic cascade in Escherichia coli envelop. We achieved an efficient whole-cell catalyst that produces only D-allulose from cheap glycerol feedstock, eliminating the involvement of purified enzymes. Detailed process optimization improved the D-allulose titer by 1500.00%. Finally, the production was validated in 3-L scale using a 5-L fermenter, and 5.67 g/L D-allulose was produced with a molar yield of 31.43%. This study provided a facile approach to produce D-allulose from glycerol feedstock.
It is common practice in the development of bioprocesses to genetically modify a microorganism and study a large number of resulting mutants in order to select the ones that perform best for use at the industrial scale. At industrial scale, strict nutrient-controlled growth conditions are imposed to control the metabolic activity and growth rate of the microorganism, thereby enhancing the expression of the product of interest. Although it is known that microorganisms that perform best under these strictly controlled conditions are not the same as the ones that perform best under uncontrolled batch conditions, screening, and selection is predominantly performed under batch conditions. Tools that afford high throughput on the one hand and dynamic control over cultivation conditions on the other hand are not yet available. Microbioreactors offer the potential to address this problem, resolving the gap between bioprocess development and industrial scale use. In this review, we highlight the current state-of-the-art of microbioreactors that offer the potential to screen microorganisms under dynamically controlled conditions. We classify them into: (i) microtiter plate-based platforms, (ii) microfluidic chamber-based platforms, and (iii) microfluidic droplet-based platforms. We conclude this review by discussing the opportunities of nutrient-fed microbioreactors in the field of biotechnology.
Auxotrophic marker genes have widely used for genetic engineering in yeast. However, the effects of auxotrophic strains that are deficient in synthesis of amino acids or nucleotides on the growth and production are rarely reported. In this study, a total of eight auxotrophic strains with single knockout of selective markers were obtained to evaluate cell growth and free fatty acid (FFA) production in Saccharomyces cerevisiae with supplementing different concentrations of amino acids or nucleotides. Generally, except for gene ADE2, most auxotrophic strains possessed decreased cell growth and FFA production, which could be remedied by the higher concentrations of supplements. leu2Δ damaged both growth and production even with supplementation of 1000 mg/L leucine. Therefore, this study shows that auxotrophs compromise the metabolic engineering endeavor and provides a guidance in supplementing amino acids or nucleotides during fermentations for maximizing bio-productions.
N6–methyl adenosine (m6A) is the most abundant internal modification on eukaryotic mRNA and has been implicated in a wide range of fundamental cellular processes. This modification is regulated and interpreted by a set of writer, eraser, and reader proteins. To date, there have been no reports on the potential of mRNA epigenetic regulators to influence recombinant protein expression in mammalian cells. In this study we evaluated the potential of manipulating the expression of the m6A YTH domain-containing readers, YTHDF1, 2, and 3 to improve recombinant protein yield based on their role in regulating mRNA stability and promoting translation. Using siRNA-mediated gene depletion, cDNA over-expression and methylation-specific RNA immunoprecipitation, we demonstrate that (i) knock-down of YTHDF2 enhances (~2-fold) the levels of recombinant protein derived from GFP and EPO transgenes in CHO cells; (ii) the effects of YTHDF2 depletion on transgene expression is m6A-mediated and (iii) YTHDF2 depletion or over-expression of YTHDF1 increases viral protein expression and yield of infectious lentiviral particles (~2-3 fold) in HEK293 cells. We conclude that various transgenes can be subjected to regulation by m6A regulators in mammalian cell lines and that these findings demonstrate the utility of epi-transcriptomic-based approaches to host cell line engineering for improved recombinant protein and viral vector production.
Metabolic reprogramming has been coined as a hallmark of cancer, accompanied by which the alterations in metabolite levels have profound effects on gene expression, cellular differentiation and the tumor environment. Yet a systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of tumor cells is currently lacking. To achieve this, this study is aimed at establishing an unbiased and leakage-free metabolome preparation protocol for Hela carcinoma cell. We evaluated 12 combinations of quenching and extraction methods from three quenchers (liquid nitrogen, -40°C 50% methanol, 0.5°C normal saline) and four extractants (80% methanol, methanol: chloroform: water (1:1:1, v/v/v), 50% acetonitrile, 75°C 70% ethanol) for global metabolite profiling of adherent Hela carcinoma cells. Based on the isotope dilution mass spectrometry (IDMS) method, gas/liquid chromatography in tandem with mass spectrometry was used to quantitatively determine 43 metabolites including sugar phosphates, organic acids, amino acids, adenosine nucleotides and coenzymes involved in central carbon metabolism. Among 12 combinations, cells that washed twice with phosphate buffered saline (PBS), quenched with liquid nitrogen, and then extracted with 50% acetonitrile was found to be the most optimal method to acquire intracellular metabolites with minimal loss during sample preparation. Furthermore, a case study was carried out to evaluate the effect of doxorubicin (DOX) on both adherent cells and 3D tumor spheroids using quantitative metabolite profiling. Based on this, quantitative time-resolved metabolite data can serve to the generation of hypotheses on metabolic reprogramming to reveal its important role in tumor development and treatment.
Background: The identification of protein-protein interactions is of great challenge. Therefore, we conducted this study to fabricate a gold surface biochip with activated sophorolipids in combination with 16-amino-1-hexadecanethiol hydrochloride. Methods: We designed a direct on-chip immunological assay strategy for measuring ligand-receptor interactions in a forward or reverse manner, that is, a ligand was immobilized on the biochip surface and allowed to interact with its specific free receptor in the liquid phase and vice versa. The specificity of molecular interactions on the biochip was evaluated using an immunological blocking assay and a chemiluminescent immunoassay. To test the potential utilization of biochip, we used the serum of hemophagocytic lymphohistiocytosis (HLH) patients as an experimental entity. Results: The receptor CD25-based IL-2 and ligand IL-2-based CD25 assays revealed that the detection limits on the biochip were as low as 156pg/mL and 78pg/mL, respectively. Meanwhile, using the receptor- or ligand-based platforms, we found that the positive rates of free IL-2 and soluble CD25 (sCD25) monomers in the sera of HLH patients were 14.3% and 71.4%, respectively, like our previous specific-antibody-based biochip investigation. Also, the biochip shared a good compatibility with CLIA assay in the measurement of sCD25(r=0.77, P<0.01). Conclusions: The biochip platform can be expanded to protein-specific serological diagnosis as a potential substitute for immunoprecipitation and ELISA to understand the interactions between proteins, ligands and receptors, and enzymes and substrates.
As a rapidly developing cell engineering technique, cell electrofusion has been increasingly applied in the field of hybridoma preparation in recent years. However, electrofusion is a certain degree of difficulty to completely replace the polyethylene glycol-mediated cell fusion. The key elements limiting electrofusion in the field of hybridoma preparation are practical complicated. This review summarizes the state of art of cell electrofusion in hybridoma preparation based on recent published literatures, mainly focusing on electrofusion instruments and their components, process control, cell treatment, and process characterization. The review provides new information and insightful commentary, critically important to the promotion of further electrofusion development in the field of hybridoma preparation.