Development of disposable, rapid and convenient biosensor with high sensitivity and reliability is the most desired method of viral disease prevention. To achieve this goal, in this work, a practical impedimetric biosensor has been implemented into a disposable electrode on a screen-printed carbon electrode (SPCE) for the detection of two mosquito-borne viruses. The biosensor fabrication has step-wisely carried out on the disposable electrode surface at room temperature: starting from conductive film formation, physical binding of the gold nanoparticles (AuNPs)-polyaniline (PAni) into the conductive film, and biofunctionalization. To get the maximum efficiency of the antibody, biotinylated antibody has been conjugated on the surface of AuNP-PAni /PAni-SPCE via the streptavidin-biotin conjugation method which is a critical factor for the high sensitivity. Using the antibody-antigen interaction, this disposable electrode has designed to detect mosquito-borne infectious viruses, Chikungunya virus (CHIKV) and Zika Virus (ZIKV) separately in a wide linear range of 100 fg/mL to 1 ng/mL with a low detection limit of 1.33 fg/mL and 12.31 fg/mL, respectively.
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.
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.
Functional interaction between cancer cells and the surrounding microenvironment is still not sufficiently understood, which motivates the tremendous interest for the development of numerous in vitro and in vivo tumor models. Diverse parameters, e.g., transport of nutrients and metabolites, availability of space in the confinement, interaction with scaffolds, etc. make an impact on the size, shape, and metabolism of the tumoroids. Herein, we demonstrate the fluidics-based low-cost methodology to reproducibly generate the alginate and alginate-chitosan microcapsules and apply it to grow human hepatoma (HepG2) tumoroids of different dimensions and geometries. Focusing specifically on the composition and thickness of the hydrogel shell, permeability of the microcapsules is selectively tuned. The diffusion of the selected benchmark molecules through the shell has been systematically investigated using both, experiments and simulations, which is essential to ensure efficient mass transfer of small molecules and prevent large substances from reaching the encapsulated cells. Depending on available space, phenotypically different 3D cell assemblies have been observed inside the capsules, varying in the tightness of cell aggregations and their shapes. Metabolic activity of tumoroids in microcapsules was confirmed by tracking the turnover of testosterone to androstenedione with chromatography studies in a metabolic assay. Because of the high reproducibility, compartmentalization, and facile tuning of the shell thickness and permeability, our system is not only a great platform for the formation of cancer tumoroids, but also a promising tool for the design and engineering of other cells.
Previous work developed a quantitative model using capacitance spectroscopy in an at-line setup to predict the dying cell percentage measured from a flow cytometer. This work aimed to transfer the at-line model to monitor lab-scale bioreactors in real-time, waiving the need for frequent sampling and enabling precise controls. Due to the difference between the at-line and in-line capacitance probes, direct application of the at-line model resulted in poor accuracy and high prediction bias. A new model with a variable range that had similar spectra shape across all probes was first constructed, which improved the prediction accuracy. Moreover, the global calibration method included the variance of different probes and scales into the model, reducing the prediction bias. External parameter orthogonalization also mitigated the interference from feeding, which further improved the model performance. The culture evolution trajectory predicted by the in-line model captured the cell death and alarmed cell death onset earlier than the trypan blue exclusion test. In addition, incorporation of at-line spectra following orthogonal design into the calibration set is more likely to generate robust calibration models than the calibration models constructed using the in-line spectra only. This is advantageous, as at-line spectra collection is easier, faster, and more material-sparing than in-line spectra collection. The root-mean-square error of prediction of the final model was 6.56% (8.42% of the prediction range) with an R2 of 92.4%.
Raman spectroscopy has gained popularity to monitor multiple process indicators simultaneously in biopharmaceutical processes. However, robust and specific model calibration remains a challenge due to insufficient analyte variability to train the models and high cross-correlation of various media components and artefacts throughout the process. Therefore, a systematic Raman calibration workflow for perfusion processes enabling highly specific and fast model calibration was developed. A harvest library consisting of frozen harvest samples from multiple CHO cell culture bioreactors collected at different process times was established, capturing process variability as widely as possible. Model calibration was subsequently performed in an offline setup using a flow cell by spiking process harvest with various sugars known to modulate glycosylation patterns of monoclonal antibodies. In a screening phase, Raman spectroscopy was proven capable not only to distinguish glucose, raffinose, galactose, mannose, and fructose in perfusion harvest, but also to quantify them independently in process relevant concentrations. In a second phase, a robust and highly specific calibration model for simultaneous glucose (RMSEP = 0.32 g/L) and raffinose (RMSEP = 0.17 g/L) real-time monitoring was generated and verified in a third phase during a perfusion process. The proposed offline calibration workflow allowed proper Raman peak decoupling, reduced calibration time from months down to days and can potentially be applied to other analytes of interest including lactate, ammonia, amino acids, or product titer.
The scale-up of bioprocesses is still one of the major obstacles in biotechnological industry. Scale-down bioreactors were identified as valuable tools to investigate the heterogeneities observed in large-scale tanks in laboratory-scale. Additionally, computational fluid dynamics (CFD) simulations can be used to gain information about fluid flow in tanks used for production. Here we present the rational design and comprehensive characterization of a scale-down setup, in which a flexible and modular plug-flow reactor is connected to a stirred tank bioreactor. With the help of CFD the mixing time difference between differently scaled bioreactors were evaluated and used as scale-down criterium. Additionally, it was used to characterize the setup at conditions were experiments could technically not be performed. This was the first time a scale-down setup was tested on high cell density Escherichia coli cultivations to produce industrial relevant antigen-binding fragments (Fab). Reduced biomass and product yields were observed during the scale-down cultivations. Additionally, the intracellular Fab fraction was increased by using the setup. The results show that including CFD in the design and characterization of a scale-down reactor can help to keep a connection to production scale and also gain intensive knowledge about the setup, which enhances usability.
Protein-based condensates have been proposed to accelerate biochemical reactions by enriching reactants and enzymes simultaneously. Here, we engineered those condensates into a Photo-Activated Switch in E. coli (PhASE) to regulate enzymatic reactions via tuning the spatial correlation of enzymes and substrates. In this system, scaffold proteins undergo liquid-liquid phase separation (LLPS) to form light-responsive compartments. Tethered with a light-responsive protein, enzymes of interest (EOIs) can be recruited by those compartments from cytosol within only a few seconds after a pulse of light induction and fully released in 15 minutes. Furthermore, we managed to enrich small molecular substrates simultaneously with enzymes in the compartments and achieved the acceleration of luciferin and catechol oxidation by 2.3 folds and 1.6 folds, respectively. We also developed a quantitative model to guide the further optimization of this de-mixed regulatory system. Our tool can thus be used to study the rapid redistribution of proteins, and reversibly regulate enzymatic reactions in E. coli.
Immobilized enzymes have drawn widespread attention due to the enhanced stability, easy separation from reaction mixture, and the prominent recyclability. Nevertheless, it is still an ongoing challenge to develop potent immobilization techniques which are capable of stable enzyme encapsulation, minimal loss of activity, and modulability for various enzymes and applications. Here, microfibers with tunable size and composition were fabricated using a home-made microfluidic device. These microfibers were able to efficiently encapsulate bovine serum albumin (BSA), glucose oxidase (GOX) and horseradish peroxidase (HRP). But the physically adsorbed enzymes readily diffused from microfibers into the catalytic reaction system. The leakage of enzymes could be substantially inhibited by conjugating to polyacrylic acid (PAA) and incorporating into the alginate-based microfibers, enabling stable immobilization, improved recyclability, and enhanced thermostability. In addition, GOX and HRP-loaded microfibers were fabricated under the optimized conditions for the visual detection of glucose using the cascade reaction of these enzymes, showing sensitive color change to glucose with concentration range of 0-2 mM. Due to the tunability and versatility, this microfluidic-based microfiber platform may provide a valuable approach to the enzyme immobilization for the cascade catalysis and diagnoses with multiple clinical markers.