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.

Laccases have shown to be efficient biocatalysts for the removal of recalcitrant pollutants from wastewater. Thus, they catalyse the oxidation of a wide variety of organic compounds by reducing molecular oxygen to water. However, the use of free laccases holds several drawbacks such as poor reusability, high cost, low stability and sensibility to different denaturing agents that may occur in wastewater. Such drawbacks can be circumvented by immobilising laccase enzymes in/on solid carriers. Hence, during the last decades different approaches considering various techniques and solid carriers to immobilise laccase enzymes have been developed and tested for the removal of pollutants from wastewater. To scale up wastewater treatment bioprocesses, the immobilised laccases are placed in different reactor configurations.

Recent technological advancements in synthetic and systems biology have enabled the construction of microbial cell factories expressing diverse heterologous pathways in unprecedentedly short time scales. However, the translation of such laboratory scale breakthroughs to industrial bioprocesses remains a major bottleneck. In this study, an accelerated bioprocess development approach was employed to optimize the biosynthetic pathway of the blockbuster chemotherapy drug, Taxol. Statistical design of experiments approaches were coupled with an industrially relevant high-throughput microbioreactor system to optimize production of key Taxol intermediates, Taxadien-5α-ol and Taxadien-5α-yl-acetate, in engineered yeast cell factories. The optimal factor combination was determined via data driven statistical modelling and validated in 1L bioreactors leading to a 2.1-fold improvement in taxane production compared to a typical defined media. Elucidation and mitigation of a nutrient limitation enhanced product titers a further two-fold and titers of the critical Taxol precursors, Taxadien-5α-ol and Taxadien-5α-yl-acetate were improved to 34 and 11 mg/L, representing a three-fold improvement compared to the highest literature titers in S. cerevisiae. Comparable titers were obtained when the process was scaled up a further five-fold using 5 L bioreactors. The results of this study highlight the benefits of a holistic design of experiments guided approach to expedite early stage bioprocess development.

Peptide drugs are developed from endogenous or synthetic peptides with specific biological activities. They have advantages of strong target specificity, high efficacy and low toxicity, thus showing great promise in the treatment of many diseases such as cancer, infections and diabetes. Although an increasing number of peptide drugs have entered market in recent years, the preparation of peptide drug substances is yet a bottleneck problem for their industrial production. Comparing to the chemical synthesis method, peptide biosynthesis has advantages of simple synthesis, low cost, and low contamination. Therefore, the biosynthesis technology of peptide drugs has been widely used for manufacturing. Herein, we reviewed the development of peptide drugs and recent advances in peptide biosynthesis technology, in order to shed a light to the prospect of industrial production of peptide drugs based on biosynthesis technology.

Statins as a lipid-lowering drug can selectively inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and decrease cholesterol synthesis effectively. With the improvement of nutritional conditions, the demand for statins is increasing in global market. Due to the rapid development of modern biotechnologies, the biosynthesis of stains by microbial cell factory appears great advantages. It has the advantages of simple operation and easy separation of products. This review summarized the strategies on statins production via microbial cell factory, including both traditional fermentation culture and modern synthetic biology manufacture. Firstly, the complex fermentation parameters and process control technology on submerged fermentation (SmF) and solid-state fermentation (SSF) were introduced in detail. Especially, the possibility of recoverable agricultural wastes/(Biomass) as fermentation substrate on solid-state fermentation to produce statins was emphasized. Besides, metabolic engineering strategies to construct robust engineering strains and strains evolution were also discussed. The review highlights the potential and challenge of microbial cell factory to yield the statins. Thus, it will facilitate the production of statins in more green production mode.

Microtiter plates are suitable for screening and process development of most microorganisms. They are currently the container of choice for high-throughput and small-scale microbial culture, but require optimization for specific work. This research presents a novel type of microtiter plate was developed using computational fluid dynamics (CFD) technology. The new plate provides high oxygen supply and optimal mixing effects for the fermentation culture of docosahexaenoic acid (DHA) producing strains, surpassing the conventional method of strains screening with shake flasks, which is insufficient. the shape of the microtiter plate was modified, and baffles were introduced to improve mass transfer and oxygen supply effects in the vibrating bioreactor. CFD technology was used to model the new plate’s characteristics, establishing the superiority of hexagonal microtiter plates with six baffles. Parameters in the incubation process, such as vibration frequency and liquid load, were optimized, and the final result achieved a KLa of 0.61s-1 and a volume power input of 2364 w/m3, which was 4-5 times better than the original 96-well plate. The culture results optimized by the model were also verified. Therefore, this new microtiter plate provides a powerful tool for future high-throughput screening of strains.

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.

Drug-metabolizing enzymes play an important role in the metabolism of drugs in vivo. Their activity is an important factor affecting the rate of drug metabolism, which directly determines the intensity and persistence of drug action. Patients taking medication can be divided into different metabolic types through detection of CYP2C19 drug-metabolizing enzyme gene polymorphisms, which can then be used for medication guidance for clopidogrel. Here, we describe a detection method based on real-time polymerase chain reaction. This method uses multicolor melting curve analysis to accurately identify different mutation sites and genotypes of CYP2C19 * 2, CYP2C19 * 3, and CYP2C19 * 17. The detection limit of plasmid samples was 1 copies/µl; that of genomic samples was 0.1 ng/µl. The system can detect nine types of CYP2C19 * 2/3/17 at three sites in one tube, quickly achieving detection within 1 h. Combined with the sample release agent, sample extraction was completed in 5 s, achieving rapid diagnosis without extraction for timely diagnosis and treatment. Furthermore, the system is not limited to blood samples and can also be applied to oropharyngeal and saliva samples, increasing sampling diversity and convenience. When using clinical blood samples (n=93), the detection system we established was able to quickly and accurately identify different genotypes, and the accuracy and effectiveness of the detection were confirmed by Sanger sequencing.

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.

In recent years Next-Generation Sequencing (NGS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ~60% down to ~0.3% frequencies. We subsequently tested the effect of 16 different sample and NGS library preparation protocols on the assay’s ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ~0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.

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.

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.

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.

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.

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.

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.

Tuberculosis (TB) and its emerged drug resistance exert huge threats on the global health, therefore development of novel anti-TB antibiotics is very essential. Ilamycin-E1/E2 is a pair of cycloheptapeptide enantiomers obtained from a marine-derived Streptomyces atratus SCSIO ZH16-ΔilaR mutant, and become promising anti-TB lead compounds due to their significant anti-TB activities, but their low titer hampered the further clinical development. In this work, the statistical Plackett-Burman design (PBD) model was applied to screen out bacterial peptone as the only significant but negative factor affecting the ilamycin-E1/E2 production. Subsequent single factor optimization revealed that replacement of bacterial peptone with malt extract eliminated the accumulation of porphyrin-type competitive byproduct, and the titer of ilamycin-E1/E2 in shaking flasks was improved from original 13.6±0.8 to 142.7±5.7 mg/L for about 10.5 folds. Furthermore, a pH coordinated feeding strategy was first adopted in scaled-up production of ilamycin-E1/E2. The obtained titer of ilamycin-E1/E2 in 30L was 169.8±2.5 mg/L, while in 300L fermentor was only 131.5±7.5 mg/L due to the unsynchronization of feeding response and pH change. Therefore, the continuous pulse feeding strategy was further applied in 300L fermentor and finally achieved 415.7±29.2 mg/L ilamycin-E1/E2, which represented about 30.5 folds improvement at last. Our work provided the solid basis to achieve sufficient ilamycin-E1/E2 lead compounds and support their potential anti-TB drug development.

The combination of single-cell RNA sequencing and microdissection techniques that preserves positional information has become a major tool for spatial transcriptome analyses. However, high costs and time requirements, especially for experiments at the single cell scale, make it challenging for this approach to meet the demand for increased throughput. Therefore, we proposed combinational DNA barcode (CDB)-seq as a medium-throughput, multiplexed approach combining Smart-3SEQ and CDB magnetic microbeads for transcriptome analyses of microdissected tissue samples. We conducted a comprehensive comparison of conditions for CDB microbead preparation and related factors and then applied CDB-seq to RNA extracts, fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) mouse brain tissue samples. CDB-seq transcriptomic profiles of tens of microdissected samples could be obtained in a simple, cost-effective way, providing a promising method for future spatial transcriptomics.