Background: Epitope mapping is an increasingly important aspect of biotherapeutic and vaccine development. Recent advances in therapeutic antibody design and production has enabled candidate mAbs to be identified at a rapidly increasing rate resulting in a significant bottleneck in the characterization of ‘structural’ epitopes, that are challenging to determine using existing high throughput epitope mapping tools. Here, Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) epitope screening workflow was introduced that is well suited for accelerated characterization of epitopes with a common antigen. Main methods and major results: The method is demonstrated on set of 6 candidate mAbs targeting Pertactin (PRN). Using this approach, five of the six epitopes was unambiguously determined using two HDX mixing timepoints in 24 hours total run time, corresponding to substantial decrease in the instrument time required to map a single epitope using conventional HDX workflows. Conclusion: An accelerated HDX-MS epitope screening workflow was developed. The two-timepoint ‘screening’ workflow mapped all six mAbs and generated high confidence epitopes for five of the six mAbs assayed. The substantial improvement in the rate of data collection can advance HDX-MS for higher throughput investigations supporting the ability to evaluate a broader number of mAb candidates at an earlier stage of vaccine development.
Carotenoids and tocopherols are health-promoting metabolites in livestock and human diets. Some important crops have been genetically modified to increase their content. Although the usefulness of transgenic plants to alleviate nutritional deficiencies is obvious, their social acceptance has been controversial. Here, we demonstrate an alternative biotechnological strategy for carotenoid and tocopherol fortification of edible fruits in which no transgenic DNA is involved. A viral RNA vector derived from Zucchini yellow mosaic virus (ZYMV) was modified to express a bacterial phytoene synthase (crtB), and inoculated in zucchini (Cucurbita pepo L.) leaves nurturing pollinated flowers. After the viral vector moved to the developing fruit and expressed crtB, the rind and flesh of the fruits developed yellow-orange rather than green color. Metabolite analyses showed a substantial enrichment in health-promoting carotenoids, such as α- and β-carotene (pro-vitamin A), lutein and phytoene, in both rind and flesh. Considerably higher accumulation of α- and γ-tocopherol was also detected, particularly in fruit rind. Although this strategy is perhaps not free from controversy due to the use of genetically modified viral RNA, our work does demonstrate the possibility of metabolically fortifying edible fruits using an approach in which no transgenes are involved.
Yangpumicins (YPMs), eg. YPM A, F, and G, are newly discovered enediynes from Micromonospora yangpuensis DSM 45577, which could be exploited as promising payloads of antibody-drug conjugates. However, the low yield of YPMs in the wild-type strain (~1 mg/L) significantly hampers their further drug development. In this study, a combined ribosome engineering and fermentation optimization strategy has been used for yield improvement of YPMs. One gentamycin-resistant M. yangpuensis DSM 45577 strain (MY-G-1) showed higher YPMs production (7.4 ± 1.0 mg/L), while it exhibits delayed sporulation and slender mycelium under scanning electron microscopy. Whole genome re-sequencing of MY-G-1 reveals several deletion and single nucleotide polymorphism mutations, which were confirmed by PCR and DNA sequencing. Further Box–Behnken experiment and regression analysis determined that the optimal medium concentrations of soluble starch, mannitol, and pharmamedia for YPMs production in shaking flasks (10.0 ± 0.8 mg/L). Finally, the total titer of YPM A/F/G in MY-G-1 reached to 15.0 ± 2.5 mg/L in 3-L fermenters, which was about 11-fold higher than the original titer of 1.3 ± 0.3 mg/L in wild-type strain. Our study may be instrumental to develop YPMs into a clinical anticancer drug, and inspire the use of these multifaceted strategies for yield improvement in Micromonospora species.
Many transgenic animals have been produced using CRISPR–Cas9 technology to edit specific genes. However, there are few guidelines for the application of this technique in cattle. The goal of this study was to produce trait-improved cattle using the genome editing technology CRISPR–Cas9. Myostatin (MSTN) was selected as a target locus and synthetic mRNA of sgRNA and Cas9 was microinjected into bovine in vitro fertilized embryos. As a result, 17 healthy calves were born and 3 of these showed MSTN mutation rates of 10.5%, 45.4%, and 99.9%, respectively. Importantly, the offspring with the 99.9% MSTN mutation rate had biallelic mutation (-12bp) and a doubling muscle growth phenotype. In conclusion, we showed that the genome editing technology CRISPR–Cas9 can produce genetically modified calves with improved traits.
In the development of personalized medicine, the ultrasensitive detection of point mutations that correlate with diseases is important to improve the efficacy of treatment and guide clinical medication. In this study, locked nucleic acid (LNA) was introduced as an amplification suppressor of a massive number of wild-type alleles in an amplification refractory mutation system (ARMS) to achieve the detection of low-abundance mutations with high specificity and sensitivity of at least 0.1%. By integrating the length of clamp, base type, number and position of LNA modifications, we have established a “shortest length with the fewest LNA bases” principle from which each LNA base would play a key role in the affinity and the ability of single base discrimination could be improve. Finally, based on this LNA design guideline, a series of the most important single point mutation sites of epidermal growth factor receptor (EGFR) was verified to achieve the optimal amplification state which as low as 0.1% mutation gene amplification was not affected under the wild gene amplification was completely inhibited, demonstrating that the proposed design principle has good applicability and versatility and is of great significance for the detection of circulating tumor DNA.
Abstract: 2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2G) is an important industrial derivative of L-ascorbic acid (AA), which has the distinct advantages of non-reducibility, antioxidation, and reproducible decomposition into L-ascorbic acid and glucose. Enzymatic synthesis is a preferred method for AA-2G production over alternative chemical synthesis owing to the regioselective glycosylation reaction. α-Glucosidase, an enzyme classed into O- glycoside hydrolases, may be used in glycosylation reactions to synthesize AA-2G. Here, one α-glucosidase from Oryza sativa (rAGL) was recombinantly produced in Pichia pastoris GS115 and used for biosynthesis of AA-2G with few intermediates and byproducts. The extracellular rAGL reached 9.11 U/mL after fed-batch cultivation for 102 h in a 5-L fermenter. The specific activity of purified rAGL is 49.83 U/mg at 37 °C and pH 4.0. The optimal temperature of rAGL was 65 °C, and it was stable below 55 °C. rAGL was active over the range of pH 3.0–7.0, with the maximal activity at pH 4.0. Under the condition of 37 °C , pH 4.0, equimolar maltose and AA·Na, 8.7±0.4 g/L of AA-2G was synthesized by rAGL. These studies lay the basis for the industrial application of recombinant α-glucosidase. Keywords: α-Glucosidase; Oryza sativa; 2-O-α-D-glucopyranosyl-L-ascorbic acid; Transglycosylation; Pichia pastoris
Recent noteworthy advances in the development of high-performing microbial and mammalian strains have enabled the sustainable production of bio-economically valuable substances such as bio-compounds, biofuels, and biopharmaceuticals. However, to obtain an industrially viable mass-production scheme, much time and effort are required. The robust and rational design of fermentation processes requires analysis and optimization of different extracellular conditions and medium components, which have a massive effect on growth and productivity. In this regard, knowledge- and data-driven modeling methods have received much attention. Constraint-based modeling (CBM) is a knowledge-driven mathematical approach that has been widely used in fermentation analysis and optimization due to its capabilities of predicting the cellular phenotype from genotype through high-throughput means. On the other hand, machine learning (ML) is a data-driven statistical method that identifies the data patterns within sophisticated biological systems and processes, where there is inadequate knowledge to represent underlying mechanisms. Furthermore, ML models are becoming a viable complement to constraint-based models in a reciprocal manner when one is used as a pre-step of another. As a result, more predictable models are produced. This review highlights the applications of CBM and ML independently and the combination of these two approaches for analyzing and optimizing fermentation parameters.
In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the backbone for the mature LASSO probe through Cre-Loxp intramolecular recombination. This strategy generates high quality LASSO probes libraries (~46% of probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 E.coli ORFs spanning from 400- to 4,000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, are able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.
Viral surrogates to screen for virus inactivation (VI) can be a faster, cheaper and safer alternative to third-party testing of pathogenic BSL2 (Biosafety Level 2) model viruses. Although the bacteriophage surrogate, Ø6, has been used to assess low pH BSL2 VI, it has not been used for evaluation of detergent-mediated VI. Furthermore, Ø6 is typically assayed through host cell infectivity which introduces the risk of cross-contaminating other cell lines in the facility. To circumvent contamination, we developed an in-house RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) assay for selective detection of active Ø6 from a population of live and dead phage. The RT-qPCR assay was used to evaluate Ø6 inactivation in cell culture fluid of monoclonal antibody and fusion protein. Complementary Ø6 infectivity was also conducted at a third-party testing facility. The Ø6 RT-qPCR and infectivity data was modeled against VI of three BSL2 viruses, X- MuLV, A- MuLV and HSV-1 in corresponding therapeutics. Both Ø6 methods demonstrate that any VI agent showing Ø6 clearance of ≥ 2.5 logs would demonstrate complete BSL2 VI of ≥ 4.0 logs. Compared to BSL2 virus testing, this in-house Ø6 RT-qPCR tool can screen VI agents at 5% the cost and a turnaround time of 2-3 days versus 4-7 months.
Because of their excellent therapeutic potential, mesenchymal stem cells (MSCs) have been used as cell therapeutics for various diseases. However, the survival rate and duration of MSCs after transplantation are extremely low and short, respectively. To solve these problems, in this study, we prepared multicellular spheroids of MSCs and investigated their survival and function after intravenous injection in mice. The murine adipose-derived MSC line m17.ASC was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids of an average diameter and cell number of approximately 170 μm and 1100 cells/spheroid, respectively. The intravenously injected m17.ASC spheroids mainly accumulated in the lung and showed a higher survival rate than suspended m17.ASC cells during the experimental period of 7 days. m17.ASC spheroids efficiently reduced the lipopolysaccharide-induced increase in plasma concentrations of interleukin-6 and tumor necrosis factor-α. These results indicate that spheroid formation improved the pulmonary delivery and survival of MSCs, as well as their therapeutic potential against inflammatory pulmonary diseases.
We herein describe an ingenious centrifugal microfluidic system to accomplish a fully automated serial dilution. The liquid flow on the disc was automatically regulated by utilizing ferrowax microvalves systematically integrated into the channels within the specially designed metering structure. By opening the differently positioned microvalves through irradiation of IR laser to allow metering, the same amount of diluent was serially eluted to the dilution chamber from the same diluent chamber. After dilution, the diluted samples were automatically delivered to the respective final product chambers by appropriately opening or closing the microvalves in the connecting channels, followed by rotating the disc. Based on this unique design principle, six consecutive two-fold and ten-fold dilutions were successfully achieved, yielding excellent accuracy in a wide dynamic range up to six orders of magnitude. Very importantly, the overall serial dilution process, including the diluent addition, mixing, and product transfer steps, was completed very rapidly within five minutes, due to the minimized procedures enabled by the automated actuation of the ferrowax microvalves at the rationally designed positions. The centrifugal microfluidic system would serve as a powerful elemental tool to realize the fully automated diagnostic microsystem involving the serial dilution process.
Difference between two strong cation-exchange resins, namely sulfonium type and sulfate type regarding both their salt tolerance and hydrophobicity were investigated. There is only tiny variation between sulfate and sulfonic group and at the first glance it seems unlikely that it could be the reason for changed selectivity and salt tolerance that was detected in our preliminary experiments. For that reason salt tolerance and hydrophobicity of both ligands was investigated by using two representative polymethacrylate-based ion exchangers as for the sulfonium type TOYOPEARL GigaCap S-650M and for the sulfate type TOYOPEARL Sulfate-650F. In addition some in-silico calculations were performed for model substances representing the sulfonium and sulfate group, and significant differences were calculated regarding their hydrophobicity. These experiments confirmed the working hypothesis that salt tolerance and higher affinity and selectivity for some human plasma derived vitamin K dependent clotting factors and inhibitors are interrelated and dependent from the presence of the sulfate group. The affinity for these proteins was experimentally verified by separation of clotting factor IX from the prothrombin complex concentrate. Presented results show that a simple and fast separation between clotting factor IX and other vitamin K dependent clotting factors II, VII and X is possible, only if the resin with the sulfate, and not with sulfonic acid ligand was applied. Consequently, an immediate application of undiluted feedstock or the eluate from previous isolation step to sulfate resin is possible, and a significant optimization of downstream process can be achieved.
Current production costs of microalgal biomass indicate that only highly-productive cultivation facilities will approach commercial feasibility. Geographical site selection for siting those facilities is critical for achieving target productivities. The aim of this study was to provide a semi-empirical estimation of microalgal biomass and lipids productivity in South America. Simulated-climate was programed in environmental photobioreactors (PhenometricsTM) for a simulation of cultivation in open raceway ponds at different geographical sites. The mean annual South American biomass productivity of 20-cm deep ponds was 12 ± 4 g · m- 2 · d-1. The most productive regions were clustered in the subtropical and tropical regions of the continent. Fortaleza (Brazil) showed a low seasonality and a high annual mean productivity of 23 g · m-2 · d-1 in 5-cm deep ponds. Lipids accumulation and productivity in Fortaleza showed a high microalgal oil accumulation up to 46% (w/w) and a maximum oil productivity of 5 g · m-2 · d-1 for biomass containing around 20% lipids (w/w). This study provides the first semi-empirical estimation of microalgal productivity in South America and supports a high potential of a vast region of the continent.
Microfluidic devices have been introduced for phenotypic screening of zebrafish larvae in both fundamental and pre-clinical research. One of the remaining challenges for the broad use of microfluidic devices is their limited throughput, especially in behavioural assays. Previously, we introduced the tail locomotion of a semi-mobile zebrafish larva evoked on-demand with electric signal in a microfluidic device. Here, we report the lessons learned for increasing the number of specimens from one to four larvae in this device. Multiple parameters including loading and testing time per fish and loading and orientation efficiencies were refined to optimize the performance of modified designs. Simulations of the flow and electric field within the final device provided insight into the flow behavior and functionality of traps when compared to previous single-larva devices. Outcomes led to a new design which decreased the testing time per larva by approximately 60%. Further, loading and orientation efficiencies increased by more than 80%. Critical behavioural parameters such as response duration and tail beat frequency were similar in both single and quadruple-fish devices. The optimized microfluidic device has significant advantages for greater throughput and efficiency when behavioral phenotyping is required in various applications, including chemical testing in toxicology and gene screening.
Given the potential applications of gas vesicles (GVs) in multiple fields including antigen-displaying and imaging, heterologous reconstitution of synthetic GVs is an attractive and interesting study that has translational potential. Here, we attempted to express and assemble GV proteins (GVPs) into GVs using the model eukaryotic organism Saccharomyces cerevisiae. We first selected and expressed two core structural proteins, GvpA and GvpC from cyanobacteria Anabaena flos-aquae and Planktothrix rubescens, respectively. We then optimized the protein expression conditions and validated GV assembly in the context of cell flotation and GV shapes. We found that when two copies of AnaA were integrated into the genome, it resulted in cell floatation and GV production regardless of GvpC expression. Next, we co-expressed chaperone-RFP with the GFP-AnaA to aid the AnaA aggregation. The co-expression of individual chaperones (Hsp42, Sis1, Hsp104, and GvpN) with AnaA led to the formation of larger inclusions and enhanced the sequestration of AnaA into the perivacuolar site. To our knowledge, this represents the first study on reconstitution of GVs in S. cerevisiae. Our results could provide insights into optimizing conditions for heterologous protein expressions as well as the reconstitution of other synthetic microcompartments in yeast.
Failure in the prevention of cross-transmission from contaminated gloves has been recognized as an important factor that contributes to the spread of several healthcare-associated infections. Ex situ coating process with silver nanoparticles (AgNPs) using Eucalyptus citriodora ethanolic leaf extract as reducing and capping agents to coat glove surfaces has been developed to prevent this mode of transmission. Elemental analysis of coated gloves showed 24.8 Wt% silver densely adhere on the glove surface. The coated gloves fully eradicated important hospital-acquired pathogens including Gram-positive bacteria, Gram-negative bacteria, and yeasts within 1 h. The coated gloves showed significant reduction, an average of 5 logs when tested against all standard strains and most clinical isolates (p < 0.01). Following prolonged exposure, the coating significantly reduced the numbers of most adhered pathogenic species, compared with uncoated gloves (p < 0.0001), which was observed by fluorescence microscopy. Scanning electron microscopy further confirmed that AgNPs coated-gloves reduced microbial adhesion of mixed-species biofilms, compared with uncoated gloves. A series of contamination and transmission assays demonstrated no transmission of viable organisms. Biocompatibility analysis confirmed high cell viability of HaCaT and L929 cells at all concentrations of AgNPs tested. The coated gloves were non-toxic with direct contact with L929 cells.
The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. Fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened individually and in combinations. A titer of 29.2±1.6 mg/L Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene’s overexpression led to a further 1.5-fold titer increase. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1±13.4 mg/L of Z11-14:OH was obtained. Fatty alcohols were chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis at a level similar to that of the chemically synthesized pheromone used as a control, although full precopulatory behavior was observed less often. The study paves the way for the production of ECB pheromone by fermentation.
Ursolic acid (UA) is a ursane-type pentacyclic triterpenoid compound, naturally produced in plants via specialized metabolism and exhibits vast range of remarkable physiological activities and pharmacological manifestations. Owing to significant safety and efficacy in different medical conditions, UA may serve as a backbone to produce its derivatives with novel therapeutic functions. This review systematically provides an overview of the pharmacological activities, acquisition methods and structural modification methods of UA. In addition, we focused on the synthetic modifications of UA to yield its valuable derivatives with enhanced therapeutic potential. Furthermore, harnessing the essential advances for green synthesis of UA and its derivatives by advent of metabolic engineering and synthetic biology are highlighted. In combination with the advantages of UA biosynthesis and transformation strategy, large-scale production and applications of UA is a promising platform for further exploration.