Eminli, SarahKwieder, BaraaYi, KevinHuang, Christopher J. Z....
9页
查看更多>>摘要：The promise of using induced pluripotent stem cells (iPSCs) for cellular therapies has been hampered by the lack of easily isolatable and well characterized source cells whose genomes have undergone minimal changes during their processing. Blood-derived late-outgrowth endothelial progenitor cells (EPCs) are used for disease modeling and have potential therapeutic uses including cell transplantation and the translation of induced pluripotent stem cell (iPSC) derivatives. However, the current isolation of EPCs has been inconsistent and requires at least 40-80 mL of blood, limiting their wider use. In addition, previous EPC reprogramming methods precluded the translation of EPC-derived iPSCs to the clinic. Here a series of clinically-compatible advances in the isolation and reprogramming of EPCs is presented, including a reduction of blood sampling volumes to 10 mL and use of highly efficient RNA-based reprogramming methods together with autologous human serum, resulting in clinically relevant iPSCs carrying minimal copy number variations (CNVs) compared to their parent line.
Campo-Perez, Victordel Mar Cendra, MariaJulian, EstherTorrents, Eduard...
9页
查看更多>>摘要：Electroporation is the most widely used and efficient method to transform mycobacteria. Through this technique, fast- and slow-growing mycobacteria with smooth and rough morphotypes have been successfully transformed. However, transformation efficiencies differ widely between species and strains. In this study, the smooth and rough morphotypes of Mycobacteroides abscessus and Mycolicibacterium brumae were used to improve current electroporation procedures for fast-growing rough mycobacteria. The focus was on minimizing three well-known and challenging limitations: the mycobacterial restriction-modification systems, which degrade foreign DNA; clump formation of electrocompetent cells before electroporation; and electrical discharges during pulse delivery, which were reduced by using salt-free DNA solution. Herein, different strategies are presented that successfully address these three limitations and clearly improve the electroporation efficiencies over the current procedures. The results demonstrated that combining the developed strategies during electroporation is highly recommended for the transformation of fast-growing rough mycobacteria.
查看更多>>摘要：The synthesis of aldol adduct (3S,4R)-6-[(benzyloxycarbonyl)amino]-5,6-dideoxyhex-2-ulose, a precursor of the interesting dietary supplement, iminosugar D-fagomine, was studied in a cascade reaction with three enzymes starting from Cbz-N-3-aminopropanol. This system was studied previously using a statistical optimization method which enabled a 79 % yield of the aldol adduct with a 10 % yield of the undesired amino acid byproduct. Here, a kinetic model of the cascade, including enzyme operational stability decay rate and the undesired overoxidation of the intermediate product, was developed. The validated model was instrumental in the optimization of the cascade reaction in the batch reactor. Simulations were carried out to determine the variables with the most significant impact on substrate conversion and product yield. As a result, process conditions were found that provided the aldol adduct in 92 % yield with only 0.7 % yield of the amino acid in a one-pot one-step reaction. Additionally, compared to previous work, this improved process outcome was achieved at lower concentrations of two enzymes used in the reaction. With this study the advantages are demonstrated of a modelling approach in developing complex biocatalytical processes. Mathematical models enable better understanding of the interactions of variables in the investigated system, reduce cost, experimental efforts in the lab and time necessary to obtain results since the simulations are carried out in silico.
Park, YoungminMin, KyungminKim, Nam HyungKim, Ji-hwan...
8页
查看更多>>摘要：Porcine circovirus type 2 (PCV2) is a non-enveloped, icosahedral virus of the Circoviridae family, with a small, circular, single-stranded DNA genome. PCV2 infections cause substantial economic losses in the pig industry worldwide. Currently, commercially produced PCV2 vaccines are expensive, whereas plant-based expression systems can produce recombinant proteins at low cost for use as vaccines. In this study, recombinant PCV2 capsid protein (rCap) was transiently expressed in Nicotiana benthamiana and purified by metal affinity chromatography, with a yield of 102 mg from 1 kg plant leaves. Electron microscopy confirmed that purified rCap selfassembled into virus-like particles (VLPs) at neutral pH. It was shown to provoke a strong immune response in guinea pigs. The results indicate that plant systems can enable production of large amounts of proteins to serve as candidates for subunit vaccines.
查看更多>>摘要：As next-generation sequencing technology has become more advanced, research on microbial 16S ribosomal DNA sequences has developed rapidly. Sequencing of 16S ribosomal DNA allows the composition of bacteria and archaea in a sample to be obtained and many analytical tools related to 16S ribosomal DNA sequences have been proposed; however, most do not include a user-friendly platform with a graphical user interface. Here, a comprehensive and easy-to-use online platform, Easy Microbiome Analysis Platform (EasyMAP), has been developed for analysis of 16S ribosomal DNA sequencing data. EasyMAP integrates the QIIME2, LefSe, and PICRUSt pipelines and includes temporal profiling analysis. Users can perform quality checks, taxonomy differential abundance analysis, microbial gene function prediction and longitudinal analysis with step-by-step guidance. EasyMAP is a user-friendly tool for comprehensive analysis of 16S ribosomal DNA sequencing data. The web server and documentation are freely available at http://easymap.cgm.ntu.edu.tw/.
查看更多>>摘要：?-Galactosidases are key enzymes in the food industry. Apart from the hydrolysis of the saccharide bond of lactose, they also catalyze transgalactosylation reactions, producing galactooligosaccharides (GOS) with prebiotic activity. Here we report the heterologous production in Pichia pastoris of a novel ?-galactosidase from the fungus Thermothielavioides terrestris. The enzyme (TtbGal1) was purified and characterized, showing optimal activity at 60 ?C and pH 4. TtbGal1 is thermostable, retaining almost full activity for 24 h at 50 ?C. It was applied to the production of GOS from defined lactose solutions and acid whey, a liquid waste from the Greek yoghurt industry, reaching yields of 19.4 % and 14.8 %, respectively. HILIC?ESI-QTOF-MS analysis revealed the production of GOS with up to 4 saccharide monomers. The results demonstrate efficient GOS production catalyzed by TtbGal1, valorizing acid whey, a waste with a heavy polluting load from the dairy industry.
Mirzaee, HoomanPeralta, Noelia L. NeiraCarvalhais, Lilia C.Dennis, Paul G....
8页
查看更多>>摘要：Bacteriocins are a diverse group of bacterial antimicrobial peptides (AMPs) that represent potential replacements for current antibiotics due to their novel modes of action. At present, production costs are a key constraint to the use of bacteriocins and other AMPs. Here, we report the production of bacteriocins in planta ? a potentially scalable and cost-effective approach for AMP production. Nine bacteriocin genes with three different modes of action and minimal or no post-translational modifications were synthesized, cloned and used to transform Arabidopsis thaliana. To confirm bacteriocin functionality and the potential to use these plants as biofactories, Arabidopsis T3 crude leaf extracts were subjected to inhibition assays against the bacterial pathogens Clavibacter michiganensis subsp. michiganensis (Cmm) and Pseudomonas syringae pv. tomato DC3000 (Pst). Six and seven of nine extracts significantly inhibited Cmm and Pst, respectively. Three bacteriocin genes (plantaricin, enteriocin, and leucocin) were then selected for over-expression in tomato (Solanum lycopersicum). In vitro plant pathogen inhibition assays of T0, T1 and T2 transgenic tomato leaf extracts confirmed antimicrobial activity against both pathogens for all three generations of plants, indicating their potential use as stable biopesticide biofactories. Plantaricin and leucocin-expressing T2 tomato plants were resistant to Cmm, and leucocin-expressing T2 plants were resistant to Pst. This study highlights that plants can be used as biofactories for AMP production and that the expression of bacteriocins in planta may offer new opportunities for disease control in agriculture.
NSTL
Elsevier