The present study aims to immobilize the uricase enzyme on magnetic nanowires and to examine its potential for use in the treatment of gout. For this, Au/Ni/Au nanowires were synthesized using a polycarbonate membrane template by the sequential electrodeposition of Au, Ni, and Au, respectively. The uricase enzyme was covalently attached to these nanowires and was also coated with PEG. Optimum enzymatic conditions, kinetic parameters, thermal, storage, and operational stability were determined by performing enzymatic activity tests of free and immobilized uricase. Additionally, the efficacy of both enzyme preparations in artificial human serum and the presence of protease was also investigated. Experimental results showed that immobilized uricase showed higher stability than free uricase in all studied conditions. The potential of immobilized uricase to oxidize uric acid in artificial serum was also investigated and it was found that immobilized preparation demonstrated approximately 6 times higher activity than that of the free enzyme. The results of this study showed that uricase-attached nanowires oxidized uric acid effectively and are promising in the treatment of gout.

Exosomes have recently been considered ideal biotherapeutic nanocarriers that broaden the frontiers of current drug delivery systems to overcome the shortcomings associated with cytokine-based immunotherapy. Using this approach, the current study aimed to assess anti-proliferative activity of purified IL-29 and exosomes encapsulated IL-29. The IL-29+pET-28a construct was transformed into Rosetta 2(DE3) cells which was used for the large-scale production of IL-29. Exosomes isolated from H1HeLa, and SF-767 cells using Total Exosome Isolation reagent were loaded with IL-29 via sonication. Isolation of exosomes was validated using their core protein signature by western blotting and specific miRNA profiles by RT-PCR. The drug loading efficiency of exosomes derived from H1HeLa cells was higher than that of SF-767-derived exosomes. The drug release kinetics of IL-29 encapsulated exosomes exhibited stable release of the recombinant drug. Around 50% of all cancer cell lines survived when IL-29 was administered at a concentration of 20 µg/mL. A survival rate of less than 10% was observed when cells were treated with 20 µg/mL IL-29 loaded exosomes. It was concluded that IL-29 loaded exosomes had a more significant cytotoxic effect against cancer cells, which might be attributed to sustained drug release, improved half-life, superior targeting efficacy, capacity to harness endogenous intracellular trafficking pathways, and heightened biocompatibility of exosomes.

The use of transposase in cell line development (CLD) programs has experienced increased popularity over the past decade. However, few studies have described the mechanism of action and the genomic and phenotypic characteristics of clones derived from transposase. Additionally, how these traits impact long-term bioproduction is unknown. Here, we use chromosome painting, deep sequencing, and ddPCR to characterize the unique fingerprints associated with transposase-derived clones. Transposase reduces the cellular pool of transient vector as early as three days post transfection following transfection and expedites stable pool establishment by up to two weeks. Furthermore, recombinant DNA expression is significantly improved up to ∼3 fold along with a greater balance of antibody heavy and light chain transcripts, resulting in higher titers in transposase generated pools. Transposase derived pools contained an often innumerable number of integration sites, representing a vast increase in integration site diversity over randomly generated pools, which were bottlenecked at 1-3 integration sites per pool. These transposase mediated integrations typically occurred in clean singlets, free of genomic scars such as deletions, inversions, and other modifications associated with legacy transfection methods which exhibited higher copy numbers per integration site. Relative declines in gene expression occur with copy number increase in the randomly generated, but not the transposase derived clones. Furthermore, transposase-derived clones were more likely to exhibit enhanced a long term stability profile, including product quality attributes such as mannose-5. This improved stability may result from circumventing mechanisms associated with the silencing of tandem repeats. Thus, transposase-mediated approaches can provide multifaceted molecular and phenotypic advantages in cell line development when compared to legacy random-integration methods.

Chronic myeloid leukemia (CML) accounts for approximately 15% of leukemias. LukS-PV, a Panton-Valentine leucocidin (PVL) component, is secreted by Staphylococcus aureus. Silver nanoparticles have increasingly been used for different purposes, most notably for drug delivery and anticancer agents. In this work, the cytotoxicity effect of recombinant LukS-PV protein, chemically synthesized AgNPs, and recombinant LukS-PV protein-loaded silver nanoparticles was investigated on human Chronic myeloid leukemia K562 cells and human normal embryonic kidney HEK293 cells. Cell apoptosis was investigated by staining with Annexin V/propidium iodide. The recombinant LukS-PV protein-loaded silver nanoparticles exhibited dose-dependent cytotoxicity and induced apoptosis in the K562 cells but had little effect on normal HEK293 cells. After 24 h of exposure to recombinant LukS-PV protein-loaded silver nanoparticles (IC50 concentration), flow cytometry showed that 31.17% of K562 cells were apoptotic. These results indicate that recombinant LukS-PV protein-loaded silver nanoparticles maybe are a potential chemotherapeutic agent candidate against K562 cells. Hence, silver nanoparticles could be used as drug carriers for toxin release to cancer cells.

The loss of mixing efficiency inherent to bioreactor process operated at large-scale yields to the formation of concentration gradient and thus to heterogeneous culture conditions. For processes operated with methanol feeding, P. pastoris faces oscillatory culture conditions that significantly affect the cell ability to produce secretory recombinant proteins at high yield. Extended cell residence time in microenvironments of high methanol concentration and low oxygen availability that are typically found in the upper part of the bioreactor near the feeding point, triggers the unfolded protein response (UPR) and thus impairs proper protein secretion. Methanol co-feeding with sorbitol was shown herein to reduce the UPR response and to restore productivity of secreted protein.

D-pantolactone (D-PL) is one of the important chiral intermediates in the synthesis of D-pantothenic acid. Our previous study has revealed that ketopantolactone (KPL) reductase in Saccharomyces cerevisiae (SceCPR) could asymmetrically reduce KPL to D-PL with a relatively weak activity. In this study, engineering of SceCPR was performed using a semi-rational design to enhance its catalytic activity. Based on the computer-aided design including phylogenetic analysis and molecular dynamics simulation, Ser158, Asn159, Gln180, Tyr208, Tyr298 and Trp299 were identified as the potential sites. Semi-saturation, single and combined-site mutagenesis was performed on all six residues, and several mutants with improved enzymatic activities were obtained. Among them, the mutant SceCPRS158A/Y298H exhibited the highest catalytic efficiency in which the kcat/Km value is 2466.22 s-1·mM-1, 18.5 times higher than that of SceCPR. The 3D structural analysis showed that the mutant SceCPRS158A/Y298H had an expanded and increased hydrophilicity catalytic pocket, and an enhanced π-π interaction which could contribute to faster conversion efficiency and higher catalytic rate. The whole cell system containing SceCPRS158A/Y298H and glucose dehydrogenase (GDH), under the optimized condition, could reduce 490.21 mM D-PL with e.e.≧ 99%, conversion rate = 98%, and the space-time yield = 382.80 g·L-1·d-1, which is the highest level reported so far.

Recently, concern on several environmental issues including the pollutant discharge and high concentration of CO2 have gained high interest due to its impact on ecosystem and global warming effect, respectively. Implementation of photosynthetic microorganism carries out numerous advantages including high efficiency of CO2 fixation, the great endurance under extreme conditions and generation of valuable bioproducts. Thermosynechococcus sp. CL-1 (TCL-1), a cyanobacterium, has the ability to perform CO2 fixation and accumulation of various byproducts under extreme conditions like high temperature and alkalinity, presence of estrogen, or even using swine wastewater. This study aimed to assess TCL-1 performance under various endocrine disruptor compounds (bisphenol-A, 17-β-estradiol/E2, and 17-α-ethynilestradiol/EE2), concentrations (0-10 mg/L), light intensities (500-2000 µE/m2/s), and dissolved inorganic carbon/DIC levels (0-113.2 mM). Addition of E2 content even until 10 mg/L carried out insignificant biomass growth interruption along with the improvement in CO2 fixation rate (79.8 ± 0.1 mg/L/h). Besides the influence of E2, application of higher DIC level and light intensity also enhanced the CO2 fixation rate and biomass growth. The highest biodegradation of E2 at 71% was achieved by TCL-1 in the end of 12 h cultivation period. TCL-1 dominantly produced protein (46.7% ± 0.2%), however, production of lipid and carbohydrate (39.5 ± 1.5 and 23.3 ± 0.9%, respectively) also could be considered as the potential source for biofuel production. Thus, this study can provide an efficient strategy in simultaneously dealing with environmental issues with side advantage in production of macromolecules.

High-speed allosteric hammerhead ribozymes can be engineered to distinguish well between a perfectly matching effector and the nucleic acid sequences with a few mismatches under physiologically relevant conditions. Such ribozymes can be designed to control the expression of exogenous mRNAs and can be used to develop new gene therapies, including anticancer treatments. The in vivo selection of such ribozymes is a complicated and lengthy procedure with no guarantee of success. Thus, in silico selection of high-speed ribozymes can be employed using secondary RNA structure computation based on the partition function of the RNA folding in combination with random search algorithms. This approach has already been proven very accurate in designing allosteric hammerhead ribozymes. Herein, we present two programs for the computational design of allosteric ribozymes sensing randomized oligonucleotides based on the extended version of the hammerhead ribozyme. A Generator for High-speed Oligonucleotide Sensing allosteric ribozymes with NOT logic function (GHOST-NOT) and a Generator for High-speed Oligonucleotide Sensing allosteric ribozymes with YES logic function (GHOST-YES) for computational design of high-speed allosteric ribozymes are described. The allosteric hammerhead ribozymes had a high self-cleavage rate of about 1.8 per minute and were very selective in sensing an effector sequence.

Green technology has been developed for the quick production of stabilized silver nanoparticles (AgNPs), with the assistance of nitrate reductase from an isolated culture of Aspergillus terreus N4. The organism's intracellular and periplasmic fractions contained nitrate reductase, with the former demonstrating the highest activity of 0.20 IU/g of mycelium. When the fungus was cultivated in a medium comprising 1.056% glucose, 1.836% peptone, 0.3386% yeast extract, and 0.025% KNO3, the greatest nitrate reductase productivity of 0.3268 IU/g was achieved. Statistical modeling via response surface methodology was used to optimize the enzyme production. The periplasmic and intracellular enzyme fractions were found to convert Ag+ to Ag0, initiating synthesis within 20 min, with predominant nanoparticle sizes between 25 and 30 nm. By normalizing the effects of temperature, pH, AgNO3 concentration, and mycelium age with a variable shaking period for enzyme release, the production of AgNPs with the periplasmic fraction was optimized. The synthesis of nanoparticles occurred at temperatures of 30, 40, and 50 °C, with the highest yield observed at 40 and 50 °C during shorter incubation periods. Similarly, the nanoparticles were synthesized at pH levels of 7.0, 8.0, and 9.0, with the greatest production observed at pH 8.0 and 9.0 at lower incubation periods. The antimicrobial activity of AgNPs was demonstrated against common foodborne pathogens, including Staphylococcus aureus and Salmonella typhimurium, indicating their potential as non-alcoholic disinfectants.

In this study, we report high molecular weight (HMW) levan production by whole cells of Microbacterium paraoxydans, previously reported to be a good producer of fructooligosaccharides. Structural analysis of the extracellularly produced fructan indicated the glycosidic bonds between the adjacent fructose to be of β-(2, 6) linkage with over 90% of the fructan to have molecular weight around 2 × 108 Da and 10% with a molecular weight of ∼20 kDa. Immobilization of the cells in Ca-alginate led to the production of 44.6 g/L levan with a yield of 0.29 g/g sucrose consumed. Factors affecting the conversion rate were identified by One-Factor-At-a-Time (OFAT) analysis and the combination of these (initial sucrose concentration of 400 g/L, 100 mM buffer pH 7, the temperature of 37 °C and 20 mM CaCl2) led to the production of ∼129 g/L of levan with a yield of ∼0.41 g/g sucrose consumed and volumetric productivity of 1.8 g/L/h.