Engineering in Life Sciences最新文献

The combined effect of surface topography and substrate rigidity in stem cell cultures is still under-investigated, especially when biodegradable polymers are used. Herein, we assessed human bone marrow stem cell response on aliphatic polyester substrates as a function of anisotropic grooved topography and rigidity (7 and 12 kPa). Planar tissue culture plastic (TCP, 3 GPa) and aliphatic polyester substrates were used as controls. Cell morphology analysis revealed that grooved substrates caused nuclei orientation/alignment in the direction of the grooves. After 21 days in osteogenic and chondrogenic media, the 3 GPa TCP and the grooved 12 kPa substrate induced significantly higher calcium deposition and alkaline phosphatase (ALP) activity and glycosaminoglycan (GAG) deposition, respectively, than the other groups. After 14 days in tenogenic media, the 3 GPa TCP upregulated four and downregulated four genes; the planar 7 kPa substrate upregulated seven genes and downregulated one gene; and the grooved 12 kPa substrate upregulated seven genes and downregulated one gene. After 21 days in adipogenic media, the softest (7 kPa) substrates induced significantly higher oil droplet deposition than the other substrates and the grooved substrate induced significantly higher droplet deposition than the planar. Our data pave the way for more rational design of bioinspired constructs.

Despite its widespread usage as a chemotherapy drug in cancer treatment, doxorubicin (DOX) has limitations such as short in vivo circulation time, low solubility, and poor permeability. In this regard, a pH-responsive chitosan (CS)- montmorillonite (MMT)- nitrogen-doped carbon quantum dots (NCQDs) nanocomposite was first developed, loaded with DOX, and then incorporated into a double emulsion to further develop the sustained release. The incorporated NCQDs into the CS-MMT hydrogel exhibited enhanced loading and entrapment efficiencies. The presence of NCQDs nanoparticles in the CS-MMT hydrogel also resulted in an extended pH-responsive release of DOX over a period of 96 h compared to that of CS-MMT-DOX nanocarriers at pH 5.4. Based on the Korsmeyer-Peppas model, there was a controlled DOX release at pH 5.4, while no diffusion was observed at pH 7.4, indicating fewer side effects. MTT assay showed that the cytotoxicity of DOX-loaded CS-MMT-NCQDs hydrogel nanocomposite was significantly higher than those of free DOX (p < 0.001) and CS-MMT-NCQDs (p < 0.001) on MCF-7 cells. Flow cytometry results demonstrated that a higher apoptosis induction achieved after incorporating NCQDs nanoparticles into CS-MMT-DOX nanocarrier. These findings suggest that the DOX-loaded nanocomposite is a promising candidate for the targeted treatment of cancer cells.

The present study reports the mixed culture acidogenic production of biohydrogen and carboxylic acids (CA) from brewery spent grains (BSG) in the presence of high concentrations of cobalt, iron, nickel, and zinc. The metals enhanced biohydrogen output by 2.39 times along with CA biosynthesis by 1.73 times. Cobalt and iron promoted the acetate and butyrate pathways, leading to the accumulation of 5.14 gCOD/L of acetic and 11.36 gCOD/L of butyric acid. The production of solvents (ethanol + butanol) was higher with zinc (4.68 gCOD/L) and cobalt (4.45 gCOD/L). A combination of all four metals further enhanced CA accumulation to 42.98 gCOD/L, thus surpassing the benefits accrued from supplementation with individual metals. Additionally, 0.36 and 0.31 mol green ammonium were obtained from protein-rich brewery spent grain upon supplementation with iron and cobalt, respectively. Metagenomic analysis revealed the high relative abundance of Firmicutes (>90%), of which 85.02% were Clostridium, in mixed metal-containing reactors. Finally, a significant correlation of dehydrogenase activity with CA and biohydrogen evolution was observed upon metal addition.

Bio surfactants are natural surfactants that induce emulsification, displacement, increased solubility, and mobility of hydrophobic organic compounds. In this study, the gene expression of biosurfactant production genes by Pseudomonas aeruginosa in the presence of sodium dodecyl sulfate coated iron nanostructure (Fe/SDS) were evaluated. Emulsification Index and Surface Tension reduction test to check stability and emulsification the rhamnolipid were done. Purification was evaluated using thin layer chromatography (TLC) and expression of rhlA, mvfR, lasR, rhlR genes was determined using q-PCR technique. Binding of nanoparticles to bio surfactants was confirmed by TEM. The best emulsification index, was by the sample that exposed to 1 mg/L Fe/SDS nanoparticles for 2 days. Rhamnolipid produced in the presence of nanoparticles had an acceptable ability to reduce surface tension. The Rf (retention factor) value obtained was 0.63 by chromatography. q-PCR results showed that the expression of rhlA, mvfR, lasR, rhlR genes was significantly increased in Fe/SDS treated cells, which indicates the significant positive effect (P < 0.05) of nanoparticles on biosurfactant production of treated cells. While, SDS and Fe alone were not affected significantly (P > 0.05) on the expression of these genes. Our findings indicated the importance of nanoparticles in increasing the expression of genes involved in the bio surfactant production pathway of Pseudomonas aeruginosa.

Animal-derived anti-IgG secondary antibodies are currently employed to stain and screen of human monoclonal antibody(mAb)-producing cells, but using animal-derived antibodies may raise the concerns of high cost, complicated operations and biological safety issues in biopharmaceutical manufacturing. Nanobodies(VHHs) are attractive forms of antibodies for their straightforward engineering and expression in both eukaryotic and prokaryotic systems. Using phage-displayed immune llama VHH library, we identified new anti-Fc VHHs that could bind to human Fc with high affinity. In GFP fusion format, the anti-Fc VHH-GFP generated dramatically stronger FACS signals than AF488 conjugated anti-IgG antibodies when used for staining mAb-producing CHO cells. Furthermore, preparative sorting of CHO cells based on anti-Fc VHH-GFP staining resulted in the enrichment of cell lines capable of synthesizing mAb at high productivity. This safe and cost-efficient anti-Fc VHH-GFP may optimize the process of generating highly productive cell lines for therapeutic mAb production compared to conventional animal-derived fluorescent antibodies.

The Australian tobacco plant Nicotiana benthamiana is becoming increasingly popular as a platform for protein production and metabolic engineering. In this system, gene expression is achieved transiently by infiltrating N. benthamiana plants with suspensions of Agrobacterium tumefaciens carrying vectors with the target genes. To infiltrate larger numbers of plants, vacuum infiltration is the most efficient approach known, which is already used on industrial scale. Current laboratory-scale solutions for vacuum infiltration, however, either require expensive custom-tailored equipment or produce large amounts of biologically contaminated waste. To overcome these problems and lower the burden to establish vacuum infiltration in new laboratories, we present here 3D-printed plant holders for vacuum infiltration. We demonstrate that our plant holders are simple to use and enable a throughput of around 40 plants per hour. In addition, our 3D-printed plant holders are made from autoclavable material, which tolerate at least 12 autoclave cycles, helping to limit the production of contaminated waste and thus contributing to increased sustainability in research. In conclusion, our plant holders provide a simple, robust, safe and transparent platform for laboratory-scale vacuum infiltration that can be readily adopted by new laboratories interested in protein and metabolite production in Nicotiana benthamiana.

Practical application

Transient expression in Nicotiana benthamiana provides a popular and rapid system for producing proteins in a plant host. To infiltrate larger numbers of plants (typically >20), vacuum infiltration is the method of choice. However, no system has been described so far which is robust to use and can be used without expensive and complex equipment. Our autoclavable 3D-printed plant holders presented here will greatly reduce the efforts required to adopt the vacuum infiltration technique in new laboratories. They are easy to use and can be autoclaved at least 12 times, which contributes to waste reduction and sustainability in research laboratories. We anticipate that the 3D printing design provided here will drastically lower the bar for new groups to employ vacuum infiltration for producing proteins and metabolites in Nicotiana benthamiana.

Removal efficiency of gold from a solution of pure tetrachloroaurate ions was investigated using microbial fuel cell (MFC) technology. The effects of type of catholyte solution and initial gold concentration on the removal efficiency were considered. Due to its presence at high levels in the gold wastewater, the effect of copper ions on the removal efficiency of the gold ions was also studied. The effects of pH and initial biomass concentration on the gold removal efficiency was also determined. The results showed that after 5 h contact time, 95% of gold removal efficiency from a wastewater containing 250 ppm of initial gold ions at ambient temperature using 80 g/L yeast concentration was achieved. After 48 h of the cell's operation under the same condition, 98.86% of AuCl₄^– ions were successfully removed from the solution. At initial gold concentration in the waste solution of 250 ppm, pH 2, and initial yeast concentration of 80 g/L, 100% removal efficiency of the gold was achieved. On the other hand, the most suitable condition for copper removal was found at a pH of 5.2, where 53% removal efficiency from the waste solution was accomplished.