Targeted, Stimuli-Responsive Delivery of Plasmid DNA and miRNAs Using a Facile Self-Assembled Supramolecular Nanoparticle System.

Gene therapy is rapidly regaining traction in terms of research activity and investment across the globe, with clear potential to revolutionize medicine and tissue regeneration. Viral vectors remain the most commonly utilized gene delivery vehicles, due to their high efficiency, however, they are acknowledged to have numerous drawbacks, including limited payload capacity, lack of cell-type specificity, and risk of possible mutations in vivo, hence, patient safety. Synthetic nanoparticle gene delivery systems can offer substantial advantages over viral vectors. They can be utilized as off-the-shelf components to package genetic material, display targeting ligands, and release payloads upon environmental triggers and enable the possibility of programmed cell-specific uptake and transfection. In this study, we have synthesized three functional polymeric building blocks that, in a rapid, facile, tailorable, and stage-wise manner, associate through both electrostatic and noncovalent hydrophobic "host-guest" interactions to form monodisperse self-assembled nanoparticles (SaNP). We show that these SaNPs successfully package significant amounts of microRNA through to plasmid DNA, present desired ligands on their outer surface for targeted receptor-mediated cell-specific uptake and affect efficient translation of packaged plasmids. We confirm that these SaNPs outperform commercially available, gold standard transfection agents in terms of in vitro transfection efficiencies and have very low cytotoxicity. With facile self-assembly and tailorable composition, our SaNP gene delivery system has significant potential in targeted gene therapy applications.

Characterization of a serine hydroxymethyltransferase for L-serine enzymatic production from Pseudomonas plecoglossicida.

Pseudomonas plecoglossicida, a bacterium strain that exhibits high Serine hydroxymethyltransferase (SHMT) activity, was isolated from the seawater. A full-length glyA encoding SHMT was obtained by a modified thermal asymmetric interlaced-PCR (TRIL-PCR), which consisted of 1,254 bp, encoded a 417 amino acid polypeptide, and shared the highest identity (75 %) with a glyA gene from Acinetobacter radioresistens CMC-1. Recombinant glyA gene was expressed in Escherichia coli BL21 (DE3) and purified by electrophoretic homogeneity. The enzyme showed the optimal activity at pH 8.0 and 40 °C, and remained stable in high alkali conditions. Using SHMT to produce L-serine by catalyzing the reaction of glycine and tetrahydrofolate is one of the most promising routes to synthesize L-serine, achieving 33.4 mM L-serine at the 12th h of the enzymatic reaction with the substrates of glycine (133 mM) and formaldehyde (13.3 mM). The properties make the SHMT a candidate for further enzymatic studies and industrial applications.

A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation, purification, characterization and l-serine production.

Currently, l-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT) is the key enzyme, suggesting the importance of searching for a SHMT with high activity. Shewanella algae, a methanol-utilizing marine bacterium showing high SHMT activity, was selected based on screening bacterial strains and comparison of the activities of SHMTs. A glyA was isolated from the S. algae through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a 417 amino acid polypeptide. The SaSHMT, encoded by the glyA, showed the optimal activity at 50°C and pH 7.0, and retained over 45% of its maximal activity after incubation at 40°C for 3h. The enzyme showed better stability under alkaline environment (pH 6.5-9.0) than Hyphomicrobium methylovorum GM2's SHMT (pH 6.0-7.5). The SaSHMT can produce 77.76mM of l-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to l-serine being 1.41-fold higher than that of Escherichia coli. Therefore, the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate.