Victoria S. Haritos

Messenger RNA (mRNA)-based therapeutics have emerged as a new class of biological medicines, clearly exemplified by the global deployment of mRNA vaccines against the COVID-19 pandemic. Currently, therapeutic mRNA is primarily produced through in vitro transcription that suffers high production costs. Until now, intracellular manufacture of mRNA has been challenging due to the presence of ubiquitous RNases in vivo . Here, we have developed a new approach that protects eukaryotic mRNA from RNase degradation ensuring longevity and integrity of mRNA inside microbial cells. Through targeted strain and molecular engineering, our approach involves specially designed inserts in mRNA that facilitate formation of stabilized and protected protein-mRNA complexes. In addition to vastly improved stability, the protein-mRNA complexes enable convenient purification of mRNA from cell lysate with high purity using conventional chromatography. The work reported here promises a scalable, rapid, and low-cost approach to produce fully functional eukaryotic mRNA using well-known microbial systems.

Polydopamine (PDA) coatings are widely used to functionalize filtration membranes due to their ability to adhere to a broad range of surfaces and enhance biocompatibility and surface hydrophilicity. Co-adsorption of PDA with polyethylenimine (PEI) is investigated here to fabricate an enzyme-entrapped membrane, enabling the segregation of two incompatible enzymes, a protease (from B. licheniformis) and a ω-transaminase (from N. fischeri), within a nanoporous membrane using reverse filtration. PDA and PEI were coadsorbed on the membrane under varying dopamine and PEI concentrations, solution volumes, and applied pressures. Membrane permeance reduction and transaminase (TA) retention were used to evaluate the deposition effectiveness. Response surface methodology (RSM) revealed the PDA/PEI concentration to be the main driver of permeance reduction and enzyme retention, with nonlinear effects of volume and pressure and significant interactions between concentration and pressure. FTIR and AFM-IR analyses provided the chemical and spatial characterization of the coatings, showing that higher dopamine/PEI concentrations and longer residence times increased the polymer deposition and coating thickness. AFM-IR mapping at nanoscale confirmed the correlation among deposition parameters, coating distribution, and permeance reduction. This study demonstrates that membrane performance can be tuned by optimizing key deposition parameters, achieving high enzyme retention without compromising the permeance. Combining an RSM approach with micro- and nanoscale IR spectroscopy provides a robust framework for designing and characterizing enzyme-entrapped membranes, offering a reliable and quantitative approach for optimizing membrane-based biocatalysis applications.

Biobased, biodegradable, and biocompatible polymers provide a sustainable alternative to petrochemical-derived stabilisers, introducing green chemistries for advanced nanomedicine applications.

Atomic force microscopy-infrared spectroscopy can simultaneously acquire topographical, chemical, and stiffness information with nanoscale resolution. One approach to measuring relative stiffness is to use the phase-locked loop frequency channel to assess the probe-sample contact resonance. A stiffer sample domain results in higher contact resonance frequency, and more compliant domains result in lower frequency contact resonance; however, in practice, these data are difficult to compare between samples and can even be problematic from tip-engagement to tip-engagement on the same sample. This challenge is due to cantilever and tip variations that give rise to contact resonance frequency offsets, which can be large compared to the width of the frequency manifold associated with the surface. To overcome this challenge, we present a standard procedure for contact resonance frequency comparison between samples that circumvents the problem of the absolute frequency variation. Application of the technique is demonstrated on a carbon-epoxy composite, an enzyme-functionalized cellulose amide compressed disk, and human cadaver ligament samples.

Escherichica coli, engineered for electroactivity and butanol production, generate electricity and biofuel via electrofermentation when cultured on organic byproducts.