Plasmonic activation of gold nanorods for remote stimulation of calcium signaling and protein expression in HEK 293T cells.

Remote activation of specific cells of a heterogeneous population can provide a useful research tool for clinical and therapeutic applications. Here, we demonstrate that photostimulation of gold nanorods (AuNRs) using a tunable near-infrared (NIR) laser at specific longitudinal surface plasmon resonance wavelengths can induce the selective and temporal internalization of calcium in HEK 293T cells. Biotin-PEG-Au nanorods coated with streptavidin Alexa Fluor-633 and biotinylated anti-His antibodies were used to decorate cells genetically modified with His-tagged TRPV1 temperature-sensitive ion channel and AuNRs conjugated to biotinylated RGD peptide were used to decorate integrins in unmodified cells. Plasmonic activation can be stimulated at weak laser power (0.7-4.0 W/cm(2) ) without causing cell damage. Selective activation of TRPV1 channels could be controlled by laser power between 1.0 and 1.5 W/cm(2) . Integrin targeting robustly stimulated calcium signaling due to a dense cellular distribution of nanoparticles. Such an approach represents a functional tool for combinatorial activation of cell signaling in heterogeneous cell populations. Our results suggest that it is possible to induce cell activation via NIR-induced gold nanorod heating through the selective targeting of membrane proteins in unmodified cells to produce calcium signaling and downstream expression of specific genes with significant relevance for both in vitro and therapeutic applications. Biotechnol. Bioeng. 2016;113: 2228-2240. © 2016 Wiley Periodicals, Inc.

Biochemical strategies for enhancing the in vivo production of natural products with pharmaceutical potential.

Natural products have been associated with significant health benefits in preventing and treating various chronic human diseases such as cancer, cardiovascular diseases, diabetes, Alzheimer's disease, and pathogenic infections. However, the isolation, characterization and evaluation of natural products remain a challenge, mainly due to their limited bioavailability. Metabolic engineering and fermentation technology have emerged as alternative approaches for generating natural products under controlled conditions that can be optimized to maximize yields. Optimization of these processes includes the evaluation of factors such as host selection, product biosynthesis interaction with the cell's central metabolism, product degradation, and byproduct formation. This review summarizes the most recent biochemical strategies and advances in expanding and diversifying natural compounds as well as maximizing their production in microbial and plants cells.

Metabolic engineering and in vitro biosynthesis of phytochemicals and non-natural analogues.

Over the years, natural products from plants and their non-natural derivatives have shown to be active against different types of chronic diseases. However, isolation of such natural products can be limited due to their low bioavailability, and environmental restrictions. To address these issues, in vivo and in vitro reconstruction of plant metabolic pathways and the metabolic engineering of microbes and plants have been used to generate libraries of compounds. Significant advances have been made through metabolic engineering of microbes and plant cells to generate a variety of compounds (e.g. isoprenoids, flavonoids, or stilbenes) using a diverse array of methods to optimize these processes (e.g. host selection, operational variables, precursor selection, gene modifications). These approaches have been used also to generate non-natural analogues with different bioactivities. In vitro biosynthesis allows the synthesis of intermediates as well as final products avoiding post-translational limitations. Moreover, this strategy allows the use of substrates and the production of metabolites that could be toxic for cells, or expand the biosynthesis into non-conventional media (e.g. organic solvents, supercritical fluids). A perspective is also provided on the challenges for generating novel chemical structures and the potential of combining metabolic engineering and in vitro biocatalysis to produce metabolites with more potent biological activities.

Human parvovirus B19 virus-like particles: In vitro assembly and stability.

Virus-like particles (VLPs) are biological nanoparticles identical to the natural virions, but without genetic material. VLPs are suitable for the analysis of viral infection mechanisms, vaccine production, tissue-specific drug delivery, and as biological nanomaterials. Human parvovirus B19 (B19) infects humans; therefore VLPs derived from this virus have enormous potential in medicine and diagnostics. Current production of self-assembled VLPs derived from B19 is typically carried out in eukaryotic expression systems. However many applications of VLPs require access to its internal core. Consequently, the processes of disassembly and further reassembly of VLPs are critical both for purification of viral proteins, and for encapsulation purposes. Herein we report the in vitro self-assembly of B19 VLPs derived from the recombinant VP2 protein expressed in Escherichia coli and the effects of pH and ionic strength on the assembly process. Our results demonstrate that VP2 is able to form VLPs completely in vitro. At neutral pH, homogeneous VLPs assemble, while at acidic and basic pHs, with low ionic strength, the major assemblies are small intermediates. The in vitro self-assembled VLPs are highly stable at 37°C, and a significant fraction of particles remain assembled after 30min at 80°C.