Assessment of hepatocyte functional viability post-incubation with acetylated trehalose.

<p>(A) Overall morphology 1 and 4 days after incubation with 30 mM 6-O-Ac-Tre (scale bar = 20 μm), and (B) a 14-day follow-up of metabolism in rat hepatocytes by measuring daily albumin secretion showed a slow recovery of metabolism in trehalose-loaded cells compared to the control group. (C) The MTT viability assay showed no significant difference in the viabilities of the experimental and the control groups 48 h after trehalose loading with 30 mM 6-O-Ac-Tre (p>0.05). (D) Confocal imaging of mitochondria stained using Mito-Tracker Red 12 h after incubation with 6-O-Ac-Tre showed the presence of mitochondria competent to accumulate dye (dependent on mitochondrial membrane potential, ΔΨ) in both the control (top) and the experiment (bottom) groups. Scale bar = 10 μm (E) JC-1 staining after 6 h incubation with 6-O-Ac-Tre indicates similar mitochondrial ΔΨ in experimental and control groups. A positive control group incubated with the uncoupler CCCP during staining is shown for comparison. Error bars represent SD (n = 3).</p

Uptake of acetylated trehalose in hepatocytes.

<p>(A) Intracellular accumulation of trehalose, and (B) intracellular accumulation of (n)-O-Ac-Tre, after incubation with 30 mM concentration of trehalose, 2-, 4-, and 6-O-Ac-Tre. 8-O-Ac-Tre was not studied as it was not water soluble. Error bars represent SD (n = 3). (* denote p>0.05 and # denote p<0.05 between respective groups).</p

Best fit model parameters.

<p>Best fit model parameters.</p

Schematic of a proposed diffusion-reaction model for de-acetylation of 6-Ac-Tre in cells.

<p>Schematic of a proposed diffusion-reaction model for de-acetylation of 6-Ac-Tre in cells.</p

The synthesis steps for trehalose diacetate (2-O-Ac-Tre), trehalose tetraacetate (4-O-Ac-Tre) and trehalose hexaacetate (6-O-Ac-Tre).

<p>The synthesis steps for trehalose diacetate (2-O-Ac-Tre), trehalose tetraacetate (4-O-Ac-Tre) and trehalose hexaacetate (6-O-Ac-Tre).</p

Fitting results of the proposed diffusion-reaction model to the data.

<p>With 3 fitting parameters, the model (solid lines) closely followed the averaged uptake data for trehalose (squares) and total (n)-O-Ac-Tre (circles), which were obtained by incubation with (A) 5mM, (B) 15 mM, and (C) 30 mM 6-O-Ac-Tre. Inset Figures: Predicted increase in the concentration of 6- (black solid line), 5- (dark-gray solid line), 4- (gray solid line), 3- (gray dashed line), 2- (gray dash-dotted line) and 1-O-Ac-Tre (gray dotted line). These results were obtained by averaging the data from at least 3 animals and the error bars represent the animal-to-animal variability.</p

Ubiquitous Detection of Gram-Positive Bacteria with Bioorthogonal Magnetofluorescent Nanoparticles

The ability to rapidly diagnose gram-positive pathogenic bacteria would have far reaching biomedical and technological applications. Here we describe the bioorthogonal modification of small molecule antibiotics (vancomycin and daptomycin), which bind to the cell wall of gram-positive bacteria. The bound antibiotics conjugates can be reacted orthogonally with tetrazine-modified nanoparticles, <i>via</i> an almost instantaneous cycloaddition, which subsequently renders the bacteria detectable by optical or magnetic sensing. We show that this approach is specific, selective, fast and biocompatible. Furthermore, it can be adapted to the detection of intracellular pathogens. Importantly, this strategy enables detection of entire classes of bacteria, a feat that is difficult to achieve using current antibody approaches. Compared to covalent nanoparticle conjugates, our bioorthogonal method demonstrated 1–2 orders of magnitude greater sensitivity. This bioorthogonal labeling method could ultimately be applied to a variety of other small molecules with specificity for infectious pathogens, enabling their detection and diagnosis