Interactions of Carbohydrate-conjugated Nanoparticles with Mycobacterium Smegmatis

Mycobacterium smegmatis is a non-pathogenic microorganism and has been widely used as a model organism to study infections caused by M. tuberculosis and other mycobacterial pathogens. We report that nanoparticles conjugated with selected carbohydrate show a striking increase in the surface adherence by M. smegmatis. This applies to silica nanoparticles and magnetic nanoparticles ranging from 100 nm to 5 nm. Under the same experimental conditions, minimum adhesion was observed for unfunctionalized nanoparticles. The synthesis and characterization of the glyconanoparticles will be presented. The finding is applied to imaging M. smegmatis infected lung epithelial cells, and the results will be discussed

Glyconanoparticle Uptake Profile in Lung Carcinoma Cells

Non-small cell lung carcinoma (NSCLC) is responsible for nearly 85% of lung cancer, and early diagnosis and treatment of lung cancer can circumvent possible death. We focus on glyconanoparticles with a magnetic or a fluorescent core that act as multivalent glyco-scaffold to study cell surface interaction and internalization. The glyconanoparticles were synthesized by conjugating various carbohydrates on magnetic nanoparticles and fluorescent silica nanoparticles by a photocoupling technique developed in our laboratory. The size of nanoparticles used varies from 6 nm to 60 nm. The resulting glyconanoparticles were treated with human adenocarcinoma non-small lung epithelial cells (A549) and the primary small airway epithelial cells (PCS-301-010). The cellular uptake was studied and quantified by confocal fluorescence microscopy, flow cytometry, thin section TEM, and prussian blue staining. We found that the extent of cellular uptake was dependent on the type of carbohydrate ligands and the nature of the nanoparticles used. Experiments were conducted to investigate the mechanism of the uptake, and results will be discussed

Enhancing Antibiotic Activity Using Nanomaterial-Antibiotic Conjugates

We demonstrate that streptomycin conjugated on silica nanoparticles (SNP-Str) can be used to effectively target streptomycin-resistant Escherichia coli (E. coli) bacteria by lowering the minimum inhibitory concentration (MIC) of streptomycin up to 2 log folds. Silica nanoparticles were synthesized with an average diameter of 80, 50 and 30 nm, respectively. Streptomycin was then covalently conjugated to SNP using efficient photocoupling chemistry. The MIC for free streptomycin sulfate was recorded as a high 2.0 mg/mL for an engineered Strr mutant E. coli ORN 208. Conjugating the streptomycin to SNP resulted in the decrease in MIC to 161 μg/mL, 63 μg/mL, and 19 μg/mL for SNP of 80, 50 and 30 nm, respectively. In this poster, the synthesis, characterization, and evaluation of SNP-Str will be presented and discussed

Hypomodified tRNA in evolutionarily distant yeasts can trigger rapid tRNA decay to activate the general amino acid control response, but with different consequences.

All tRNAs are extensively modified, and modification deficiency often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of any of several tRNA body modifications results in rapid tRNA decay (RTD) of certain mature tRNAs by the 5'-3' exonucleases Rat1 and Xrn1. As tRNA quality control decay mechanisms are not extensively studied in other eukaryotes, we studied trm8Δ mutants in the evolutionarily distant fission yeast Schizosaccharomyces pombe, which lack 7-methylguanosine at G46 (m7G46) of their tRNAs. We report here that S. pombe trm8Δ mutants are temperature sensitive primarily due to decay of tRNATyr(GUA) and that spontaneous mutations in the RAT1 ortholog dhp1+ restored temperature resistance and prevented tRNA decay, demonstrating conservation of the RTD pathway. We also report for the first time evidence linking the RTD and the general amino acid control (GAAC) pathways, which we show in both S. pombe and S. cerevisiae. In S. pombe trm8Δ mutants, spontaneous GAAC mutations restored temperature resistance and tRNA levels, and the trm8Δ temperature sensitivity was precisely linked to GAAC activation due to tRNATyr(GUA) decay. Similarly, in the well-studied S. cerevisiae trm8Δ trm4Δ RTD mutant, temperature sensitivity was closely linked to GAAC activation due to tRNAVal(AAC) decay; however, in S. cerevisiae, GAAC mutations increased tRNA loss and exacerbated temperature sensitivity. A similar exacerbated growth defect occurred upon GAAC mutation in S. cerevisiae trm8Δ and other single modification mutants that triggered RTD. Thus, these results demonstrate a conserved GAAC activation coincident with RTD in S. pombe and S. cerevisiae, but an opposite impact of the GAAC response in the two organisms. We speculate that the RTD pathway and its regulation of the GAAC pathway is widely conserved in eukaryotes, extending to other mutants affecting tRNA body modifications

A connection between the ribosome and two S. pombe tRNA modification mutants subject to rapid tRNA decay.

tRNA modifications are crucial in all organisms to ensure tRNA folding and stability, and accurate translation. In both the yeast Saccharomyces cerevisiae and the evolutionarily distant yeast Schizosaccharomyces pombe, mutants lacking certain tRNA body modifications (outside the anticodon loop) are temperature sensitive due to rapid tRNA decay (RTD) of a subset of hypomodified tRNAs. Here we show that for each of two S. pombe mutants subject to RTD, mutations in ribosomal protein genes suppress the temperature sensitivity without altering tRNA levels. Prior work showed that S. pombe trm8Δ mutants, lacking 7-methylguanosine, were temperature sensitive due to RTD, and that one class of suppressors had mutations in the general amino acid control (GAAC) pathway, which was activated concomitant with RTD, resulting in further tRNA loss. We now find that another class of S. pombe trm8Δ suppressors have mutations in rpl genes, encoding 60S subunit proteins, and that suppression occurs with minimal restoration of tRNA levels and reduced GAAC activation. Furthermore, trm8Δ suppression extends to other mutations in the large or small ribosomal subunit. We also find that S. pombe tan1Δ mutants, lacking 4-acetylcytidine, are temperature sensitive due to RTD, that one class of suppressors have rpl mutations, associated with minimal restoration of tRNA levels, and that suppression extends to other rpl and rps mutations. However, although S. pombe tan1Δ temperature sensitivity is associated with some GAAC activation, suppression by an rpl mutation only modestly inhibits GAAC activation. We propose a model in which ribosomal protein mutations result in reduced ribosome concentrations, leading to both reduced ribosome collisions and a reduced requirement for tRNA, with these effects having different relative importance in trm8Δ and tan1Δ mutants. This model is consistent with our results in S. cerevisiae trm8Δ trm4Δ mutants, known to undergo RTD, fueling speculation that this model applies across eukaryotes