Gopal Iyer

Particle Size, Surface Coating, and PEGylation Influence the Biodistribution of Quantum Dots in Living Mice

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

microPET-Based Biodistribution of Quantum Dots in Living Mice

This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice. Methods: 64Cu-Labeled 800- or 525-nm emission wavelength QD (21- or 12-nm diameter), with or without 2,000 MW (molecular weight) polyethylene glycol (PEG), were injected intravenously into mice (5.55 MBq/25 pmol QD) and studied using well counting or by serial microPET and region-of-interest analysis. Results: Both methods show rapid uptake by the liver (27.4–38.9 %ID/g) (%ID/g is percentage injected dose per gram tissue) and spleen (8.0–12.4 %ID/g). Size has no influence on biodistribution within the range tested here. Pegylated QD have slightly slower uptake into liver and spleen (6 vs. 2 min) and show additional low-level bone uptake (6.5–6.9 %ID/g). No evidence of clearance from these organs was observed. Conclusion: Rapid reticuloendothelial system clearance of QD will require modification of QD for optimal utility in imaging living subjects. Formal quantitative biodistribution/imaging studies will be helpful in studying many types of nanoparticles, including quantum dots.

Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking.

Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft‐associated glycosyl‐phosphatidyl‐inositol‐anchored avidin test probe (Av‐GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high‐resolution and long‐term tracking of individual Av‐GPI and the classification of their various diffusive behaviors. Using dual‐color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av‐GPI and the location of glycosphingolipid GM1‐rich microdomains and caveolae. We show that Av‐GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1‐rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av‐GPI dynamically partition in and out of these microdomains in a cholesterol‐dependent manner. Our results provide direct evidence that cholesterol‐/sphingolipid‐rich microdomains can compartmentalize the diffusion of GPI‐anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft‐associated proteins across the plasma membrane.

VIB-1 is required for expression of genes necessary for programmed cell death in Neurospora crassa.

ABSTRACT Nonself recognition during somatic growth is an essential and ubiquitous phenomenon in both prokaryotic and eukaryotic species. In filamentous fungi, nonself recognition is also important during vegetative growth. Hyphal fusion between genetically dissimilar individuals results in rejection of heterokaryon formation and in programmed cell death of the fusion compartment. In filamentous fungi, such as Neurospora crassa, nonself recognition and heterokaryon incompatibility (HI) are regulated by genetic differences at het loci. In N. crassa, mutations at the vib-1 locus suppress nonself recognition and HI mediated by genetic differences at het-c/pin-c, mat, and un-24/het-6. vib-1 is a homolog of Saccharomyces cerevisiae NDT80, which is a transcriptional activator of genes during meiosis. For this study, we determined that vib-1 encodes a nuclear protein and showed that VIB-1 localization varies during asexual reproduction and during HI. vib-1 is required for the expression of genes involved in nonself recognition and HI, including pin-c, tol, and het-6; all of these genes encode proteins containing a HET domain. vib-1 is also required for the production of downstream effectors associated with HI, including the production of extracellular proteases upon carbon and nitrogen starvation. Our data support a model in which mechanisms associated with starvation and nonself recognition/HI are interconnected. VIB-1 is a major regulator of responses to nitrogen and carbon starvation and is essential for the expression of genes involved in nonself recognition and death in N. crassa.

Purification and characterization of laccase from the rice blast fungus, Magnaporthe grisea

A 70-kDa extracellular laccase was purified from the rice blast fungus Magnaporthe grisea using gel filtration and ion exchange chromatography The procedure provided 282-fold purification with a specific enzyme activity of 225.91 U mg(-1) and a yield of 11.92%. The enzyme oxidized a wide range of substrates. The highest level of oxidation was detected with syringaldazine as the substrate. Using syringaldazine as the substrate, the enzyme exhibited a pH optimum of 6 and temperature optimum of 30 degrees C, and its K(m) was 0.118 mM. The enzyme was strongly inhibited by Cu-chelating agents.

Microfluidic enrichment for the single cell analysis of circulating tumor cells

Resistance to drug therapy is a major concern in cancer treatment. To probe clones resistant to chemotherapy, the current approach is to conduct pooled cell analysis. However, this can yield false negative outcomes, especially when we are analyzing a rare number of circulating tumor cells (CTCs) among an abundance of other cell types. Here, we develop a microfluidic device that is able to perform high throughput, selective picking and isolation of single CTC to 100% purity from a larger population of other cells. This microfluidic device can effectively separate the very rare CTCs from blood samples from as few as 1 in 20,000 white blood cells. We first demonstrate isolation of pure tumor cells from a mixed population and track variations of acquired T790M mutations before and after drug treatment using a model PC9 cell line. With clinical CTC samples, we then show that the isolated single CTCs are representative of dominant EGFR mutations such as T790M and L858R found in the primary tumor. With this single cell recovery device, we can potentially implement personalized treatment not only through detecting genetic aberrations at the single cell level, but also through tracking such changes during an anticancer therapy.

Nonself recognition is mediated by HET‐C heterocomplex formation during vegetative incompatibility

Nonself recognition during vegetative growth in filamentous fungi is mediated by heterokaryon incompatibility (het) loci. In Neurospora crassa, het‐c is one of 11 het loci. Three allelic specificity groups, termed het‐cOR, het‐cPA and het‐cGR, exist in natural populations. Heterokaryons or partial diploids that contain het‐c alleles of alternative specificity show severe growth inhibition, repression of conidiation and hyphal compartmentation and death (HCD). Using epitope‐tagged HET‐C, we show that nonself recognition is mediated by the presence of a heterocomplex composed of polypeptides encoded by het‐c alleles of alternative specificity. The HET‐C heterocomplex localized to the plasma membrane (PM); PM‐bound HET‐C heterocomplexes occurred in all three het‐c incompatible allelic interactions. Strains containing het‐c constructs deleted for a predicted signal peptide sequence formed HET‐C heterocomplexes in the cytoplasm and showed a growth arrest phenotype. Our finding is a step towards understanding nonself recognition mechanisms that operate during vegetative growth in filamentous fungi, and provides a model for investigating relationships between recognition mechanisms and cell death.

Aromatic Aldehyde and Hydrazine Activated Peptide Coated Quantum Dots for Easy Bioconjugation and Live Cell Imaging

We present a robust scheme for preparation of semiconductor quantum dots (QDs) and cognate partners in a conjugation ready format. Our approach is based on bis-aryl hydrazone bond formation mediated by aromatic aldehyde and hydrazinonicotinate acetone hydrazone (HyNic) activated peptide coated quantum dots. We demonstrate controlled preparation of antibody--QD bioconjugates for specific targeting of endogenous epidermal growth factor receptors in breast cancer cells and for single QD tracking of transmembrane proteins via an extracellular epitope. The same approach was also used for optical mapping of RNA polymerases bound to combed genomic DNA in vitro.

High affinity scFv-hapten pair as a tool for quantum dot labeling and tracking of single proteins in live cells.

We describe a general approach to label cell surface proteins using quantum dots (QD) for single-molecule tracking. QDs coated with small-hapten modified peptides are targeted to cell surface fusion proteins containing the corresponding single-chain fragment antibody (scFv). The approach is illustrated with the small hapten fluorescein (FL) and a high-affinity anti-FL scFv fused to two different proteins in yeast and murine neuronal cell line N2a.

Peptide Coated Quantum Dots for Biological Applications

Quantum dots (QDOTs) have been widely recognized by the scientific community and the biotechnology industry, as witnessed by the exponential growth of this field in the past several years. We describe the synthesis and characterization of visible and near infrared QDots-a critical step for engineering organic molecules like proteins and peptides for building nanocomposite materials with multifunctional properties suitable for biological applications