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1.  Identification of an ABCB1 (P-glycoprotein)-positive carfilzomib-resistant myeloma subpopulation by the pluripotent stem cell fluorescent dye CDy1 
American journal of hematology  2013;88(4):265-272.
Multiple myeloma (MM) is characterized by the malignant expansion of differentiated plasma cells. Although many chemotherapeutic agents display cytotoxic activity toward MM cells, patients inevitably succumb to their disease because the tumor cells become resistant to the anticancer drugs. The cancer stem cell hypothesis postulates that a small subpopulation of chemotherapy-resistant cancer cells is responsible for propagation of the tumor. Herein we report that efflux of the pluripotent stem cell dye CDy1 identifies a subpopulation in MM cell lines characterized by increased expression of P-glycoprotein, a member of the ABC (ATP-binding cassette) superfamily of transporters encoded by ABCB1. We also demonstrate that ABCB1-overexpressing MM cells are resistant to the second-generation proteasome inhibitor carfilzomib that recently received accelerated approval for the treatment of therapy-refractive MM by the U.S. Food and Drug Administration. Moreover, increased resistance to carfilzomib in sensitive MM cells following drug selection was associated with upregulation of ABCB1 cell-surface expression which correlated with increased transporter activity as measured by CDy1 efflux. We further show that chemosensitization of MM cells to carfilzomib could be achieved in vitro by cotreatment with vismodegib, a hedgehog pathway antagonist which is currently in MM clinical trials. CDy1 efflux may therefore be a useful assay to determine whether high expression of ABCB1 is predictive of poor clinical responses in MM patients treated with carfilzomib. Our data also suggest that inclusion of vismodegib might be a potential strategy to reverse ABCB1-mediated drug resistance should it occur.
doi:10.1002/ajh.23387
PMCID: PMC3608751  PMID: 23475625
CDy1; ABCB1; multiple myeloma; carfilzomib; vismodegib; ASPM; KIF14; TMPO
2.  Actively Targeted In Vivo Multiplex Detection of Intrinsic Cancer Biomarkers Using Biocompatible SERS Nanotags 
Scientific Reports  2014;4:4075.
Surface-enhanced Raman scattering (SERS) technique is becoming highly popular for multiplex biosensing due to the ‘fingerprint’ Raman spectra from every molecule. As a proof-of-concept, we demonstrated the actively targeted multiplex in vitro and in vivo detection of three intrinsic cancer biomarkers - EGFR, CD44 and TGFβRII in a breast cancer model using three multiplexing capable, biocompatible SERS nanoparticles/nanotags. Intra-tumorally injected antibody conjugated nanotags specifically targeting the three biomarkers exhibited maximum signal at 6 hours and no detectable signal at 72 hours. However, nanotags without antibodies showed no detectable signal after 6 hours. This difference could be due to the specific binding of the bioconjugated nanotags to the receptors on the cell surface. Thus, this study establishes SERS nanotags as an ultrasensitive nanoprobe for the multiplex detection of biomarkers and opens up its potential application in monitoring tumor progression and therapy and development into a theranostic probe.
doi:10.1038/srep04075
PMCID: PMC3921631  PMID: 24518045
3.  Discovery of a Structural-Element Specific G-Quadruplex “Light-Up” Probe 
Scientific Reports  2014;4:3776.
The development of a fluorescent probe capable of detecting and distinguishing the wide diversity of G-quadruplex structures is particularly challenging. Herein, we report a novel BODIPY-based fluorescent sensor (GQR) that shows unprecedented selectivity to parallel-stranded G-quadruplexes with exposed ends and four medium grooves. Mechanistic studies suggest that GQR associates with G-quadruplex grooves close to the end of the tetrad core, which may explain the dye's specificity to only a subset of parallel structures. This specific recognition favours the disaggregation of GQR in aqueous solutions thereby recovering the inherent fluorescence of the dye. Due to its unique features, GQR represents a valuable tool for basic biological research and the rapid discovery of novel, specific ligands that target similar structural features of G-quadruplexes.
doi:10.1038/srep03776
PMCID: PMC3895904  PMID: 24441075
4.  A Single Subset of Dendritic Cells Controls the Cytokine Bias of Natural Killer T Cell Responses to Diverse Glycolipid Antigens 
Immunity  2014;40(1):105-116.
Summary
Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α+ DEC-205+ dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α+ dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.
Graphical Abstract
Highlights
•Complexes of antigenic glycolipids bound to CD1d have been visualized in situ•A single DC subset predominates in presentation of a variety of glycolipids•Antigen presentation to iNKT cells rapidly alters accessory molecules on APCs•Reciprocal induction of CD70 and PD-L2 controls cytokine bias of iNKT cell responses
doi:10.1016/j.immuni.2013.12.004
PMCID: PMC3895174  PMID: 24412610
5.  Make Caffeine Visible: a Fluorescent Caffeine “Traffic Light” Detector 
Scientific Reports  2013;3:2255.
Caffeine has attracted abundant attention due to its extensive existence in beverages and medicines. However, to detect it sensitively and conveniently remains a challenge, especially in resource-limited regions. Here we report a novel aqueous phase fluorescent caffeine sensor named Caffeine Orange which exhibits 250-fold fluorescence enhancement upon caffeine activation and high selectivity. Nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy indicate that π-stacking and hydrogen-bonding contribute to their interactions while dynamic light scattering and transmission electron microscopy experiments demonstrate the change of Caffeine Orange ambient environment induces its fluorescence emission. To utilize this probe in real life, we developed a non-toxic caffeine detection kit and tested it for caffeine quantification in various beverages. Naked-eye sensing of various caffeine concentrations was possible based on color changes upon irradiation with a laser pointer. Lastly, we performed the whole system on a microfluidic device to make caffeine detection quick, sensitive and automated.
doi:10.1038/srep02255
PMCID: PMC3719075  PMID: 23877095
6.  Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling 
Cell  2012;149(4):832-846.
SUMMARY
We report a novel mechanism of ribonucleoprotein (RNP) nucleocytoplasmic export by nuclear envelope budding. During development of Drosophila synapses, a fragment of the Wnt-1 receptor, DFrizzled2, is imported into postsynaptic nuclei where it forms prominent foci. We now show these foci to be composed of large RNP granules harboring synaptic protein transcripts. These RNPs exit the nucleus via a budding mechanism akin to nuclear egress of Herpes-type viruses, a process previously thought to be exclusive to these viruses. During this mechanism, RNP granules bud into the space between the inner and the outer nuclear membranes (the perinuclear space), in a manner dependent on Lamin C, a nuclear protein linked to muscular dystrophies. Like herpes virus nuclear egress, this process requires protein kinase C, which is known to disrupt the lamin through phosphorylation. We suggest that nuclear budding is an endogenous nuclear export pathway for large RNP granules.
doi:10.1016/j.cell.2012.03.032
PMCID: PMC3371233  PMID: 22579286
7.  Prospecting for Live Cell BioImaging Probes With Cheminformatic Assisted Image Arrays (CAIA) 
High-throughput microscopic screening instruments can generate huge collections of images of live cells incubated with combinatorial libraries of fluorescent molecules. Organizing and visualizing these images to discern biologically important patterns that link back to chemical structure is a challenge. We present an analysis and visualization methodology - Cheminformatic Assisted Image Array (CAIA) - that greatly facilitates data mining efforts. For illustration, we considered a collection of microscopic images acquired from cells incubated with each member of a combinatorial library of styryl molecules being screened for candidate bioimaging probes. By sorting CAIAs based on quantitative image features, the relative contribution of each combinatorial building block on probe intracellular distribution could be visually discerned. The results revealed trends hidden in the dataset: most interestingly, the building blocks of the styryl molecules appeared to behave as chemical address tags, additively and independently encoding spatial patterns of intracellular fluorescence. Translated into practice, CAIA facilitated discovery of several outstanding styryl molecules for live cell nuclear imaging applications.
doi:10.1109/ISBI.2007.357050
PMCID: PMC3592986  PMID: 23482717
Cheminformatics; high content screening; combinatorial library; styryl; fluorescence; bioimaging; chemical address tags; QSAR; CAIA
8.  Identification of Cancer Cell-Line Origins Using Fluorescence Image-Based Phenomic Screening 
PLoS ONE  2012;7(2):e32096.
Universal phenotyping techniques that can discriminate among various states of biological systems have great potential. We applied 557 fluorescent library compounds to NCI's 60 human cancer cell-lines (NCI-60) to generate a systematic fluorescence phenotypic profiling data. By the kinetic fluorescence intensity analysis, we successfully discriminated the organ origin of all the 60 cell-lines.
doi:10.1371/journal.pone.0032096
PMCID: PMC3285665  PMID: 22384151
9.  Styryl-Based and Tricyclic Compounds as Potential Anti-Prion Agents 
PLoS ONE  2011;6(9):e24844.
Prion diseases currently have no effective therapy. These illnesses affect both animal and human populations, and are characterized by the conformational change of a normal self protein PrPC (C for cellular) to a pathological and infectious conformer, PrPSc (Sc for scrapie). We used a well characterized tissue culture model of prion infection, where mouse neuroblastoma cells (N2a) were infected with 22L PrPSc, to screen compounds for anti-prion activity. In a prior study we designed a library of styryl based, potential imaging compounds which were selected for high affinity binding to Alzheimer's disease β-amyloid plaques and good blood-brain barrier permeability. In the current study we screened this library for activity in the N2a/22L tissue culture system. We also tested the anti-prion activity of two clinically used drugs, trimipramine and fluphenazine, in the N2a/22L system. These were selected based on their structural similarity to quinacrine, which was previously reported to have anti-prion activity. All the compounds were also screened for toxicity in tissue culture and their ability to disaggregate amyloid fibrils composed of PrP and β-amyloid synthetic peptides in vitro. Two of the imaging agents, 23I and 59, were found to be both effective at inhibiting prion infection in N2a/22L tissue culture and to be non-toxic. These two compounds, as well as trimipramine and fluphenazine were evaluated in vivo using wild-type CD-1 mice infected peripherally with 139A PrPSc. All four agents significantly prolonged the asymptomatic incubation period of prion infection (p<0.0001 log-rank test), as well as significantly reducing the degree of spongiform change, astrocytosis and PrPSc levels in the brains of treated mice. These four compounds can be considered, with further development, as candidates for prion therapy.
doi:10.1371/journal.pone.0024844
PMCID: PMC3172287  PMID: 21931860
10.  A Chemical Screen Identifies Novel Compounds That Overcome Glial-Mediated Inhibition Of Neuronal Regeneration 
A major barrier to regeneration of central nervous system (CNS) axons is the presence of growth-inhibitory proteins associated with myelin and the glial scar. To identify chemical compounds with the ability to overcome the inhibition of regeneration, we screened a novel triazine library, based on the ability of compounds to increase neurite outgrowth from cerebellar neurons on inhibitory myelin substrates. The screen produced 4 “hit compounds”, which act with nM potency on several different neuronal types, and on several distinct substrates relevant to glial inhibition. Moreover, the compounds selectively overcome inhibition rather than promote growth in general. The compounds do not affect neuronal cAMP levels, PKC activity, or EGFR activation. Interestingly, one of the compounds alters microtubule dynamics and increases microtubule density in both fibroblasts and neurons. This same compound promotes regeneration of dorsal column axons after acute lesions, and potentiates regeneration of optic nerve axons after nerve crush in vivo. These compounds should provide insight into the mechanisms through which glial-derived inhibitors of regeneration act, and could lead to the development of novel therapies for CNS injury.
doi:10.1523/JNEUROSCI.0302-10.2010
PMCID: PMC2855497  PMID: 20357120
axon growth; chemical genetics; chondroitin sulfate proteoglycan; glial scar; myelin; triazine
11.  Imaging metabolic syndrome 
EMBO Molecular Medicine  2010;2(6):196-210.
Metabolic syndrome is a fast growing public health burden for almost all the developed countries and many developing nations. Despite intense efforts from both biomedical and clinical scientists, many fundamental questions regarding its aetiology and development remain unclear, partly due to the lack of suitable imaging technologies to visualize lipid composition and distribution, insulin secretion, β-cell mass and functions in vivo. Such technologies would not only impact on our understanding of the complexity of metabolic disorders such as obesity and diabetes, but also aid in their diagnosis, drug development and assessment of treatment efficacy. In this article we discuss and propose several strategies for visualization of physiological and pathological changes that affect pancreas and adipose tissue as a result of the development of metabolic diseases.
doi:10.1002/emmm.201000074
PMCID: PMC3377322  PMID: 20533426
diabetes; dynamic nuclear polarization; magnetic resonance imaging; magnetic resonance spectroscopy; obesity
12.  Discovery of Small-Molecule HIV-1 Fusion and Integrase Inhibitors Oleuropein and Hydroxytyrosol: I. Fusion Inhibition 
We have identified oleuropein (Ole) and hydroxytyrosol (HT) as a unique class of HIV-1 inhibitors from olive leaf extracts effective against viral fusion and integration. We used molecular docking simulation to study the interactions of Ole and HT with viral targets. We find that Ole and HT bind to the conserved hydrophobic pocket on the surface of the HIV-gp41 fusion domain by hydrogen bonds with Q577 and hydrophobic interactions with I573, G572, and L568 on the gp41 N-terminal heptad repeat peptide N36, interfering with formation of the gp41 fusion-active core. To test and confirm modeling predications, we examined the effect of Ole and HT on HIV-1 fusion complex formation using native polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Ole and HT exhibit dose dependent inhibition on HIV-1 fusion core formation with EC50s of 66–58 nM, with no detectable toxicity. Our findings on effects on HIV-1 integrase are reported separately.
doi:10.1016/j.bbrc.2007.01.071
PMCID: PMC2790717  PMID: 17275783
HIV-1; AIDS; natural product; small molecule HIV-1 inhibitors; HIV-1 entry inhibitor; Olive Leaf Extract (OLE); Oleuropein (Ole); Hydroxytyrosol (HT); structure-function; molecular modeling
13.  Kinetics and Cellular Site of Glycolipid Loading Control the Outcome of Natural Killer T Cell Activation 
Immunity  2009;30(6):888-898.
Summary
CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating α galactosylceramide (αGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for αGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing αGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.
doi:10.1016/j.immuni.2009.03.022
PMCID: PMC2719696  PMID: 19538930
MOLIMMUNO; CELLIMMUNO
14.  High-content live cell imaging with RNA probes: advancements in high-throughput antimalarial drug discovery 
BMC Cell Biology  2009;10:45.
Background
Malaria, a major public health issue in developing nations, is responsible for more than one million deaths a year. The most lethal species, Plasmodium falciparum, causes up to 90% of fatalities. Drug resistant strains to common therapies have emerged worldwide and recent artemisinin-based combination therapy failures hasten the need for new antimalarial drugs. Discovering novel compounds to be used as antimalarials is expedited by the use of a high-throughput screen (HTS) to detect parasite growth and proliferation. Fluorescent dyes that bind to DNA have replaced expensive traditional radioisotope incorporation for HTS growth assays, but do not give additional information regarding the parasite stage affected by the drug and a better indication of the drug's mode of action. Live cell imaging with RNA dyes, which correlates with cell growth and proliferation, has been limited by the availability of successful commercial dyes.
Results
After screening a library of newly synthesized stryrl dyes, we discovered three RNA binding dyes that provide morphological details of live parasites. Utilizing an inverted confocal imaging platform, live cell imaging of parasites increases parasite detection, improves the spatial and temporal resolution of the parasite under drug treatments, and can resolve morphological changes in individual cells.
Conclusion
This simple one-step technique is suitable for automation in a microplate format for novel antimalarial compound HTS. We have developed a new P. falciparum RNA high-content imaging growth inhibition assay that is robust with time and energy efficiency.
doi:10.1186/1471-2121-10-45
PMCID: PMC2702272  PMID: 19515257
15.  Machine vision assisted analysis of structure-localization relationships in a combinatorial library of prospective bioimaging probes 
With a combinatorial library of bioimaging probes, it is now possible to use machine vision to analyze the contribution of different building blocks of the molecules to their cell-associated visual signals. For athis purpose, cell-permeant, fluorescent styryl molecules were synthesized by condensation of 168 aldehyde with 8 pyridinium/quinolinium building blocks. Images of cells incubated with fluorescent molecules were acquired with a high content screening instrument. Chemical and image feature analysis revealed how variation in one or the other building block of the styryl molecules led to variations in the molecules' visual signals. Across each pair of probes in the library, chemical similarity was significantly associated with spectral and total signal intensity similarity. However, chemical similarity was much less associated with similarity in subcellular probe fluorescence patterns. Quantitative analysis and visual inspection of pairs of images acquired from pairs of styryl isomers confirm that many closely-related probes exhibit different subcellular localization patterns. Therefore, idiosyncratic interactions between styryl molecules and specific cellular components greatly contribute to the subcellular distribution of the styryl probes' fluorescence signal. These results demonstrate how machine vision and cheminformatics can be combined to analyze the targeting properties of bioimaging probes, using large image data sets acquired with automated screening systems.
doi:10.1002/cyto.a.20713
PMCID: PMC2692593  PMID: 19243023
Cheminformatics; machine vision; bioimaging; fluorescence; styryl; high content screening; image cytometry; combinatorial chemistry
16.  Discovery of Small-Molecule HIV-1 Fusion and Integrase Inhibitors Oleuropein and Hydroxytyrosol: II. Integrase Inhibition 
We report molecular modeling and functional confirmation of Ole and HT binding to HIV-1 integrase. Docking simulations identified two binding regions for Ole within the integrase active site. Region I encompasses the conserved D64-D116-E152 motif, while region II involves the flexible loop region formed by amino acid residues 140–149. HT, on the other hand, binds to region II. Both Ole and HT exhibit favorable interactions with important amino acid residues through strong H-bonding and van der Waals contacts, predicting integrase inhibition. To test and confirm modeling predictions, we examined the effect of Ole and HT on HIV-1 integrase activities including 3′-processing, strand transfer and disintegration. Ole and HT exhibit dose-dependent inhibition on all three activities, with EC50s in the nM range. These studies demonstrate that molecular modeling of target–ligand interaction coupled with structural-activity analysis should facilitate the design and identification of innovative integrase inhibitors and other therapeutics.
doi:10.1016/j.bbrc.2007.01.058
PMCID: PMC1857318  PMID: 17261269
HIV-1; AIDS; natural product; small molecule HIV-1 inhibitors; HIV-1 integrase inhibitor; Olive Leaf Extract (OLE); Oleuropein (Ole); Hydroxytyrosol (HT); structure-function; molecular modeling
17.  Identification of 12Cysβ on tubulin as the binding site of tubulyzine 
Bioorganic & medicinal chemistry  2005;14(4):1169-1175.
We have undertaken quantitative binding site studies in order to identify the binding site of the known microtubule destabilizing agents, the tubulyzines, in the tubulin dimer. Two different approaches were employed that utilized the tubulyzines and their derivatives. The first approach was based on a chemical affinity labeling method using tubulyzine affinity derivatives, and the second approach employed the mass spectrometric measurement of the differential reactivity of cysteines using the tubulyzines and monobromobimane. Based on overlapping data from these two approaches, we propose that the tubulyzines bind at the guanosine-5′-triphosphate binding site of β-tubulin. Interestingly, we also show that the tubulyzines’ binding to tubulin induces a conformational change in tubulin that prevents further interaction of the 239Cysβ with other reagents.
doi:10.1016/j.bmc.2005.09.069
PMCID: PMC1408322  PMID: 16266809
Tublin; Tubulyzine; Binding site; MS-DRC; MS-DRC, mass spectrometric measurement of the differential reactivity of cysteines; mBrB, monobromobimane; GTP, guanosine-5′-triphosphate; rt, room temperature; LC—MS, liquid chromatography—mass spectrometry; THF, tetrahydrofuran; DIPEA, diisopropylethylamine; EtOAc, ethyl acetate; TEA, triethylamine; T-LC, thin-layer chromatography; PIPES, piperazine-N,N-bis(ethane-sulfonic acid); TCEP, tris(2-carboxyethyl)phosphine hydrochloride; Tn, tubulyzine affinity derivatives; ESI, electrospray ionization; HP-LC, high-performance liquid chromatography; CID, collision induced dissociation; MS, mass spectrometry; SIC, single ion current; TIC, total ion current; FWHM, full width at half maximum; GDP, guanosine di phosphate; TA, tubulyzine A; TB, tubulyzine B
18.  Bioactive small molecules reveal antagonism between the integrated stress response and sterol-regulated gene expression 
Cell metabolism  2005;2(6):361-371.
Summary
Phosphorylation of translation initiation factor 2α (eIF2α ) coordinates a translational and transcriptional program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum (ER) stress. A screen for small molecule activators of the ISR identified two related compounds that also activated sterol-regulated genes by blocking cholesterol biosynthesis at the level of CYP51. Ketoconazole, a known CYP51 inhibitor, had similar effects, establishing that perturbed flux of precursors to cholesterol activates the ISR. Surprisingly, compound-mediated activation of sterol-regulated genes was enhanced in cells with an ISR-blocking mutation in the regulatory phosphorylation site of eIF2α . Furthermore, induction of the ISR by an artificial drug-activated eIF2α kinase reduced the level of active sterol regulatory element binding protein (SREBP) and sterol-regulated mRNAs. These findings suggest a mechanism by which interactions between sterol metabolism, the ISR, and the SREBP pathway affect lipid metabolism during ER stress.
doi:10.1016/j.cmet.2005.11.005
PMCID: PMC1361344  PMID: 16330322
19.  A Rapid Fluorescence-Based Assay for Classification of iNKT Cell Activating Glycolipids 
Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.
doi:10.1021/ja200070u
PMCID: PMC3072113  PMID: 21425779
20.  Identification of Fluorescent Compounds with Non-Specific Binding Property via High Throughput Live Cell Microscopy 
PLoS ONE  2012;7(1):e28802.
Introduction
Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved.
Method
Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties.
Results
The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i) mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii) retention and spatial localization of chemical compounds vary within and between each cell line; and (iii) the structural similarities of compounds can infer their non-specific binding properties.
Conclusion
We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.
doi:10.1371/journal.pone.0028802
PMCID: PMC3252290  PMID: 22242152
21.  α-Galactosylceramide Analogs with Weak Agonist Activity for Human iNKT Cells Define New Candidate Anti-Inflammatory Agents 
PLoS ONE  2010;5(12):e14374.
CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.
doi:10.1371/journal.pone.0014374
PMCID: PMC3003687  PMID: 21179412

Results 1-21 (21)