Herein, we report fifty four membered, a new set of novel NIR Raman reporters and CyRLA-572 has been selected to be the best among them considering the signal intensity and stability. This new reporter molecule is an excellent partner with our in house Raman reporters (Cy7LA and Cy7.5LA). These three NIR Raman reporters are adsorbed on the gold nanoparticles to obtain their corresponding unique SERS fingerprints in which three individual characteristic peaks are capable to multiplex among them. These multiplexed Raman reporters are applied to develop biocompatible and specific targeting SERS nanotags after tagging with specific antibodies. These multiplex targeted SERS nanotags are applied to detect three targeting receptors in differentiated mouse embryonic stem cells (mESCs) consisting three germ layers such as ectoderm, mesoderm and endoderm. After successful recognition of cells by SERS techniques, we detect simultaneously three germ layers in teratoma which is a monster tumor formed from mESC cells in animal xenograft model.
Gold nanoparticles; multiplexing; teratoma; biocompatible; nanotags; in vivo; SERS; cell mapping; differentiated germ layers; mesoderm; endoderm; ectoderm
Aggregation of amyloid β-peptide (Aβ) is implicated in the pathology of Alzheimer’s disease (AD), with the soluble, Aβ oligomeric species thought to be the critical pathological species. Identification and characterization of intermediate species formed during the aggregation process is crucial to the understanding of the mechanisms by which oligomeric species mediate neuronal toxicity and following disease progression. Probing these species proved to be extremely challenging, as evident by the lack of reliable sensors, due to their heterogeneous and transient nature. We describe here an oligomer-specific fluorescent chemical probe, BoDipy-Oligomer (BD-Oligo), developed through the use of the diversity-oriented fluorescent library approach (DOFLA) and high-content, imaging-based screening. This probe enables dynamic oligomer monitoring during fibrillogenesis in vitro and shows in vivo Aβ oligomers staining possibility in the AD mice model.
Screening of bead-based peptide libraries against fluorescence-labeled target proteins was found significantly influenced by the dye characteristics. Commercially available red fluorescence dyes with net negative charges adversely showed strong interactions with library beads. The introduction of zwitterionic dyes significantly reduced the unwanted interactions, which sheds light upon using the right fluorescence probe for acquisition of reliable results in various fluorescence-assisted applications.
Stem cell research has gathered immense attention in the past decade due to the remarkable ability of stem cells for self-renewal and tissue-specific differentiation. Despite having numerous advancements in stem cell isolation and manipulation techniques, there is a need for highly reliable probes for the specific detection of live stem cells. Herein we developed a new fluorescence probe (CDy9) with high selectivity for mouse embryonic stem cells. CDy9 allows the detection and isolation of intact stem cells with marginal impact on their function and capabilities.
Mouse embryonic stem cells; Fluorescence imaging; Single cell PCR; Fluorescence-assisted cell sorting; Three germ layers
Recent evidence suggests that tau aggregates are not only neurotoxic, but also propagate in neurons acting as a seed for native tau aggregation. Prion-like tau transmission is now considered as an important pathogenic mechanism driving the progression of tau pathology in the brain. However, prion-like tau species have not been clearly characterized. To identify infectious tau conformers, here we prepared diverse tau aggregates and evaluated the effect on inducing intracellular tau-aggregation. Among tested, tau dimer containing P301L-mutation is identified as the most infectious form to induce tau pathology. Biochemical analysis reveals that P301L-tau dimer is covalently cross-linked with a disulfide bond. The relatively small and covalently cross-linked tau dimer induced tau pathology efficiently in primary neurons and also in tau-transgenic mice. So far, the importance of tau disulfide cross-linking has been overlooked in the study of tau pathology. Here our results suggested that tau disulfide cross-linking might play critical role in tau propagation by producing structurally stable and small tau conformers.
The dynamics of cellular heat production and propagation remains elusive at a subcellular level. Here we report the first small molecule fluorescent thermometer selectively targeting the endoplasmic reticulum (ER thermo yellow), with the highest sensitivity reported so far (3.9%/°C). Unlike nanoparticle thermometers, ER thermo yellow stains the target organelle evenly without the commonly encountered problem of aggregation, and successfully demonstrates the ability to monitor intracellular temperature gradients generated by external heat sources in various cell types. We further confirm the ability of ER thermo yellow to monitor heat production by intracellular Ca2+ changes in HeLa cells. Our thermometer anchored at nearly-zero distance from the ER, i.e. the heat source, allowed the detection of the heat as it readily dissipated, and revealed the dynamics of heat production in real time at a subcellular level.
Macrophages are an essential component of the immune system and have protective and pathogenic functions in various diseases. Imaging of macrophages in vivo could furnish new tools to advance evaluation of disease and therapies. Critical limb ischemia is a disease in which macrophages have considerable pathogenic roles, and are potential targets for cell-based immunotherapy. We sought to develop a new near-infrared fluorescence (NIRF) imaging probe to target macrophages specifically in
vivo in various pathological states, including hind-limb ischemia. We rapidly screened the photostable cyanine-based NIRF library against different blood cell lines. The identified monocyte/macrophage-selective hit was tested in
vitro in live-cell labeling assay. Non-invasive NIRF imaging was performed with murine models of paw inflammation by lipopolysaccharide challenge and hind-limb ischemia with femoral artery ligation. in
vivo macrophage targeting was further evaluated using intravital microscopy with Csf1r-EGFP transgenic mice and immunofluorescent staining with macrophage-specific markers. We discovered MF800, a Macrophage-specific near-infrared Fluorophore, which showed selective live-cell imaging performance in a panel of cell lines and primary human blood samples. MF800 outperforms the clinically-available NIRF contrast agent ICG for in
vivo specificity in paw inflammation and hind-limb ischemia models. We observed a marked overlap of MF800-labeled cells and EGFP-expressing macrophages in intravital imaging of Csf1r-EGFP transgenic mice. In the histologic analysis, MF800-positive cells also expressed the macrophage markers CD68 and CD169. NIRF imaging showcased the potential of using MF800 to understand macrophage behavior in
vivo, characterize macrophage-associated diseases, and may help in assessing therapeutic responses in the clinic.
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.
CDy1; ABCB1; multiple myeloma; carfilzomib; vismodegib; ASPM; KIF14; TMPO
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.
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.
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.
•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
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.
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.
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.
Cheminformatics; high content screening; combinatorial library; styryl; fluorescence; bioimaging; chemical address tags; QSAR; CAIA
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.
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.
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.
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.
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.
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.
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.
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.
axon growth; chemical genetics; chondroitin sulfate proteoglycan; glial scar; myelin; triazine
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.
diabetes; dynamic nuclear polarization; magnetic resonance imaging; magnetic resonance spectroscopy; obesity
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.
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
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.
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.
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.
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.
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.
Cheminformatics; machine vision; bioimaging; fluorescence; styryl; high content screening; image cytometry; combinatorial chemistry