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1.  Chemical Address Tags of Fluorescent Bioimaging Probes 
Chemical address tags can be defined as specific structural features shared by a set of bioimaging probes having a predictable influence on cell-associated visual signals obtained from these probes. Here, using a large image dataset acquired with a high content screening instrument, machine vision and cheminformatics analysis have been applied to reveal chemical address tags. With a combinatorial library of fluorescent molecules, fluorescence signal intensity, spectral, and spatial features characterizing each one of the probes' visual signals were extracted from images acquired with the three different excitation and emission channels of the imaging instrument. With multivariate regression, the additive contribution from each one of the different building blocks of the bioimaging probes towards each measured, cell-associated image-based feature was calculated. In this manner, variations in the chemical features of the molecules were associated with the resulting staining patterns, facilitating quantitative, objective analysis of chemical address tags. Hierarchical clustering and paired image-cheminformatics analysis revealed key structure-property relationships amongst many building blocks of the fluorescent molecules. The results point to different chemical modifications of the bioimaging probes that can exert similar (or different) effects on the probes' visual signals. Inspection of the clustered structures suggests intramolecular charge migration or partial charge distribution as potential mechanistic determinants of chemical address tag behavior.
PMCID: PMC2907078  PMID: 20104576
Cheminformatics; machine vision; bioimaging; fluorescence; high content screening; image cytometry; combinatorial chemistry
2.  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.
PMCID: PMC3592986  PMID: 23482717
Cheminformatics; high content screening; combinatorial library; styryl; fluorescence; bioimaging; chemical address tags; QSAR; CAIA
3.  Visualizing chemical structure-subcellular localization relationships using fluorescent small molecules as probes of cellular transport 
To study the chemical determinants of small molecule transport inside cells, it is crucial to visualize relationships between the chemical structure of small molecules and their associated subcellular distribution patterns. For this purpose, we experimented with cells incubated with a synthetic combinatorial library of fluorescent, membrane-permeant small molecule chemical agents. With an automated high content screening instrument, the intracellular distribution patterns of these chemical agents were microscopically captured in image data sets, and analyzed off-line with machine vision and cheminformatics algorithms. Nevertheless, it remained challenging to interpret correlations linking the structure and properties of chemical agents to their subcellular localization patterns in large numbers of cells, captured across large number of images.
To address this challenge, we constructed a Multidimensional Online Virtual Image Display (MOVID) visualization platform using off-the-shelf hardware and software components. For analysis, the image data set acquired from cells incubated with a combinatorial library of fluorescent molecular probes was sorted based on quantitative relationships between the chemical structures, physicochemical properties or predicted subcellular distribution patterns. MOVID enabled visual inspection of the sorted, multidimensional image arrays: Using a multipanel desktop liquid crystal display (LCD) and an avatar as a graphical user interface, the resolution of the images was automatically adjusted to the avatar’s distance, allowing the viewer to rapidly navigate through high resolution image arrays, zooming in and out of the images to inspect and annotate individual cells exhibiting interesting staining patterns. In this manner, MOVID facilitated visualization and interpretation of quantitative structure-localization relationship studies. MOVID also facilitated direct, intuitive exploration of the relationship between the chemical structures of the probes and their microscopic, subcellular staining patterns.
MOVID can provide a practical, graphical user interface and computer-assisted image data visualization platform to facilitate bioimage data mining and cheminformatics analysis of high content, phenotypic screening experiments.
PMCID: PMC3852740  PMID: 24093553
Machine vision; Cheminformatics; Virtual reality; Data mining; Optical probes; Multivariate analysis; Human-computer interaction; Graphical user interface
4.  RAMTaB: Robust Alignment of Multi-Tag Bioimages 
PLoS ONE  2012;7(2):e30894.
In recent years, new microscopic imaging techniques have evolved to allow us to visualize several different proteins (or other biomolecules) in a visual field. Analysis of protein co-localization becomes viable because molecules can interact only when they are located close to each other. We present a novel approach to align images in a multi-tag fluorescence image stack. The proposed approach is applicable to multi-tag bioimaging systems which (a) acquire fluorescence images by sequential staining and (b) simultaneously capture a phase contrast image corresponding to each of the fluorescence images. To the best of our knowledge, there is no existing method in the literature, which addresses simultaneous registration of multi-tag bioimages and selection of the reference image in order to maximize the overall overlap between the images.
Methodology/Principal Findings
We employ a block-based method for registration, which yields a confidence measure to indicate the accuracy of our registration results. We derive a shift metric in order to select the Reference Image with Maximal Overlap (RIMO), in turn minimizing the total amount of non-overlapping signal for a given number of tags. Experimental results show that the Robust Alignment of Multi-Tag Bioimages (RAMTaB) framework is robust to variations in contrast and illumination, yields sub-pixel accuracy, and successfully selects the reference image resulting in maximum overlap. The registration results are also shown to significantly improve any follow-up protein co-localization studies.
For the discovery of protein complexes and of functional protein networks within a cell, alignment of the tag images in a multi-tag fluorescence image stack is a key pre-processing step. The proposed framework is shown to produce accurate alignment results on both real and synthetic data. Our future work will use the aligned multi-channel fluorescence image data for normal and diseased tissue specimens to analyze molecular co-expression patterns and functional protein networks.
PMCID: PMC3280195  PMID: 22363510
5.  A Cell-based Computational Modeling Approach for Developing Site-Directed Molecular Probes 
PLoS Computational Biology  2012;8(2):e1002378.
Modeling the local absorption and retention patterns of membrane-permeant small molecules in a cellular context could facilitate development of site-directed chemical agents for bioimaging or therapeutic applications. Here, we present an integrative approach to this problem, combining in silico computational models, in vitro cell based assays and in vivo biodistribution studies. To target small molecule probes to the epithelial cells of the upper airways, a multiscale computational model of the lung was first used as a screening tool, in silico. Following virtual screening, cell monolayers differentiated on microfabricated pore arrays and multilayer cultures of primary human bronchial epithelial cells differentiated in an air-liquid interface were used to test the local absorption and intracellular retention patterns of selected probes, in vitro. Lastly, experiments involving visualization of bioimaging probe distribution in the lungs after local and systemic administration were used to test the relevance of computational models and cell-based assays, in vivo. The results of in vivo experiments were consistent with the results of in silico simulations, indicating that mitochondrial accumulation of membrane permeant, hydrophilic cations can be used to maximize local exposure and retention, specifically in the upper airways after intratracheal administration.
Author Summary
We have developed an integrative, cell-based modeling approach to facilitate the design and discovery of chemical agents directed to specific sites of action within a living organism. Here, a computational, multiscale transport model of the lung was adapted to enable virtual screening of small molecules targeting the epithelial cells of the upper airways. In turn, the transport behaviors of selected candidate probes were evaluated to establish their degree of retention at a site of absorption, using computational simulations as well as two in vitro cell-based assay systems. Lastly, bioimaging experiments were performed to examine candidate molecules' distribution in the lungs of mice after local and systemic administration. Based on computational simulations, the higher mitochondrial density per unit absorption surface area is the key parameter determining the higher retention of small molecule hydrophilic cations in the upper airways, relative to lipophilic weak bases, specifically after intratracheal administration.
PMCID: PMC3285574  PMID: 22383866
6.  P2X antagonists inhibit styryl dye entry into hair cells 
Neuroscience  2009;161(4):1144-1153.
The styryl pyridinium dyes, FM1-43 and AM1-43, are fluorescent molecules that can permeate the mechanotransduction channels of hair cells, the sensory receptors of the inner ear. When these dyes are applied to hair cells, they enter the cytoplasm rapidly, resulting in a readily detectable increase in intracellular fluorescence that is often used as a molecular indication of mechanotransduction channel function. However, such dyes can also permeate the ATP receptor, P2X2. Therefore, we explored the contribution of P2X receptors to the loading of hair cells with AM1-43. The chick inner ear was found to express P2X receptors and to release ATP, similar to the inner ear of mammals, allowing for the endogenous stimulation of P2X receptors. The involvement of these receptors was evaluated pharmacologically, by exposing the sensory epithelium of the chick inner ear to 5 μM AM1-43 under different experimental conditions and measuring the fluorescence in hair cells after fixation of the tissue. Pre-exposure of the tissue to 5 mM EGTA for 15 minutes, which should eliminate most of the gating “tip links” of the mechanotransduction channels, deceased fluorescence by only 44%. In contrast, P2X receptor antagonists (PPADS, suramin, TNP-ATP, and d-tubocurarine) had greater effects on dye loading. PPADS, suramin, and TNP-ATP all decreased intracellular AM1-43 fluorescence in hair cells by at least 69% when applied at a concentration of 100 μM. The difference between d-tubocurarine-treated and control fluorescence was statistically insignificant when d-tubocurarine was applied at a concentration that blocks the mechanotransduction channel (200 μM). At a concentration that also blocks P2X2 receptors (2 mM), d-tubocurarine decreased dye loading by 72%. From these experiments, it appears that AM1-43 can enter hair cells through endogenously activated P2X receptors. Thus, the contribution of P2X receptors to dye entry must be considered when using styryl pyridinium dyes to detect hair cell mechanotransduction channel activity in the absence of explicit mechanical stimulation of stereocilia.
PMCID: PMC2846836  PMID: 19272432
mechanotransduction; basilar papilla; auditory; AM1-43; FM1-43; pannexin
7.  rsEGFP2 enables fast RESOLFT nanoscopy of living cells 
eLife  2012;1:e00248.
The super-resolution microscopy called RESOLFT relying on fluorophore switching between longlived states, stands out by its coordinate-targeted sequential sample interrogation using low light levels. While RESOLFT has been shown to discern nanostructures in living cells, the reversibly photoswitchable green fluorescent protein (rsEGFP) employed in these experiments was switched rather slowly and recording lasted tens of minutes. We now report on the generation of rsEGFP2 providing faster switching and the use of this protein to demonstrate 25–250 times faster recordings.
eLife digest
For decades it was assumed that the diffraction of light meant that optical microscopy could not resolve features that were smaller than about the half the wavelength of the light being used to create an image. However, various ‘super-resolution’ methods have allowed researchers to overcome this diffraction limit for fluorescence imaging, which is the most popular form of microscopy used in the life sciences. This approach involves tagging the biomolecules of interest with fluorescent molecules, such as green fluorescent protein (GFP), so that they can be identified in cells. An excitation laser then drives the fluorescent molecule, which is also known as a fluorophore, into an excited state: after a short time, the fluorophore can return to its ground state by releasing a fluorescence photon. Images of the sample are built up by detecting these photons.
In STED super-resolution microscopy a second laser is used to instantly send the molecules from their excited or ‘on’ states back to their ground or ‘off’ states before any fluorescence can occur. The second laser beam is usually shaped like a doughnut, with a small region of low light intensity surrounded by a region of much higher intensity. STED microscopy is able to beat the diffraction limit because the second laser turns all the fluorophores ‘off’ except those in the small sub-wavelength region at the centre of the doughnut. The image is build up by scanning both lasers over the sample so that the small region in which the fluorophores are ‘on’ probes the entire cell.
RESOLFT is a similar technique that employs fluorescent molecules with ‘on’ and ‘off’ times that are much longer than those used in STED microscopy. In particular, RESOLFT uses fluorescent molecules that can be rapidly switched back and forth between long-lived ‘on’ and ‘off’ states many times by the two lasers. The fact that both these states are long-lived states means that RESOLFT requires much lower laser intensities than STED, which makes it attractive for imaging biological samples over large areas or long times.
RESOLFT demonstrated its suitability for bioimaging for the first time last year, with a protein called rsEGFP (reversibly switchable enhanced GFP) being employed as the fluorophore. However, the time needed to switch this protein between the ‘on state’ and the ‘off state’ was relatively long, and it took about an hour to record a typical image. Now, Grotjohann et al. have modified this protein to make a new fluorophore called rsEGFP2 with a shorter switching time, and have used it to image various structures—including Vimentin, a protein that forms part of the cytoskeleton in many cells, and organelles called peroxisomes—inside live mammalian cells. They were able to record these images some 25–250 times faster than would have been possible with previous RESOLFT approaches. The combination of RESOLFT and rsEGFP2 should allow researchers to image a wide variety of structures and processes in living cells that have not been imaged before.
PMCID: PMC3534202  PMID: 23330067
confocal microscopy; fluorescent probes; GFP; nanoscopy; superresolution; live-cell imaging; None
8.  Highly sensitive detection of cancer cells using femtosecond dual-wavelength near-IR two-photon imaging 
Biomedical Optics Express  2012;3(7):1534-1547.
We describe novel imaging protocols that allow detection of small cancer cell colonies deep inside tissue phantoms with high sensitivity and specificity. We compare fluorescence excited in Styryl-9M molecules by femtosecond pulses at near IR wavelengths, where Styryl-9M shows the largest dependence of the two-photon absorption (2PA) cross section on the local environment. We show that by calculating the normalized ratio of the two-photon excited fluorescence (2PEF) intensity at 1200 nm and 1100 nm excitation wavelengths we can achieve high sensitivity and specificity for determining the location of cancer cells surrounded by normal cells. The 2PEF results showed a positive correlation with the levels of MDR1 proteins expressed by the cells, and, for high MDR1 expressors, as few as ten cancer cells could be detected. Similar high sensitivity is also demonstrated for tumor colonies induced in mouse external ears. This technique could be useful in early cancer detection, and, perhaps, also in monitoring dormant cancer deposits.
PMCID: PMC3395479  PMID: 22808426
(170.3880) Medical and biological imaging; (190.4180) Multiphoton processes
9.  Development of fluorescent probes for bioimaging applications 
Fluorescent probes, which allow visualization of cations such as Ca2+, Zn2+ etc., small biomolecules such as nitric oxide (NO) or enzyme activities in living cells by means of fluorescence microscopy, have become indispensable tools for clarifying functions in biological systems. This review deals with the general principles for the design of bioimaging fluorescent probes by modulating the fluorescence properties of fluorophores, employing mechanisms such as acceptor-excited Photoinduced electron Transfer (a-PeT), donor-excited Photoinduced electron Transfer (d-PeT), and spirocyclization, which have been established by our group. The a-PeT and d-PeT mechanisms are widely applicable for the design of bioimaging probes based on many fluorophores and the spirocyclization process is also expected to be useful as a fluorescence off/on switching mechanism. Fluorescence modulation mechanisms are essential for the rational design of novel fluorescence probes for target molecules. Based on these mechanisms, we have developed more than fifty bioimaging probes, of which fourteen are commercially available. The review also describes some applications of the probes developed by our group to in vitro and in vivo systems.
PMCID: PMC3037519  PMID: 20948177
probe; bioimaging; photoinduced electron transfer; fluorescence; spirocyclization
10.  Analysis of multiple compound–protein interactions reveals novel bioactive molecules 
The authors use machine learning of compound-protein interactions to explore drug polypharmacology and to efficiently identify bioactive ligands, including novel scaffold-hopping compounds for two pharmaceutically important protein families: G-protein coupled receptors and protein kinases.
We have demonstrated that machine learning of multiple compound–protein interactions is useful for efficient ligand screening and for assessing drug polypharmacology.This approach successfully identified novel scaffold-hopping compounds for two pharmaceutically important protein families: G-protein-coupled receptors and protein kinases.These bioactive compounds were not detected by existing computational ligand-screening methods in comparative studies.The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes.
The discovery of novel bioactive molecules advances our systems-level understanding of biological processes and is crucial for innovation in drug development. Perturbations of biological systems by chemical probes provide broader applications not only for analysis of complex systems but also for intentional manipulations of these systems. Nevertheless, the lack of well-characterized chemical modulators has limited their use. Recently, chemical genomics has emerged as a promising area of research applicable to the exploration of novel bioactive molecules, and researchers are currently striving toward the identification of all possible ligands for all target protein families (Wang et al, 2009). Chemical genomics studies have shown that patterns of compound–protein interactions (CPIs) are too diverse to be understood as simple one-to-one events. There is an urgent need to develop appropriate data mining methods for characterizing and visualizing the full complexity of interactions between chemical space and biological systems. However, no existing screening approach has so far succeeded in identifying novel bioactive compounds using multiple interactions among compounds and target proteins.
High-throughput screening (HTS) and computational screening have greatly aided in the identification of early lead compounds for drug discovery. However, the large number of assays required for HTS to identify drugs that target multiple proteins render this process very costly and time-consuming. Therefore, interest in using in silico strategies for screening has increased. The most common computational approaches, ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS; Oprea and Matter, 2004; Muegge and Oloff, 2006; McInnes, 2007; Figure 1A), have been used for practical drug development. LBVS aims to identify molecules that are very similar to known active molecules and generally has difficulty identifying compounds with novel structural scaffolds that differ from reference molecules. The other popular strategy, SBVS, is constrained by the number of three-dimensional crystallographic structures available. To circumvent these limitations, we have shown that a new computational screening strategy, chemical genomics-based virtual screening (CGBVS), has the potential to identify novel, scaffold-hopping compounds and assess their polypharmacology by using a machine-learning method to recognize conserved molecular patterns in comprehensive CPI data sets.
The CGBVS strategy used in this study was made up of five steps: CPI data collection, descriptor calculation, representation of interaction vectors, predictive model construction using training data sets, and predictions from test data (Figure 1A). Importantly, step 1, the construction of a data set of chemical structures and protein sequences for known CPIs, did not require the three-dimensional protein structures needed for SBVS. In step 2, compound structures and protein sequences were converted into numerical descriptors. These descriptors were used to construct chemical or biological spaces in which decreasing distance between vectors corresponded to increasing similarity of compound structures or protein sequences. In step 3, we represented multiple CPI patterns by concatenating these chemical and protein descriptors. Using these interaction vectors, we could quantify the similarity of molecular interactions for compound–protein pairs, despite the fact that the ligand and protein similarity maps differed substantially. In step 4, concatenated vectors for CPI pairs (positive samples) and non-interacting pairs (negative samples) were input into an established machine-learning method. In the final step, the classifier constructed using training sets was applied to test data.
To evaluate the predictive value of CGBVS, we first compared its performance with that of LBVS by fivefold cross-validation. CGBVS performed with considerably higher accuracy (91.9%) than did LBVS (84.4%; Figure 1B). We next compared CGBVS and SBVS in a retrospective virtual screening based on the human β2-adrenergic receptor (ADRB2). Figure 1C shows that CGBVS provided higher hit rates than did SBVS. These results suggest that CGBVS is more successful than conventional approaches for prediction of CPIs.
We then evaluated the ability of the CGBVS method to predict the polypharmacology of ADRB2 by attempting to identify novel ADRB2 ligands from a group of G-protein-coupled receptor (GPCR) ligands. We ranked the prediction scores for the interactions of 826 reported GPCR ligands with ADRB2 and then analyzed the 50 highest-ranked compounds in greater detail. Of 21 commercially available compounds, 11 showed ADRB2-binding activity and were not previously reported to be ADRB2 ligands. These compounds included ligands not only for aminergic receptors but also for neuropeptide Y-type 1 receptors (NPY1R), which have low protein homology to ADRB2. Most ligands we identified were not detected by LBVS and SBVS, which suggests that only CGBVS could identify this unexpected cross-reaction for a ligand developed as a target to a peptidergic receptor.
The true value of CGBVS in drug discovery must be tested by assessing whether this method can identify scaffold-hopping lead compounds from a set of compounds that is structurally more diverse. To assess this ability, we analyzed 11 500 commercially available compounds to predict compounds likely to bind to two GPCRs and two protein kinases. Functional assays revealed that nine ADRB2 ligands, three NPY1R ligands, five epidermal growth factor receptor (EGFR) inhibitors, and two cyclin-dependent kinase 2 (CDK2) inhibitors were concentrated in the top-ranked compounds (hit rate=30, 15, 25, and 10%, respectively). We also evaluated the extent of scaffold hopping achieved in the identification of these novel ligands. One ADRB2 ligand, two NPY1R ligands, and one CDK2 inhibitor exhibited scaffold hopping (Figure 4), indicating that CGBVS can use this characteristic to rationally predict novel lead compounds, a crucial and very difficult step in drug discovery. This feature of CGBVS is critically different from existing predictive methods, such as LBVS, which depend on similarities between test and reference ligands, and focus on a single protein or highly homologous proteins. In particular, CGBVS is useful for targets with undefined ligands because this method can use CPIs with target proteins that exhibit lower levels of homology.
In summary, we have demonstrated that data mining of multiple CPIs is of great practical value for exploration of chemical space. As a predictive model, CGBVS could provide an important step in the discovery of such multi-target drugs by identifying the group of proteins targeted by a particular ligand, leading to innovation in pharmaceutical research.
The discovery of novel bioactive molecules advances our systems-level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound–protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold-hopping compounds. Through a machine-learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G-protein-coupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand-screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes.
PMCID: PMC3094066  PMID: 21364574
chemical genomics; data mining; drug discovery; ligand screening; systems chemical biology
11.  High Throughput and High Content Screening Capabilities of the University of Cincinnati Drug Discovery Center 
The Drug Discovery Center collaborates with a wide range of academic and industrial research centers to facilitate the identification of active small molecules with high potential for use as biological probes or as starting points for drug discovery programs. The DDC operates state-of-the-art high throughput and high content screening instrumentation and a diverse 350,000 compound library. The center's personnel provide collaborators with advice in assay design, analytical technology selection, and library design via cheminformatics and/or structure-based approaches. Typical programs are exemplified by an HTS program targeting the identification of novel atypical PKC inhibitors for potential use in cancer and an HCS program targeting the identification of stimulators of the differentiation of oligodendrocyte progenitor cells to oligodendrocytes for potential use in multiple sclerosis. Activities of atypical PKCs, PKCiota and PKCzeta, were measured using both fluorescent detection of ADP production and MALDI-TOF detection of substrate phosphorylation. 30,000 compounds were screened for their effect on PKC activity using these orthogonal detection methods to identify potential inhibitors. Oligodendrocyte differentiation was measured in a High Content Screen by pairing a selective marker of oligodendrocytes with Alexa Fluor 488 secondary antibody for the detection of mature ODs. DAPI stain was used for nucleus detection. Confocal microscope images were acquired and analyzed using an algorithm for neurite outgrowth adapted for the characterization of oligodendrocyte processes. Three measurements: Mean Maximum Process Length per Cell, Mean Process Signal Intensity per Cell, and Percentage Differentiated Cells per Well, were quantified by the image analysis script and afforded a statistically significant separation of promoter controls and inhibitor controls from non-treated (neutral) controls with Mean Maximum Process Length as measurement.
PMCID: PMC3635306
12.  Dual-modality molecular imaging using antibodies labeled with activatable fluorescence and a radionuclide for specific and quantitative targeted cancer detection 
Bioconjugate chemistry  2009;20(11):2177-2184.
Multimodality molecular imaging should have potential for compensating the disadvantages and enhancing the advantages of each modality. Nuclear imaging is superior to optical imaging in whole body imaging and in quantification due to good tissue penetration of gamma rays. However, target specificity can be compromised by high background signal due to the always signal ON feature of nuclear probes. In contrast, optical imaging can be superior in target specific imaging by employing target-specific signal activation systems, although it is not quantitative because of signal attenuation. In this study, to take advantage of the mutual cooperation of each modality, multimodality imaging was performed by a combination of quantitative radiolabeled probe and an activatable optical probe. The monoclonal antibodies, panitumumab (anti-HER1) and trastuzumab (anti-HER2) were labeled with 111In and ICG, and tested in both HER1 and HER2 tumor bearing mice by the cocktail injection of radiolabeled and optical probes, and by the single injection of a dual-labeled probe. The optical and nuclear images were obtained over 6 days after the conjugates injection. The fluorescence activation properties of ICG labeled antibodies were also investigated by in vitro microscopy. In vitro microscopy demonstrated that there was no fluorescence signal with either panitumumab-ICG or trastuzumab-ICG, when the probes were bound to cell surface antigens but were not yet internalized. After the conjugates were internalized into the cells, both conjugates showed bright fluorescence signal only in the target cells. These results show both conjugates work as activatable probes. In vivo multimodality imaging by injection of a cocktail of radio-optical probes, only the target specific tumor was visualized by optical imaging. Meanwhile, the biodistribution profile of the injected antibody was provided by nuclear imaging. Similar results were obtained with radio and optical dual labeled probe, and it is confirmed that pharmacokinetic properties did not affect the results above.
Here, we could characterize the molecular targets by activatable optical probes, and visualize the delivery of targeting molecules quantitatively by radioactive probes. Multimodality molecular imaging combining activatable optical and radioactive probe has great potential for simultaneous visualization, characterization, and measurement of biological processes.
PMCID: PMC2782620  PMID: 19919110
13.  Visualizing the actin cytoskeleton in living plant cells using a photo-convertible mEos::FABD-mTn fluorescent fusion protein 
Plant Methods  2008;4:21.
The actin cytoskeleton responds quickly to diverse stimuli and plays numerous roles in cellular signalling, organelle motility and subcellular compartmentation during plant growth and development. Molecular and cell biological tools that can facilitate visualization of actin organization and dynamics in a minimally invasive manner are essential for understanding this fundamental component of the living cell.
A novel, monomeric (m) Eos-fluorescent protein derived from the coral Lobophyllia hemprichii was assessed for its green to red photo-convertibility in plant cells by creating mEosFP-cytosolic. mEosFP was fused to the F-(filamentous)-Actin Binding Domain of the mammalian Talin gene to create mEosFP::FABDmTalin. Photo-conversion, visualization and colour quantification protocols were developed for EosFP targeted to the F-actin cytoskeleton. Rapid photo-conversion in the entire cell or in a region of interest was easily achieved upon illumination with an approximately 400 nm wavelength light beam using an epi-fluorescent microscope. Dual color imaging after photo-conversion was carried out using a confocal laser-scanning microscope. Time-lapse imaging revealed that although photo-conversion of single mEosFP molecules can be rapid in terms of live-cell imaging it involves a progressive enrichment of red fluorescent molecules over green species. The fluorescence of photo-converted cells thus progresses through intermediate shades ranging from green to red. The time taken for complete conversion to red fluorescence depends on protein expression level within a cell and the quality of the focusing lens used to deliver the illuminating beam. Three easily applicable methods for obtaining information on fluorescent intensity and colour are provided as a means of ensuring experimental repeatability and data quantification, when using mEosFP and similar photo-convertible proteins.
The mEosFP::FABD-mTn probe retains all the imaging qualities associated with the well tested GFP::mTn probe while allowing for non-invasive, regional photo-conversion that allows colour based discrimination within a living cell. Whereas a number of precautions should be exercised in dealing with photo-convertible probes, mEosFP::FABD-mTn is a versatile live imaging tool for dissecting the organization and activity of the actin cytoskeleton in plants.
PMCID: PMC2557004  PMID: 18803828
14.  Discovery of Selective Probes and Antagonists for G Protein-Coupled Receptors FPR/FPRL1 and GPR30 
Recent technological advances in flow cytometry provide a versatile platform for high throughput screening of compound libraries coupled with high-content biological testing and drug discovery. The G protein-coupled receptors (GPCRs) constitute the largest class of signaling molecules in the human genome with frequent roles in disease pathogenesis, yet many examples of orphan receptors with unknown ligands remain. The complex biology and potential for drug discovery within this class provide strong incentives for chemical biology approaches seeking to develop small molecule probes to facilitate elucidation of mechanistic pathways and enable specific manipulation of the activity of individual receptors. We have initiated small molecule probe development projects targeting two distinct families of GPCRs: the formylpeptide receptors (FPR/FPRL1) and G protein-coupled estrogen receptor (GPR30). In each case the assay for compound screening involved the development of an appropriate small molecule fluorescent probe, and the flow cytometry platform provided inherently biological rich assays that enhanced the process of identification and optimization of novel antagonists. The contributions of cheminformatics analysis tools, virtual screening, and synthetic chemistry in synergy with the biomolecular screening program have yielded valuable new chemical probes with high binding affinity, selectivity for the targeted receptor, and potent antagonist activity. This review describes the discovery of novel small molecule antagonists of FPR and FPRL1, and GPR30, and the associated characterization process involving secondary assays, cell based and in vivo studies to define the selectivity and activity of the resulting chemical probes
PMCID: PMC2885834  PMID: 19807662
flow cytometry; fluorescent; GPCR; formylpeptide receptor; inflammation; GPR30; GPER; estrogen; nongenomic; cancer; antidepressant
15.  Endothelial Cell-Surface Gp60 Activates Vesicle Formation and Trafficking via Gi-Coupled Src Kinase Signaling Pathway 
The Journal of Cell Biology  2000;150(5):1057-1070.
We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein Gi, and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial 125I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11–amino acid Gαi carboxyl-terminal peptide inhibited endothelial 125I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of Gαi with the caveolin-1, whereas dn-Src inhibited Gαi binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60 with caveolin-1, followed by the activation of the downstream Gi-coupled Src kinase signaling pathway.
PMCID: PMC2175246  PMID: 10973995
transcytosis; endocytosis; caveolae; microvascular endothelial cells; albumin permeability
16.  Fluorene-Based Metal-Ion Sensing Probe with High Sensitivity to Zn2+ and Efficient Two-photon Absorption 
The journal of physical chemistry. B  2010;114(28):9313-9321.
The photophysical, photochemical, two-photon absorption (2PA), and metal ion sensing properties of a new fluorene derivative (E)-1-(7-(4-(benzo[d]thiazol-2-yl)styryl)-9,9-bis(2-(2-ethoxyethoxy)ethyl)-9H-fluoren-2-yl)-3-(2-(9,10,16,17,18,19,21,22,23,24-decahydro-6H dibenzo[h,s][1,4,7,11,14,17]trioxatriazacycloicosin-20(7H)-yl)ethyl)thiourea (1) were investigated in organic and aqueous media. High sensitivity and selectivity of 1 to Zn2+ in THF and a water/ACN mixture were shown by both absorption and fluorescence titration. The observed complexation processes corresponded to 1:1 stoichiometry with the range of binding constants ~ (2–3)·105 M−1. The degenerate 2PA spectra of 1 and 1:Zn2+ complex were obtained in the 640–900 nm spectral range, with the maximum values of two-photon action cross section for ligand:metal complex ~ (90–130) GM, using a standard two-photon induced fluorescence methodology under femtosecond excitation. The nature of the 2PA bands was analyzed by quantum chemical methods and a specific dependence on metal ion binding processes was shown. Ratiometric fluorescence detection (420/650 nm) provided a good dynamic range (10−4 to 10−6 M) for detecting Zn2+, which, along with the good photostability and 2PA properties of probe 1. makes it a good candidate in two-photon fluorescence microscopy imaging and sensing of Zn ions.
PMCID: PMC2913098  PMID: 20590077
17.  PHIP Hyperpolarized MR Receptor Imaging In Vivo: A Pilot Study of 13C Imaging of Atheroma in Mice 
NMR in biomedicine  2011;24(8):1023-1028.
Magnetic resonance (MR) techniques using hyperpolarized 13C have successfully produced examples of angiography and intermediary metabolic imaging, but to date no receptor imaging has been attempted. The goal of this study is to synthesize and evaluate a novel hyperpolarizable molecule, tetrafluoropropyl 1-13C-propionate-d3 (TFPP), for detecting atheromatous plaque in vivo. TFPP binds to lipid bilayers and its use in hyperpolarized MR could prove to be a major step towards receptor imaging.
The precursor, Tetrafluoropropyl 1-13C-acrylate (TFPA) binds to dimyristoylphosphatidylcholine (DMPC) lipid bilayers with a 1.6 ppm chemical shift in the 19F MR spectrum. This molecule was designed to be hyperpolarized through addition of parahydrogen to 13C acrylate moiety by Parahydrogen Induced Polarization (PHIP). 13C TFPA was hyperpolarized to Tetrafluoropropyl 1-13C-propionate (TFPP) to a similar extent to that of hydroxyethylacrylate (HEA) to hydroxyethylpropionate (HEP); 17% +/− 4 % for TFPP vs 20% for HEP; T1 relaxation times (45s ± 2 vs 55s ± 2) were comparable and the hyperpolarized properties of TFPP were characterized. HEA, like TFPA has a chemical structure with an acrylate moiety but do not have the lipid binding Tetrafluoropropyl functional group. Hyperpolarized 13C TFPP binds to lipid bilayer appearing as a second, chemically shifted 13C hyperpolarized MR resonance with further reduction in longitudinal relaxation time (T1 = 21s ± 1). In aortas harvested from Low Density Lipoprotein Receptor (LDLR) knock-out mice fed with a high fat diet for nine months, and in which atheroma is deposited in aorta and heart, 13C TFPP showed greater binding to lipid on the intimal surface than in normal diet control mice. When 13C TFPP was hyperpolarized and administered in vivo to atheromatous mice in a pilot study, increased binding was observed on the endocardial surface of the intact heart compared to normal fed controls.
Hyperpolarized 13C TFPP has bio-sensing specificity for lipid, coupled with 42,000 fold sensitivity gain in MR signal at 4.7 Tesla. Binding of TFPP with lipids results in the formation of a characteristic second peak in MR spectroscopy. TFPP therefore has the potential to act as an in vivo molecular probe for atheromatous plaque imaging and may serve as a model of receptor targeted bioimaging with enhanced MR sensitivity.
PMCID: PMC3240663  PMID: 21538638
13C; heart; receptor imaging; atheroma; hyperpolarization; TFPP; PHIP; MR
18.  Porphyrins for Probing Electrical Potential Across Lipid Bilayer Membranes by Second Harmonic Generation** 
Neurons communicate by using electrical signals, mediated by transient changes in the voltage across the plasma membrane. Optical techniques for visualizing these transmembrane potentials could revolutionize the field of neurobiology by allowing the spatial profile of electrical activity to be imaged in real time with high resolution, along individual neurons or groups of neurons within their native networks.1, 2 Second harmonic generation (SHG) is one of the most promising methods for imaging membrane potential, although so far this technique has only been demonstrated with a narrow range of dyes.3 Here we show that SHG from a porphyrin-based membrane probe gives a fast electro-optic response to an electric field which is about 5–10 times greater than that of conventional styryl dyes. Our results indicate that porphyrin dyes are promising probes for imaging membrane potential.
PMCID: PMC3881515  PMID: 23861287
dyes; membrane potential; membranes; nonlinear optics; porphyrinoids
19.  Facile Synthesis of Amine-Functionalized Eu3+-Doped La(OH)3 Nanophosphors for Bioimaging 
Here, we report a straightforward synthesis process to produce colloidal Eu3+-activated nanophosphors (NPs) for use as bioimaging probes. In this procedure, poly(ethylene glycol) serves as a high-boiling point solvent allowing for nanoscale particle formation as well as a convenient medium for solvent exchange and subsequent surface modification. The La(OH)3:Eu3+ NPs produced by this process were ~3.5 nm in diameter as determined by transmission electron microscopy. The NP surface was coated with aminopropyltriethoxysilane to provide chemical functionality for attachment of biological ligands, improve chemical stability and prevent surface quenching of luminescent centers. Photoluminescence spectroscopy of the NPs displayed emission peaks at 597 and 615 nm (λex = 280 nm). The red emission, due to 5D0 → 7F1 and 5D0 → 7F2 transitions, was linear with concentration as observed by imaging with a conventional bioimaging system. To demonstrate the feasibility of these NPs to serve as optical probes in biological applications, an in vitro experiment was performed with HeLa cells. NP emission was observed in the cells by fluorescence microscopy. In addition, the NPs displayed no cytotoxicity over the course of a 48-h MTT cell viability assay. These results suggest that La(OH)3:Eu3+ NPs possess the potential to serve as a luminescent bioimaging probe.
PMCID: PMC3211300
Nanophosphor; Nanoparticle synthesis; Lanthanum hydroxide; Europium; Photoluminescence; Bioimaging
20.  Integrin-targeting Block Copolymer Probes for Two-photon Fluorescence Bioimaging 
Biomacromolecules  2010;12(2):441-449.
Targeted molecular imaging with two-photon fluorescence microscopy (2PFM) is a powerful technique for chemical biology and, potentially, for non-invasive diagnosis and treatment of a number of diseases. The synthesis, photophysical studies, and bioimaging are reported for a versatile norbornene-based block copolymer multifunctional scaffold containing biocompatible (PEG), two-photon fluorescent dyes (fluorenyl), and targeting (cyclic-RGD peptide) moieties. The two bioconjugates, containing two different fluorenyl dyes and cRGDfK covalently attached to the polymer probe, formed a spherical micelle and self-assembled structure in water, for which size was analyzed by TEM and DLS. Cell-viability and 2PFM imaging of human epithelial U87MG cell lines that over express αvβ3 integrin was performed via incubation with the new probes, along with negative control studies using MCF-7 breast cancer cells and blocking experiments. 2PFM microscopy confirmed the high selectivity of the biocompatible probe in the integrin rich area in the U87MF cells while blocking as well as negative control MCF-7 experiments confirmed the integrin targeting ability of the new probes.
PMCID: PMC3040259  PMID: 21190348
Water-soluble block copolymer probe; ROMP; two-photon bioimaging; integrin targeting
21.  A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems 
Scientific Reports  2014;4:6476.
Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology.
PMCID: PMC4174567  PMID: 25252596
22.  Folate Receptor-targeted Aggregation-enhanced Near-IR Emitting Silica Nanoprobe for One-photon in vivo and Two-photon ex vivo Fluorescence Bioimaging 
Bioconjugate chemistry  2011;22(7):1438-1450.
A two-photon absorbing (2PA) and aggregation-enhanced near infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong signal as the unaggregated dye, a three-fold increase in two-photon absorption relative to the DFP in solution, and approx. four-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and 1H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FRtargeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 µm deep in the HeLa tumor.
PMCID: PMC3147277  PMID: 21688841
Aggregation enhanced emission; near infrared emission; folate receptor targeting; two-photon absorption; two-photon fluorescence microscopy; silica nanoparticles; HeLa tumor; in vivo imaging; ex vivo imaging
23.  Real-time, Spatially Resolved Analysis of Serotonin Transporter Activity And Regulation Using the Fluorescent Substrate, ASP+ 
Journal of neurochemistry  2010;114(4):1019-1029.
The serotonin transporter (SERT) mediates clearance of serotonin from the synapse, thereby, regulating extracellular serotonin concentrations. Radioligand uptake techniques are typically used to assess SERT function in tissue and heterologous expression systems. The need for sufficient protein in samples, however, requires use of homogenate preparations, potentially masking effects limited to specific cell populations. 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP+) is a fluorescent monoamine transporter substrate that has been used for real-time monitoring of dopamine and norepinephrine transporter function in single cells. The present live cell imaging studies examine the utility of ASP+ for quantifying hSERT function in HEK-293 and neuroblastoma cells. We show rapid membrane binding and intracellular ASP+ accumulation in hSERT expressing cells. Accumulation is saturable; dependent on temperature and the presence of sodium and chloride in the media, and attenuated by serotonin. Acute or prolonged exposure of cells to serotonin re-uptake inhibitors produces a concentration-dependent decrease in accumulation. Similar effects are produced by PKC activation whereas p38MAPK activation increases ASP+ accumulation. These data demonstrate the validity of ASP+ as a probe for monitoring SERT function in living cells. Alterations in SERT binding and uptake can be quantified in the same cell and use of a within cell design permits analysis of time-related alterations in SERT function.
PMCID: PMC2930987  PMID: 20524964
live-cell imaging; 4-(4-(dimethylamino)-styryl)-N-methylpyridinium; p38 MAPK; monoamine transporter; PKC
24.  Evaluation of voltage-sensitive fluorescence dyes for monitoring neuronal activity in the embryonic central nervous system 
The Journal of membrane biology  2013;246(9):679-688.
Using an optical imaging technique with voltage-sensitive dyes (VSDs), we have been investigating the functional organization and architecture of the central nervous system (CNS) during embryogenesis. In the embryonic nervous system, a merocyanine-rhodanine dye, NK2761, has proved to be the most useful absorption dye for detecting neuronal activity because of its high signal-to-noise ratio (S/N), low toxicity, and small dye bleaching. In the present study, we evaluated the suitability of voltage-sensitive fluorescence dyes for optical recording in the embryonic CNS. We screened eight styryl (hemicyanine) dyes in isolated brainstem-spinal cord preparations from 7-day old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recording system. The signal size, S/N, photobleaching, effects of perfusion, and recovery of neural responses after staining were compared. We also evaluated optical responses with various magnifications. Although the S/N was lower than with the absorption dye, clear optical responses were detected with several fluorescence dyes, including di-2-ANEPEQ, di-4-ANEPPS, di-3-ANEPPDHQ, di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA, and di-2-ANEPPTEA. Di-2-ANEPEQ showed the largest S/N, whereas its photobleaching was faster and the recovery of neural responses after staining was slower. Di-4-ANEPPS and di-3-ANEPPDHQ also exhibited a large S/N, but required a relatively long time for recovery of neural activity. Di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA, and di-2-ANEPPTEA showed smaller S/Ns than di-2-ANEPEQ, di-4-ANEPPS, and di-3-ANEPPDHQ, but the recovery of neural responses after staining was faster. This study demonstrates the potential utility of these styryl dyes in optical monitoring of voltage changes in the embryonic CNS.
PMCID: PMC4096138  PMID: 23975337
optical recording; voltage-sensitive dye; embryo; nervous system; fluorescence; screening
25.  Protein localization as a principal feature of the etiology and comorbidity of genetic diseases 
Proteins localized within the same subcellular compartment tend to be functionally associated. This study shows that subcellular localization and network distance between disease-associated proteins provide complementary information explaining patterns of disease comorbidity.
A positive correlation was found between subcellular localization of disease-associated protein pairs and measures of comorbidity.A higher comorbidity tendency was found for disease-associated protein pairs that are positioned within a shorter distance in the protein interaction network.The integration of subcellular localization information with protein interaction network sheds light onto the potential molecular connections underlying comorbidity patterns and will help to understand the mechanisms of human disease.
It was shown that the emergence of phenotypically similar diseases are triggered as a result of molecular connections between disease-causing genes (Oti and Brunner, 2007; Zaghloul and Katsanis, 2010). From a genetics, perspective diseases are associated with certain genes (Goh et al, 2007; Feldman et al, 2008), whereas from a proteomics perspective phenotypically similar diseases are connected via biological modules such as protein–protein interactions (PPIs) or molecular pathways (Lage et al, 2007; Jiang et al, 2008; Wu et al, 2008; Linghu et al, 2009; Suthram et al, 2010). These molecular connections between diseases were observed on the population level as well: diseases connected through molecular connections such as shared genes, PPIs, and metabolic pathways tend to show elevated comorbidity (Rzhetsky et al, 2007; Lee et al, 2008; Zhernakova et al, 2009; Park et al, 2009a, 2009b). While these findings constitute a step toward improving our understanding of the mechanism of disease progression, there are still many more molecule-level connections between disease pairs that need to be explored in order to establish a firmer comorbidity association.
Subcellular localization provides spatial information of proteins in the cell; proteins target subcellular localizations to interact with appropriate partners and form functional complexes in signaling pathways and metabolic processes (Au et al, 2007). Abnormal protein localizations are known to lead to the loss of functional effects in diseases (Luheshi et al, 2008; Laurila and Vihinen, 2009). For example, mis-localizations of nuclear/cytoplasmic transport have been detected in many types of carcinoma cells (Kau et al, 2004). A proper identification of protein subcellular localization can hence be useful in discovering disease-associated proteins (Giallourakis et al, 2005; Calvo and Mootha, 2010). With this understanding, we postulate that disease-associated proteins connected by subcellular localizations could also explain the phenotypic similarities between diseases. Furthermore, such connections may also couple to disease progressions that contribute to multiple disease manifestation, that is, comorbidity.
Protein subcellular localization has been extensively studied through various methods to determine a variety of protein functions. To the best of our knowledge, the connection between diseases and subcellular localizations are yet to be studied systematically. To resolve this we constructed, for the first time, a human Disease-associated Protein and subcellular Localization (DPL) matrix (top panel in Box 1). Our DPL matrix provides the ‘cellular localization map of diseases' that represents the spatial index of diseases in the cell. We found that each disease shows unique characteristics of subcellular localization profile in the DPL matrix. We were interested in determining whether subsets of 1284 human diseases exhibit distinct enrichment profiles across subcellular localizations. We calculated pairwise correlations and performed a hierarchical clustering of the enrichments of the 1284 diseases across 10 different subcellular localizations.
Our DPL matrix revealed that 778 diseases (∼62%, P=1.40 × 10−3) are enriched in a single localization and 273 diseases (∼21%, P=3.45 × 10−3) are enriched in dual localizations. In the DPL matrix, certain disease-associated proteins are likely to be found in membrane-bounded organelles such as mitochondria, lysosome, and peroxisome, indicating that the mutations of proteins localized to these compartments are connected to the pathophysiological conditions of those organelles. Meanwhile, certain disease-associated proteins in the DPL matrix are enriched in dual localizations, such as extracellular/plasma membrane or endoplasmic reticulum/Golgi. Although these two pairs of subcellular localizations appear to be distinct compartments at first, they are functionally related compartments in close proximity during protein translocation process in the cell, and thus are likely to share interacting protein partners (Gandhi et al, 2006).
Comorbidity represents the co-occurrence of multiple diseases in the same individual (Lee et al, 2008; Hidalgo et al, 2009; Park et al, 2009a). Many comorbid disease pairs have been shown to share common genes in the human disease network. For example, Diabetes and Alzheimer's disease share a risk factor in angiotensin I converting enzyme, and frequently occur together in an individual. In such instances, comorbidity can be partially attributed to the disease connections on the molecular level. To explore the impact of protein subcellular localization on comorbidity, we hypothesized that certain disease pairs could also be connected via subcellular localization by the molecular connections between the disease-associated proteins (bottom panel in Box 1).
We found a positive correlation between subcellular localization similarity and relative risk (Figure 3B, Pearson's correlation coefficient between relative risk and subcellular localization similarity=0.81, P=2.96 × 10−5). The subcellular localization similarity represents the correlation of subcellular localization profiles between disease pairs. To our surprise, when we compared the relative risk of disease pairs linked via various molecular connections, we found that disease pairs connected by subcellular localization showed a near three-fold higher comorbidity tendency (with link distances equal to 2 or 3) when compared with random pairs (Figure 3E).
We then assessed quantitatively the impact of network distances and subcellular localizations on the comorbidity tendency of disease pairs. We expected the proteins associated with comorbid disease pairs to be located closely in the protein interaction network via fewer links compared with random disease pairs. Indeed, a higher comorbidity tendency was found when two disease-associated proteins were positioned within a shorter distance (gray plots in Figure 3F). Moreover, when subcellular localization information was combined with small network distances, the comorbidity tendency increased dramatically (orange plots in Figure 3F). It suggests that subcellular localization and close network distances, two conceptually distinct molecular connections, contributed synergistically to the comorbidity tendency.
Disease progression is not restricted to the mutation of disease-causing genes, but also affected by molecular connections in ‘disease modules,' resulting in comorbidity (Fraser, 2006; Lee et al, 2008). In this study, for the first time we applied subcellular localization information to elucidate the molecular connections between comorbid diseases. We believe that, based on our finding, our approach helps to define the boundaries of ‘disease modules.' Taken together, integration of diverse molecular connections should improve the molecular level understanding of hitherto unexplained comorbid disease pairs and help us in expanding the scope of our knowledge of the mechanism of human disease progression.
Proteins targeting the same subcellular localization tend to participate in mutual protein–protein interactions (PPIs) and are often functionally associated. Here, we investigated the relationship between disease-associated proteins and their subcellular localizations, based on the assumption that protein pairs associated with phenotypically similar diseases are more likely to be connected via subcellular localization. The spatial constraints from subcellular localization significantly strengthened the disease associations of the proteins connected by subcellular localizations. In particular, certain disease types were more prevalent in specific subcellular localizations. We analyzed the enrichment of disease phenotypes within subcellular localizations, and found that there exists a significant correlation between disease classes and subcellular localizations. Furthermore, we found that two diseases displayed high comorbidity when disease-associated proteins were connected via subcellular localization. We newly explained 7584 disease pairs by using the context of protein subcellular localization, which had not been identified using shared genes or PPIs only. Our result establishes a direct correlation between protein subcellular localization and disease association, and helps to understand the mechanism of human disease progression.
PMCID: PMC3130560  PMID: 21613983
cellular networks; comorbidity; human disease; subcellular localization

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