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1.  Nanoparticle Detection of Urinary Markers for Point-of-Care Diagnosis of Kidney Injury 
PLoS ONE  2015;10(7):e0133417.
The high incidence of acute and chronic kidney injury due to various environmental factors such as heavy metals or chemicals has been a major problem in developing countries. However, the diagnosis of kidney injury in these areas can be more challenging due to the lack of highly sensitive and specific techniques that can be applied in point-of-care settings. To address this, we have developed a technique called ‘micro-urine nanoparticle detection (μUNPD)’, that allows the detection of trace amounts of molecular markers in urine. Specifically, this technique utilizes an automated on-chip assay followed by detection with a hand-held device for the read-out. Using the μUNPD technology, the kidney injury markers KIM-1 and Cystatin C were detected down to concentrations of 0.1 ng/ml and 20 ng/ml respectively, which meets the cut-off range required to identify patients with acute or chronic kidney injury. Thus, we show that the μUNPD technology enables point of care and non-invasive detection of kidney injury, and has potential for applications in diagnosing kidney injury with high sensitivity in resource-limited settings.
doi:10.1371/journal.pone.0133417
PMCID: PMC4506142  PMID: 26186708
2.  Ultrafluorogenic Coumarin-Tetrazine Probes for Real-Time Biological Imaging** 
We have developed a series of new ultrafluorogenic probes in the blue-green spectrum that display fluorescence enhancement exceeding 11,000-fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal tetrazine-transcyclooctene (TCO) chemistry platform. By exploiting highly efficient through bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported thus far for any bioorthogonal fluorogenic dyes. No-wash, fluorogenic imaging of diverse targets including cell surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds with minimal background signal and no appreciable non-specific binding, opening the possibility for in vivo sensing.
doi:10.1002/anie.201403890
PMCID: PMC4122131  PMID: 24915832
click chemistry; energy transfer; fluorescence probes; fluorogenic probes; tetrazines
3.  Differential Contribution of Monocytes to Heart Macrophages in Steady-State and after Myocardial Infarction 
Circulation research  2014;115(2):284-295.
Rationale
Macrophages populate the steady-state myocardium. Previously, all macrophages were thought to arise from monocytes; however, it emerged that in several organs tissue-resident macrophages may self-maintain through local proliferation.
Objective
To study the contribution of monocytes to cardiac resident macrophages in steady-state, after macrophage depletion in CD11bDTR/+ mice and in myocardial infarction.
Methods and Results
Using in vivo fate mapping and flow cytometry, we estimated that during steady-state the heart macrophage population turns over in about one month. To explore the source of cardiac resident macrophages, we joined the circulation of mice using parabiosis. After 6 weeks, we observed blood monocyte chimerism of 35.3±3.4% while heart macrophages showed a much lower chimerism of 2.7±0.5% (p<0.01). Macrophages self renewed locally through proliferation: 2.1±0.3% incorporated BrdU 2 hours after a single injection and 13.7±1.4% heart macrophages stained positive for the cell cycle marker Ki67. The cells likely participate in defense against infection, as we found them to ingest fluorescently labeled bacteria. In ischemic myocardium, we observed that tissue resident macrophages died locally while some also migrated to hematopoietic organs. If the steady-state was perturbed by coronary ligation or diphtheria toxin-induced macrophage depletion in CD11bDTR/+ mice, blood monocytes replenished heart macrophages. However, in the chronic phase after myocardial infarction, macrophages residing in the infarct were again independent from the blood monocyte pool, returning to the steady-state situation.
Conclusions
In this study we show differential contribution of monocytes to heart macrophages during steady-state, after macrophage depletion or in the acute and chronic phase after myocardial infarction. We found that macrophages participate in the immunosurveillance of myocardial tissue. These data correspond with previous studies on tissue-resident macrophages and raise important questions on the fate and function of macrophages during the development of heart failure.
doi:10.1161/CIRCRESAHA.115.303567
PMCID: PMC4082439  PMID: 24786973
Macrophage; monocyte; heart; myocardial infarction; myocardial
4.  Magnetic Ligation Method for Quantitative Detection of MicroRNAs 
Advanced healthcare materials  2014;3(7):1015-1019.
A magnetic ligation method is utilized for the detection of microRNAs amongst a complex biological background without polymerase chain reaction or nucleotide modification. The sandwich probes assay can be adapted to analyze a panel of microRNAs associated with cardiovascular diseases in heart tissue samples.
doi:10.1002/adhm.201300672
PMCID: PMC4107040  PMID: 24532323
microRNA; enzymatic ligation; magnetic labeling; diagnostics; magnetic resonance
5.  Efficient Acid-catalyzed 18F/19F Fluoride Exchange of BODIPY Dyes 
ChemMedChem  2014;9(7):1368-1373.
Fluorine containing fluorochromes represent important validation agents for PET imaging agents as they can be easily rapidly validated in cells by fluorescence imaging. In particular, the 18F-labeled BODIPY-FL fluorophore has emerged as an important platform but little is known about alternative 18F-labeling strategies or labeling on red shifted fluorophores. Here we explore the acid-catalyzed 18F/19F exchange on a range of commercially available N-hydroxysuccinimidyl ester and maleimide BODIPY fluorophores. We show this method to be a simple and efficient 18F-labeling strategy for a diverse span of fluorescent compounds, including a BODIPY modified PARP-1 inhibitor, and amine- and thiol-reactive BODIPY fluorophores.
doi:10.1002/cmdc.201300506
PMCID: PMC4145401  PMID: 24596307
18F-Fluorine; Molecular Imaging; BODIPY; Fluorescence; Positron emission tomography (PET)
6.  Engineered Trehalose Permeable to Mammalian Cells 
PLoS ONE  2015;10(6):e0130323.
Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.
doi:10.1371/journal.pone.0130323
PMCID: PMC4482662  PMID: 26115179
7.  Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters 
Nature Communications  2015;6:7029.
Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA.
Extracellular vesicles (EVs) act as a conduit for intercellular communication through the exchange of cellular materials without direct cell-to-cell contacts. Here the authors develop a multiplexed reporter system that allows monitoring of EV exchange, cargo delivery and protein translation between different cell populations.
doi:10.1038/ncomms8029
PMCID: PMC4435621  PMID: 25967391
8.  Resolving Cancer-Stroma Interfacial Signaling and Interventions with Micropatterned Tumor-Stromal Assays 
Nature communications  2014;5:5662.
Tumor-stromal interactions are a determining factor in cancer progression. In vivo, the interaction interface is associated with spatially-resolved distributions of cancer and stromal phenotypes. Here, we establish a micropatterned tumor-stromal assay (μTSA) with laser capture microdissection to control the location of co-cultured cells and analyze bulk and interfacial tumor-stromal signaling in driving cancer progression. μTSA reveals a spatial distribution of phenotypes in concordance with human estrogen receptor-positive (ER+) breast cancer samples, and heterogeneous drug activity relative to the tumor-stroma interface. Specifically, an unknown mechanism of reversine is shown in targeting tumor-stromal interfacial interactions using ER+ MCF-7 breast cancer and bone marrow-derived stromal cells. Reversine suppresses MCF-7 tumor growth and bone metastasis in vivo by reducing tumor stromalization including collagen deposition and recruitment of activated stromal cells. This study advocates μTSA as a platform for studying tumor microenvironmental interactions and cancer field effects with applications in drug discovery and development.
doi:10.1038/ncomms6662
PMCID: PMC4261930  PMID: 25489927
9.  Platinum compounds for high-resolution in vivo cancer imaging 
ChemMedChem  2014;9(6):1131-1135.
Platinum(II) compounds, principally cisplatin and carboplatin, are commonly used front-line cancer therapeutics. Despite widespread use and the interest of developing new derivatives, including nanoformulations with improved properties, it has been difficult to visualize Pt compounds in live subjects, in real-time, and with subcellular resolution. Here we present four novel cisplatin- and carboplatin-derived fluorescent imaging compounds for quantitative intravital cancer imaging. We conjugate boron dipyromethene FL (BODIPY-FL) to PtII complexes for robust in vivo fluorescence, and show retained DNA-damaging and cytotoxic properties. We image pharmacokinetics and tumor uptake in a xenograft cancer mouse model. Finally, we present a genetic reporter of single-cell DNA damage for in vivo imaging, and simultaneously monitor Pt drug accumulation and resultant DNA damage in individual tumor cells, at subcellular resolution, and in real-time in a live animal model of cancer.
doi:10.1002/cmdc.201300502
PMCID: PMC4037345  PMID: 24504646
intravital imagingagents; pharmacology; fluorescent probes; DNA damage; pharmacokinetics
10.  Single cell resolution in vivo imaging of DNA damage following PARP inhibition 
Scientific Reports  2015;5:10129.
Targeting DNA repair pathways is a powerful strategy to treat cancers. To gauge efficacy in vivo, typical response markers include late stage effects such as tumor shrinkage, progression free survival, or invasive repeat biopsies. These approaches are often difficult to answer critical questions such as how a given drug affects single cell populations as a function of dose and time, distance from microvessels or how drug concentration (pharmacokinetics) correlates with DNA damage (pharmacodynamics). Here, we established a single-cell in vivo pharmacodynamic imaging read-out based on a truncated 53BP1 double-strand break reporter to determine whether or not poly(ADP-ribose) polymerase (PARP) inhibitor treatment leads to accumulation of DNA damage. Using this reporter, we show that not all PARP inhibitor treated tumors incur an increase in DNA damage. The method provides a framework for single cell analysis of cancer therapeutics in vivo.
doi:10.1038/srep10129
PMCID: PMC4434991  PMID: 25984718
11.  Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma 
Nature Communications  2015;6:6999.
Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O6-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.
Predicting and monitoring chemotherapy response remains a challenge for glioma treatment. Here the authors show that a microfluidic device can isolate glioma-derived exosomes from patient blood and accurately determine the levels of mRNA of key enzymes important for chemoresponsiveness.
doi:10.1038/ncomms7999
PMCID: PMC4430127  PMID: 25959588
12.  New techniques for motion-artifact-free in vivo cardiac microscopy 
Intravital imaging microscopy (i.e., imaging in live animals at microscopic resolution) has become an indispensable tool for studying the cellular micro-dynamics in cancer, immunology and neurobiology. High spatial and temporal resolution, combined with large penetration depth and multi-reporter visualization capability make fluorescence intravital microscopy compelling for heart imaging. However, tissue motion caused by cardiac contraction and respiration critically limits its use. As a result, in vitro cell preparations or non-contracting explanted heart models are more commonly employed. Unfortunately, these approaches fall short of understanding the more complex host physiology that may be dynamic and occur over longer periods of time. In this review, we report on novel technologies, which have been recently developed by our group and others, aimed at overcoming motion-induced artifacts and capable of providing in vivo subcellular resolution imaging in the beating mouse heart. The methods are based on mechanical stabilization, image processing algorithms, gated/triggered acquisition schemes or a combination of both. We expect that in the immediate future all these methodologies will have considerable applications in expanding our understanding of the cardiac biology, elucidating cardiomyocyte function and interactions within the organism in vivo, and ultimately improving the treatment of cardiac diseases.
doi:10.3389/fphys.2015.00147
PMCID: PMC4428079  PMID: 26029116
cardiac imaging; motion artifacts; laser scanning microscopy; motion compensation; image stabilization
13.  Ly-6Chigh Monocytes Depend on Nr4a1 to Balance both Inflammatory and Reparative Phases in the Infarcted Myocardium 
Circulation research  2014;114(10):1611-1622.
Rationale
Healing after myocardial infarction (MI) involves the biphasic accumulation of inflammatory Ly-6Chigh and reparative Ly-6Clow monocytes/macrophages (Mo/MΦ). According to one model, Mo/MΦ heterogeneity in the heart originates in the blood and involves the sequential recruitment of distinct monocyte subsets that differentiate to distinct macrophages. Alternatively, heterogeneity may arise in tissue from one circulating subset via local macrophage differentiation and polarization. The orphan nuclear hormone receptor, Nr4a1, is essential to Ly-6Clow monocyte production but dispensable to Ly-6Clow macrophage differentiation; dependence on Nr4a1 can thus discriminate between systemic and local origins of macrophage heterogeneity.
Objective
This study tested the role of Nr4a1 in MI in the context of the two Mo/MΦ accumulation scenarios.
Methods and Results
We show that Ly-6Chigh monocytes infiltrate the infarcted myocardium and, unlike Ly-6Clow monocytes, differentiate to cardiac macrophages. In the early, inflammatory phase of acute myocardial ischemic injury, Ly-6Chigh monocytes accrue in response to a brief Ccl2 burst. In the second, reparative phase, accumulated Ly-6Chigh monocytes give rise to reparative Ly-6Clow F4/80high macrophages that proliferate locally. In the absence of Nr4a1, Ly-6Chigh monocytes express heightened levels of Ccr2 on their surface, avidly infiltrate the myocardium, and differentiate to abnormally inflammatory macrophages, which results in defective healing and compromised heart function.
Conclusions
Ly-6Chigh monocytes orchestrate both inflammatory and reparative phases during MI and depend on Nr4a1 to limit their influx and inflammatory cytokine expression.
doi:10.1161/CIRCRESAHA.114.303204
PMCID: PMC4017349  PMID: 24625784
Monocyte; macrophage; myocardial infarction; nuclear hormone receptor; healing
14.  In Vivo Imaging of Multidrug Resistance Using a Third Generation MDR1 Inhibitor 
Bioconjugate Chemistry  2014;25(6):1137-1142.
Cellular up-regulation of multidrug resistance protein 1 (MDR1) is a common cause for resistance to chemotherapy; development of third generation MDR1 inhibitors—several of which contain a common 6,7-dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline substructure—is underway. Efficacy of these agents has been difficult to ascertain, partly due to a lack of pharmacokinetic reporters for quantifying inhibitor localization and transport dynamics. Some of the recent third generation inhibitors have a pendant heterocycle, for example, a chromone moiety, which we hypothesized could be converted to a fluorophore. Following synthesis and teasing of a small set of analogues, we identified one lead compound that can be used as a cellular imaging agent that exhibits structural similarity and behavior akin to the latest generation of MDR1 inhibitors.
doi:10.1021/bc500154c
PMCID: PMC4098115  PMID: 24806886
15.  Red Si-rhodamine drug conjugates enable imaging in GFP cells 
Here we evaluated a series of Si-derivatized rhodamine (SiR) dyes for their ability to visualize a model drug in live cells. We show that a charge neutral SiR derivative (but not others) can indeed be used to follow the intracellular location of the model therapeutic drug in GFP cells
doi:10.1039/c4cc00144c
PMCID: PMC4028821  PMID: 24663433
16.  In vivo silencing of the transcription factor IRF5 reprograms the macrophage phenotype and improves infarct healing 
Objectives
The aim of the study was to test wether silencing of the transcription factor Interferon Regulatory Factor 5 (IRF5) in cardiac macrophages improves infarct healing and attenuates post-MI remodeling.
Background
In healing wounds, M1➛M2 macrophage phenotype transition supports resolution of inflammation and tissue repair. Persistence of inflammatory M1 macrophages may derail healing and compromise organ functions. The transcription factor IRF5 promotes genes associated with M1 macrophages.
Methods
Here we used nanoparticle-delivered siRNA to silence the transcription factor IRF5 in macrophages residing in myocardial infarcts (MI) and in surgically induced skin wounds in mice.
Results
Infarct macrophages expressed high levels of IRF5 during the early inflammatory wound healing stages (day 4 after coronary ligation) whereas expression of the transcription factor decreased during the resolution of inflammation (day 8). Following in vitro screening, we identified an siRNA sequence that, when delivered by nanoparticles to wound macrophages, efficiently suppressed expression of IRF5 in vivo. Reduction of IRF5 expression, a factor that regulates macrophage polarization, reduced inflammatory M1 macrophage markers, supported resolution of inflammation, accelerated cutaneous and infarct healing and attenuated development of post-MI heart failure after coronary ligation as measured by protease targeted FMT-CT imaging and cardiac MRI (p<0.05 respectively).
Conclusion
This work identifies a new therapeutic avenue to augment resolution of inflammation in healing infarcts by macrophage phenotype manipulation. This therapeutic concept may be used to attenuate post-MI remodeling and heart failure.
doi:10.1016/j.jacc.2013.11.023
PMCID: PMC3992176  PMID: 24361318
17.  Innate Response Activator B Cells Aggravate Atherosclerosis by Stimulating TH1 Adaptive Immunity 
Circulation  2014;129(16):1677-1687.
Background
Atherosclerotic lesions grow via the accumulation of leukocytes and oxidized lipoproteins in the vessel wall. Leukocytes can attenuate or augment atherosclerosis through the release of cytokines, chemokines, and other mediators. Deciphering how leukocytes develop, oppose and complement each other’s function, and shape the course of disease, can illuminate understanding of atherosclerosis. Innate response activator (IRA) B cells are a recently described population of GM-CSF-secreting cells of hitherto unknown function in atherosclerosis.
Methods and Results
Here we show that IRA B cells arise during atherosclerosis in mice and humans. In response to high cholesterol diet, IRA B cell numbers increase preferentially in secondary lymphoid organs via Myd88-dependent signaling. Mixed chimeric mice lacking B cell-derived GM-CSF develop smaller lesions with fewer macrophages and effector T cells. Mechanistically, IRA B cells promote the expansion of classical dendritic cells, which then generate IFNγ-producing TH1 cells. This IRA B cell-dependent TH1 skewing manifests in an IgG1 to IgG2c isotype switch in the immunoglobulin response against oxidized lipoproteins.
Conclusions
GM-CSF-producing IRA B cells alter adaptive immune processes and shift the leukocyte response toward a TH1-associated mileu that aggravates atherosclerosis.
doi:10.1161/CIRCULATIONAHA.113.006381
PMCID: PMC3997655  PMID: 24488984
atherosclerosis; immunology; B cells; Dendritic cells; T cells; Granulocyte macrophage colony-stimulating factor
18.  Molecular characterization of scant lung tumor cells using iron-oxide nanoparticles and micro-nuclear magnetic resonance 
Advances in nanotechnology and microfluidics are enabling the analysis of small amounts of human cells. We tested whether recently developed micro-nuclear magnetic resonance (μNMR) technology could be leveraged for diagnosing pulmonary malignancy using fine needle aspirate (FNA) of primary lesions and/or peripheral blood samples. We enrolled a cohort of 35 patients referred for CT biopsy of primary pulmonary nodules, liver or adrenal masses and concurrently obtained FNA and peripheral blood samples. FNA sampling yielded sufficient material for μNMR analysis in 91% of cases and had a sensitivity and specificity of 91.6 and 100% respectively. Interestingly, among blood samples with positive circulating tumor cells (CTC), μNMR analysis of each patient's peripheral blood led to similar diagnosis (malignant vs benign) and differential diagnosis (lung malignancy subtype) in 100% and 90% (18/20) of samples, respectively. μNMR appears to be valuable, non-invasive adjunct in the diagnosis of lung cancer.
doi:10.1016/j.nano.2013.10.008
PMCID: PMC3966960  PMID: 24200523
Bioorthogonal click chemistry; Circulating Tumor Cell; μNMR; Iron-oxide nanoparticles; Point of care diagnosis
19.  Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor 
Nature biotechnology  2014;32(5):490-495.
Exosomes show potential for cancer diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires extensive sample purification and labeling. Here we describe a label-free, high-throughput approach for quantitative analyses of exosomes. Our nano-plasmonic exosome (nPLEX) assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. We show that this approach offers improved sensitivity over previous methods, enables portable operation when integrated with miniaturized optics and allows retrieval of exosomes for further study. Using nPLEX to analyze ascites samples from ovarian cancer patients, we find that exosomes derived from ovarian cancer cells can be identified by their expression of CD24 and EpCAM, suggesting the potential of exosomes for diagnostics.
doi:10.1038/nbt.2886
PMCID: PMC4356947  PMID: 24752081
exosomes; surface plasmon resonance; periodic nanohole arrays; cancer; label-free sensing
20.  Steady state anisotropy two-photon microscopy resolves multiple, spectrally similar fluorophores, enabling in vivo multilabel imaging 
Optics letters  2014;39(15):4482-4485.
The use of spectrally distinguishable fluorescent dyes enables imaging of multiple targets. However, in two-photon microscopy the number of fluorescent labels with distinct emission spectra that can be effectively excited and resolved is constrained by the confined tuning range of the excitation laser and the broad and overlapping nature of fluorophore two-photon absorption spectra. This limitation effectively reduces the number of available imaging channels. Here, we demonstrate that two-photon steady state anisotropy imaging (2PSSA) offers the capability to resolve otherwise unresolvable fluorescent tracers both in live cells and in mouse tumor models. This approach expands the number of biological targets that can be imaged simultaneously, increasing the total amount of information that can be obtained through imaging.
PMCID: PMC4341989  PMID: 25078208
21.  μHall Chip for Sensitive Detection of Bacteria 
Advanced healthcare materials  2013;2(9):1224-1228.
Sensitive, rapid and phenotype-specific enumeration of pathogens is essential for the diagnosis of infectious disease, monitoring of food chains, and for defense against bioterrorism. Microbiological culture and genotyping, techniques that sensitively and selectively detect bacteria in laboratory settings, have limited application in clinical environments due to high cost, slow response times, and the need for specially trained staff and laboratory infrastructure. To address these challenges, we developed a microfluidic chip-based micro-Hall (μHall) platform capable of measuring single, magnetically tagged bacteria directly in clinical specimens with minimal sample processing. We demonstrated the clinical utility of the μHall chip by enumerating Gram-positive bacteria using a two-step bioorthogonal labeling procedure. The overall detection limit of the system was similar to that of culture tests (~10 bacteria), but the assay time was 50-times faster. This low-cost, single-cell analytical technique is especially well-suited to diagnose infectious diseases in resource-limited clinical settings.
doi:10.1002/adhm.201200380
PMCID: PMC4340655  PMID: 23495188
μHall; bacterial detection; magnetic nanoparticles
22.  Comparison of select cancer biomarkers in human circulating and bulk tumor cells using magnetic nanoparticles and miniaturized micro-NMR system 
Circulating tumor cells (CTC) harvested from peripheral blood have received significant interest as sources for serial sampling to gauge treatment efficacy. Nanotechnology and microfluidic based approaches are emerging to facilitate such analyses. While of considerable clinical importance, there is little information on how similar or different CTC are from their shedding bulk tumors. In this clinical study, paired tumor fine needle aspirate and peripheral blood samples were obtained from cancer patients during image guided biopsy. Using targeted magnetic nanoparticles and a point-of-care micro-NMR system, we compared selected biomarkers (EpCAM, EGFR, HER-2 and vimentin) in both CTC and fine needle biopsies of solid epithelial cancers. We show a weak correlation between each paired sample, suggesting that use of CTC as ‘liquid biopsies’ and proxies to metastatic solid lesions could be misleading.
doi:10.1016/j.nano.2013.03.011
PMCID: PMC4330975  PMID: 23570873
Circulating tumor cells; magnetic nanoparticles; micro-NMR (µNMR); point of care diagnostics
23.  Statins Improve the Resolution of Established Murine Venous Thrombosis: Reductions in Thrombus Burden and Vein Wall Scarring 
PLoS ONE  2015;10(2):e0116621.
Despite anticoagulation therapy, up to one-half of patients with deep vein thrombosis (DVT) will develop the post-thrombotic syndrome (PTS). Improving the long-term outcome of DVT patients at risk for PTS will therefore require new approaches. Here we investigate the effects of statins—lipid-lowering agents with anti-thrombotic and anti-inflammatory properties—in decreasing thrombus burden and decreasing vein wall injury, mediators of PTS, in established murine stasis and non-stasis chemical-induced venous thrombosis (N = 282 mice). Treatment of mice with daily atorvastatin or rosuvastatin significantly reduced stasis venous thrombus burden by 25% without affecting lipid levels, blood coagulation parameters, or blood cell counts. Statin-driven reductions in VT burden (thrombus mass for stasis thrombi, intravital microscopy thrombus area for non-stasis thrombi) compared similarly to the therapeutic anticoagulant effects of low molecular weight heparin. Blood from statin-treated mice showed significant reductions in platelet aggregation and clot stability. Statins additionally reduced thrombus plasminogen activator inhibitor-1 (PAI-1), tissue factor, neutrophils, myeloperoxidase, neutrophil extracellular traps (NETs), and macrophages, and these effects were most notable in the earlier timepoints after DVT formation. In addition, statins reduced DVT-induced vein wall scarring by 50% durably up to day 21 in stasis VT, as shown by polarized light microscopy of picrosirius red-stained vein wall collagen. The overall results demonstrate that statins improve VT resolution via profibrinolytic, anticoagulant, antiplatelet, and anti-vein wall scarring effects. Statins may therefore offer a new pharmacotherapeutic approach to improve DVT resolution and to reduce the post-thrombotic syndrome, particularly in subjects who are ineligible for anticoagulation therapy.
doi:10.1371/journal.pone.0116621
PMCID: PMC4334538  PMID: 25680183
24.  Endoscopic fluorescence imaging of cathepsin activity can discriminate, dysplasia from colitis 
Inflammatory bowel diseases  2013;19(7):1339-1345.
BACKGROUND
Surveillance colonoscopy using random biopsies to detect colitis-associated cancer (CAC) suffers from poor sensitivity. Although chromo-endoscopy improves detection, acceptance in the community has been slow. Here, we examine the usefulness of near infrared fluorescence (NIRF) endoscopy to image molecular probes for cathepsin activity in colitis-induced dysplasia.
METHODS
In patient samples, cathepsin activity was correlated with colitis and dysplasia. In mice, cathepsin activity was detected as fluorescent hydrolysis product of substrate based probes (SBPs) after injection into IL-10−/− colitic mice. Fluorescence colonoscopy and colonic whole mount imaging were performed prior to complete sectioning and pathology review of resected colons.
RESULTS
Cathepsin activity was 5 and 8 – fold higher in dysplasia and CAC respectively, compared to areas of mild colitis in patient tissue sections. The signal to noise ratios for dysplastic lesions seen by endoscopy in IL-10−/− mice were 5.2 ±1.3, (P=0.0001). Lesions with increased NIRF emissions were classified as raised or flat dysplasia, lymphoid tissue and ulcers. Using images collected by endoscopy, a receiver operating characteristic curve (ROC) for correctly diagnosing dysplasia was calculated. The area under the curve was 0.927. At a cut off of 1000 MFI, the sensitivity and specificity for detecting dysplasia were 100% and 83% respectively. Analysis revealed that enhanced NIRF emissions derived from increased numbers of infiltrating myeloid derived suppressor cells and macrophages with equivalent cathepsin activity.
CONCLUSIONS
These studies indicate that cathepsin SBP imaging correctly identifies dysplastic foci within chronically inflamed colons. Cathepsin-based NIRF endoscopy presents unique advantages that may increase sensitivity and specificity of surveillance colonoscopy in patients with CUC.
doi:10.1097/MIB.0b013e318281f3f8
PMCID: PMC4306589  PMID: 23591598
Dysplasia; colitis; macrophages; cathepsin activity; fluorescence endoscopy
25.  Fluorescent Exendin-4 Derivatives for Pancreatic β-Cell Analysis 
Bioconjugate chemistry  2013;25(1):171-177.
The ability to reliably identify pancreatic β-cells would have far reaching implications for a greater understanding of β-cell biology, measurement of β-cell mass in diabetes, islet transplantation, and drug development. The glucagon-like peptide-1 receptor (GLP1R) is highly expressed on the surface of insulin producing pancreatic β-cells. Using systematic modifications of the GLP1R ligand, exendin-4, we screened over 25 compounds and identified a palette of fluorescent exendin-4 with high GLP1R binding affinity. We show considerable differences in affinity, as well as utility of the top candidates for flow cytometry and microscopy of β-cells. Some of the developed compounds should be particularly useful for basic and translational β-cell research.
doi:10.1021/bc4005014
PMCID: PMC4016126  PMID: 24328216
pancreatic beta cells; GLP1R; fluorochromes; flow cytometry

Results 1-25 (292)