Orthogonally-tunable and ER-targeting fluorophores detect avian influenza virus early infection.

Cell-based assays can monitor virus infection at a single-cell level with high sensitivity and cost-efficiency. For this purpose, it is crucial to develop molecular probes that respond selectively to physiological changes in live cells. We report stimuli-responsive light-emitters built on a T-shaped benzimidazole platform, and consecutive borylation reactions to produce a library of homologs displaying systematic changes in fluorescence quantum yield and environmental sensitivity. We find that certain fluorophores localize selectively at the endoplasmic reticulum, and interact with proteins involved in the stress signaling pathways. Notably, the mono-borylated compound responds selectively to the stress conditions by enhancing fluorescence, and detects avian influenza virus infection at the single-cell level. Our findings demonstrate the unprecedented practical utility of the stress-responsive molecular probes to differentiate cellular states for early diagnosis.

A Near-Infrared Probe Tracks and Treats Lung Tumor Initiating Cells by Targeting HMOX2.

Tumor initiating cells (TIC) are resistant to conventional anticancer therapy and associated with metastasis and relapse in cancer. Although various TIC markers and their antibodies have been proposed, it is limited to the use of antibodies for in vivo imaging or treatment of TIC. In this study, we discovered heme oxygenase 2 (HMOX2) as a novel biomarker for TIC and developed a selective small molecule probe TiNIR (tumor initiating cell probe with near infrared). TiNIR detects and enriches the functionally active TIC in human lung tumors, and through the photoacoustic property, TiNIR also visualizes lung TIC in the patient-derived xenograft (PDX) model. Furthermore, we demonstrate that TiNIR inhibits tumor growth by blocking the function of HMOX2, resulting in significantly increased survival rates of the cancer model mice. The novel therapeutic target HMOX2 and its fluorescent ligand TiNIR will open a new path for the molecular level of lung TIC diagnosis and treatment.

Differentiation of selectively labeled peptides using solid-state nanopores.

Determination of the amino acid sequence of a protein is critical for understanding various biological processes. Mass spectrometry has mainly been used for protein identification; however, there are limitations to its sensitivity when detecting low abundance proteins. In this study, we attempted to distinguish between three similar peptide sequences (∼40 amino acids, ∼5 kDa) that differed only by the location or number of cysteine residues with solid-state nanopores. The cysteine residues are located at one end, one at the center, and at both ends for each of the three peptides. We found that differentiation of the three types of peptides by nanopore signals was difficult. However, when the cysteine residue was labeled with a negatively charged molecule, Flamma® 496, the labeled peptides showed distinct signals for each peptide. Comparing the relative current blockades of labeled peptides with applied voltages, we found that the label was able to change peptide conformations and the resulting ionic current signals from the three labeled peptides were distinguished based on the relative current blockade, full width at half-maximum of the current blockade distribution, and single-molecule level peak shape analysis. Our results suggest that solid-state nanopores combined with a targeted labeling strategy could be used to obtain characteristic peptide signatures that could ultimately be used for protein identification.

Reinvestigation of an O-Salicylaldehyde Ester Functional Group in Aqueous Buffer and Discovery of a Coumarin Scaffold Probe for Selective N-Terminal Cysteine Labeling.

Many intracellular proteins are metabolically unstable, and their half-life was known to be controlled by the "N-end rule," that is, the N-terminal residue controlled protein stability. To visualize or measure the cellular stability of a protein, depending on the N-terminal residues, attention is being paid to the development of selective labeling methods for individual N-terminal amino acids. However, there are only a limited number of functional groups available for specific N-terminal amino acid labeling in a biological environment. Herein, we report a re-examination of salicylaldehyde ester for selective N-terminal residue tagging. Salicylaldehyde ester has been used for chemical ligation to N-terminal serine or threonine under pyridine/acetic acid conditions. Inspired by previous selective serine/threonine labeling, N-terminal labeling of salicylaldehyde ester in aqueous buffer has been examined by using boron-dipyrromethene (BODIPY), rhodamine, and coumarin probes. Surprisingly, the selectivity not only significantly differed, depending on the fluorophore incorporated in salicylaldehyde, but was also perturbed by the addition of a small fraction of phosphate-buffered saline. In particular, the coumarin-based salicylaldehyde ester probe showed notable selectivity against N-terminal cysteine under aqueous buffer conditions. This result reveals the serendipitous discovery of a new N-terminal cysteine labeling strategy.

Discrimination of Avian Influenza Virus Subtypes using Host-Cell Infection Fingerprinting by a Sulfinate-based Fluorescence Superoxide Probe.

The current gold-standard diagnosis method for avian influenza (AI) is an embryonic egg-based hemagglutination assay followed by immunoblotting or PCR sequencing to confirm subtypes. It requires, however, specialized facilities to handle egg inoculation and incubation, and the subtyping methods relied on costly reagents. Now, the first differential sensing approach to distinguish AI subtypes is demonstrated using series of cell lines and a fluorescent sensor. Susceptibility of AI virus differs depending on genetic backgrounds of host cells. Cells were examined from different organ origins, and the infection patterns against a panel of cells were utilized for AI virus subtyping. To quantify AI infection, a highly cell-permeable fluorescent superoxide sensor was designed to visualize infection. This differential sensing strategy successfully proved discriminations of AI subtypes and demonstrated as a useful primary screening platform to monitor a large number of samples.

Chloro-Functionalized Photo-crosslinking BODIPY for Glutathione Sensing and Subcellular Trafficking.

Glutathione (GSH) is one of major antioxidants inside cells that regulates oxidoreduction homeostasis. Recently, there have been extensive efforts to visualize GSH in live cells, but most of the probes available today are simple detection sensors and do not provide details of cellular localization. A new fluorescent probe (pcBD2-Cl), which is cell permeable and selectively reacts with GSH in situ, has been developed. The in situ GSH-labeled probe (pcBD2-GSH) exhibited quenches fluorescence, but subsequent binding to cellular abundant glutathione S-transferase (GST) recovers the fluorescence intensity, which makes it possible to image the GSH-GST complex in live cells. Interactions between probe and GST were confirmed by means of photo-crosslinking under intact live-cell conditions. Interestingly, isomers of chloro-functionalized 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) compounds behaved very distinctively inside the cells. Following co-staining imaging with MitoTracker and mitochondria fractionation upon lipopolysaccharide-mediated reactive oxygen species induction experiments showed that pcBD2-GSH accumulated in mitochondria. This is the first example of a live-cell imaging probe to visualize translocation of GSH from the cytosol to mitochondria.

Bulk Aggregation Based Fluorescence Turn-On Sensors for Selective Detection of Progesterone in Aqueous Solution.

Steroids are polycyclic compounds that share tetracyclic ring as core scaffold, and selective detection of a steroid is challenging owing to their structural similarities. The discovery of chemosensors that recognize progesterone by alteration of self-aggregation state is described, and these show significant fluorescence turn-on. A self-aggregated 48-membered dansyl library was screened against a series of metabolites in aqueous buffer and discovered two compounds (PG-1, PG-2) exhibited exceptional selectivity for progesterone. Following studies of aggregation properties of probes using dynamic light scattering and transmission electron microscopy supports progesterone recognition lead to the generation of bulk aggregates that induce fluorescence enhancement. Though many fluorescence sensing mechanisms have been proposed, a sensing mode based on the bulk aggregate formation of fluorophore has never been reported, and this may open a new avenue of chemosensor design.

Cancer-Microenvironment-Sensitive Activatable Quantum Dot Probe in the Second Near-Infrared Window.

Recent technological advances have expanded fluorescence (FL) imaging into the second near-infrared region (NIR-II; wavelength = 1000-1700 nm), providing high spatial resolution through deep tissues. However, bright and compact fluorophores are rare in this region, and sophisticated control over NIR-II probes has not been fully achieved yet. Herein, we report an enzyme-activatable NIR-II probe that exhibits FL upon matrix metalloprotease activity in tumor microenvironment. Bright and stable PbS/CdS/ZnS core/shell/shell quantum dots (QDs) were synthesized as a model NIR-II fluorophore, and activatable modulators were attached to exploit photoexcited electron transfer (PET) quenching. The quasi type-II QD band alignment allowed rapid and effective FL modulations with the compact surface ligand modulator that contains methylene blue PET quencher. The modulator was optimized to afford full enzyme accessibility and high activation signal surge upon the enzyme activity. Using a colon cancer mouse model, the probe demonstrated selective FL activation at tumor sites with 3-fold signal enhancement in 10 min. Optical phantom experiments confirmed the advantages of the NIR-II probe over conventional dyes in the first near-infrared region.

Combinatorial Dansyl Library and its Applications to pH-Responsive Probes.

Herein, we report the first 48-membered, dansyl-based, combinatorial fluorescent library. From the electronic and structural properties of the probes, we analyzed their optical properties and chemical yields, with an average of 49 %. The molecules were examined for their pH responses, and DS-2 and DS-45 showed blue-shifts, whereas DS-7 and DS-40 showed red-shifts in wavelength with increasing pH. Finally, cell permeability was investigated by treating SNU-2292 cells. Our results demonstrate the potential application of this library in biosensors, bio-imaging and pH indicators.

Photo-affinity labeling (PAL) in chemical proteomics: a handy tool to investigate protein-protein interactions (PPIs).

Protein-protein interactions (PPIs) trigger a wide range of biological signaling pathways that are crucial for biomedical research and drug discovery. Various techniques have been used to study specific proteins, including affinity chromatography, activity-based probes, affinity-based probes and photo-affinity labeling (PAL). PAL has become one of the most powerful strategies to study PPIs. Traditional photocrosslinkers are used in PAL, including benzophenone, aryl azide, and diazirine. Upon photoirradiation, these photocrosslinkers (Pls) generate highly reactive species that react with adjacent molecules, resulting in a direct covalent modification. This review introduces recent examples of chemical proteomics study using PAL for PPIs.

Magnetic-assembly mechanism of superparamagneto-plasmonic nanoparticles on a charged surface.

One-dimensional magnetoplasmonic nanochains (MPNCs) were self-assembled using Au-coated Fe3O4 core-shell superparamagnetic nanoparticles (Fe3O4@Au NPs) by applying an external static magnetic field. The assembly mechanism of the Fe3O4@Au NPs was investigated thoroughly, revealing that substrate-particle interactions, van der Waals forces, and magnetic forces play important roles in the formation and control of the MPNCs. Magnetic force microscopy (MFM) and vibrating sample magnetometry (VSM) were used to study the magnetic properties of the MPNCs, which were compared with those of Fe3O4 nanochains.

Rational design of a photo-crosslinking BODIPY for in situ protein labeling.

Photo-crosslinking agents have emerged as critical tools to investigate protein-protein interactions in complex proteomes, but there are few photocrosslinkers available at the moment. Here, we report the first rational design of a photo-crosslinking BODIPY fluorophore (pcBD) and its biological application for biomolecule labeling. As a photosensitizing functional motif, an aryl ketone group was incorporated into the BODIPY fluorophore, and a series of proteins were labeled by pcBD compounds upon UV irradiation. In order to investigate protein-protein interactions in a protein mixture, amino-functionalized pcBD was prepared and covalently attached to a ubiquitin ligase binding peptide. Upon UV irradiation, we could successfully visualize the substrates in the total lysate. These results provided a proof of concept for spatially controllable tagging via photo-activation of the pcBD scaffold, and demonstrated its potential usage for in situ labeling applications.

Lower urinary tract symptoms and erectile dysfunction in men with type 2 diabetes mellitus.

PURPOSE: To assess the prevalence of lower urinary tract symptoms (LUTS) and erectile dysfunction (ED) and the relationships between LUTS, ED, depression, and other factors in Korean men with type 2 diabetes mellitus (T2DM). METHODS: This cross-sectional study included 124 male patients with T2DM who attended a university hospital diabetes clinic between October 2010 and April 2012. Data were collected using structured interviews and chart reviews. LUTS were measured using the International Prostate Symptom Score (IPSS), ED using the five-item Korean version of the International Index of Erectile Function (IIEF), depression using the Center for Epidemiologic Studies Depression Scale, and glycosylated hemoglobin level from clinical data. RESULTS: The IPSS score was 9.2±6.6. The total IPSS scores indicated that 53.3% of the subjects had either moderate or severe symptoms. The mean IIEF score was 7.3±8.6, indicating the severity of ED to be mild, mild to moderate, moderate, and severe in 10.5%, 9.7%, 1.6%, and 66.9% of the participants, respectively. LUTS showed a significant negative correlation with ED (r=-0.26, P=0.003) and a significant positive correlation with depression (r=0.33, P<0.001). ED was negatively correlated with age (r=-0.44, P<0.001), duration of diabetes (r=-0.26, P=0.004), and depression (r=-0.24, P=0.008). CONCLUSIONS: LUTS and ED were found to have a high prevalence among Korean men with T2DM. More severe ED was associated with worse LUTS, whereas more severe depressive symptoms were found to be associated with more severe ED and LUTS.

Difference between Toxicities of Iron Oxide Magnetic Nanoparticles with Various Surface-Functional Groups against Human Normal Fibroblasts and Fibrosarcoma Cells.

Recently, many nanomedical studies have been focused on magnetic nanoparticles (MNPs) because MNPs possess attractive properties for potential uses in imaging, drug delivery, and theranostics. MNPs must have optimized size as well as functionalized surface for such applications. However, careful cytotoxicity and genotoxicity assessments to ensure the biocompatibility and biosafety of MNPs are essential. In this study, Fe3O4 MNPs of different sizes (approximately 10 and 100-150 nm) were prepared with different functional groups, hydroxyl (-OH) and amine (-NH2) groups, by coating their surfaces with tetraethyl orthosilicate (TEOS), 3-aminopropyltrimethoxysilane (APTMS) or TEOS/APTMS. Differential cellular responses to those surface-functionalized MNPs were investigated in normal fibroblasts vs. fibrosarcoma cells. Following the characterization of MNP properties according to size, surface charge and functional groups, cellular responses to MNPs in normal fibroblasts and fibrosarcoma cells were determined by quantifying metabolic activity, membrane integrity, and DNA stability. While all MNPs induced just about 5% or less cytotoxicity and genotoxicity in fibrosarcoma cells at lower than 500 µg/mL, APTMS-coated MNPs resulted in greater than 10% toxicity against normal cells. Particularly, the genotoxicity of MNPs was dependent on their dose, size and surface charge, showing that positively charged (APTMS- or TEOS/APTMS-coated) MNPs induced appreciable DNA aberrations irrespective of cell type. Resultantly, smaller and positively charged (APTMS-coated) MNPs led to more severe toxicity in normal cells than their cancer counterparts. Although it was difficult to fully differentiate cellular responses to various MNPs between normal fibroblasts and their cancer counterparts, normal cells were shown to be more vulnerable to internalized MNPs than cancer cells. Our results suggest that functional groups and sizes of MNPs are critical determinants of degrees of cytotoxicity and genotoxicity, and potential mechanisms of toxicity.

Genetic diversity of emetic toxin producing Bacillus cereus Korean strains.

Bacillus cereus is divided into emetic and enterotoxin producing strains. Emetic B. cereus showed the low level of genetic diversity and single evolutionary lineage but no specific study of the genotypic characterization of emetic B. cereus Korean strains has been conducted. The objective of this study was to investigate the genotypic diversity of emetic B. cereus Korean strains. A total 39 strains (35 clinical and 4 food isolates) was analyzed for the genotypic characterization. A total of 17 distinct patterns were obtained from the random-amplified polymorphic DNA (RAPD) banding patterns and the majority of clusters belong to group 3. The pulsed-field gel electrophoresis (PFGE) banding patterns were divided into 17 distinct pulsotypes, and groups B and C were dominated. Emetic B. cereus Korean strains showed diverse pulsotypes in contrast with previous studies. All strains were resistant to ß-lactam antibiotics such as penicillin and ampicillin. Combining biochemical properties, PFGE types, RAPD types and antibiotic resistance types, a total of 7 composite clusters were found. The majority of composite clusters were consisted with cluster 5 and 6. Enterotoxin producing reference strains belong to composite cluster 7. However, JNHE 6 (Jeollabuk-do Research Institute of Health & Environment; cluster 1) and JNHE 36 (cluster 2) which possessed the ability of starch hydrolysis and saline fermentation showed different composite clusters comparing with most emetic B. cereus. JNHE 7 and JNHE 53 formed composite cluster 3 and 4. Emetic B. cereus Korean strains showed genotypic diversity comparing with the previous studies.

Subtle cytotoxicity and genotoxicity differences in superparamagnetic iron oxide nanoparticles coated with various functional groups.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely utilized for the diagnosis and therapy of specific diseases, as magnetic resonance imaging (MRI) contrast agents and drug-delivery carriers, due to their easy transportation to targeted areas by an external magnetic field. For such biomedical applications, SPIONs must have multifunctional characteristics, including optimized size and modified surface. However, the biofunctionality and biocompatibility of SPIONs with various surface functional groups of different sizes have yet to be elucidated clearly. Therefore, it is important to carefully monitor the cytotoxicity and genotoxicity of SPIONs that are surfaced-modified with various functional groups of different sizes. In this study, we evaluated SPIONs with diameters of approximately 10 nm and 100~150 nm, containing different surface functional groups. SPIONs were covered with -O⁻ groups, so-called bare SPIONs. Following this, they were modified with three different functional groups--hydroxyl (-OH), carboxylic (-COOH), and amine (-NH2) groups--by coating their surfaces with tetraethyl orthosilicate (TEOS), (3-aminopropyl)trimethoxysilane (APTMS), TEOS-APTMS, or citrate, which imparted different surface charges and sizes to the particles. The effects of SPIONs coated with these functional groups on mitochondrial activity, intracellular accumulation of reactive oxygen species, membrane integrity, and DNA stability in L-929 fibroblasts were determined by water-soluble tetrazolium, 2',7'-dichlorodihydrofluorescein, lactate dehydrogenase, and comet assays, respectively. Our toxicological observations suggest that the functional groups and sizes of SPIONs are critical determinants of cellular responses, degrees of cytotoxicity and genotoxicity, and potential mechanisms of toxicity. Nanoparticles with various surface modifications and of different sizes induced slight, but possibly meaningful, changes in cell cytotoxicity and genotoxicity, which would be significantly valuable in further studies of bioconjugation and cell interaction for drug delivery, cell culture, and cancer-targeting applications.

Fabrication of uniform au-carbon nanofiber by two-step low temperature decomposition.

This paper presents a facile and efficient way to prepare carbon nanofibers ornamented with Au nanoparticles (Au/CNFs). Gold nanoparticles were first deposited in the channels of an anodized aluminum oxide (AAO) membrane by thermal decomposition of HAuCl4and then carbon nanofibers were produced in the same channels loaded with the Au nanoparticles by decomposition of sucrose at 230 °C. An electron microscopy study revealed that the carbon nanofibers, ~10 nm thick and 6 µm long, were decorated with Au nanoparticles with a diameter of 10 nm. This synthetic route can produce uniform Au nanoparticles on CNF surfaces without using any additional chemicals to modify the AAO channels or the CNF surfaces.