Highly Sensitive and Specific Detection of Influenza A Viruses Using Bimolecular Fluorescence Complementation (BiFC) Reporter System.

In this study, we developed a highly sensitive and specific bimolecular fluorescence complementation (BiFC)-based influenza A virus (IAV)-sensing system by combining a galactose/glucose-binding protein (GGBP) with an N-terminal large domain (YN1-172) and a C-terminal small domain (YC173-239) made up of enhanced yellow fluorescence protein (eYFP). The GGBP-based BiFC reporter exhibits the fluorescence reconstitution as a result of conformational changes in GGBP when lactose, which was derived from 6'-silalyllactose and used as a substrate for neuraminidase (NA), binds to GGBP in the presence of IAV. The system showed a linear dynamic range extending from 1 × 100 to 1 × 107 TCID50/mL, and it had a detection limit of 1.1 × 100 TCID50/mL for IAV (H1N1), demonstrating ultra-high sensitivity. Our system exhibited fluorescence intensity enhancements in the presence of IAV, while it displayed weak fluorescence signals when exposed to NA-deficient viruses, such as RSV A, RSV B, adenovirus and rhinovirus, thereby indicating selective responses for IAV detection. Overall, our system provides a simple, highly sensitive and specific IAV detection platform based on BiFC that is capable of detecting ligand-induced protein conformational changes, obviating the need for virus culture or RNA extraction processes.

Occurrence and sources of micro-plastics in various water bodies, sediments, and fishes in Ansan, South Korea.

In this study, the Pearson correlation coefficients were determined to derive correlations between micro-plastics (MPs) in carp and river crabs. MPs were detected for various water sources, including four rivers and four main waterways, sediments, and fish, using Fourier transform infrared spectrometry (FTIR), microscopic analysis, and image mapping. Carp and river crabs had coefficients of 0.888 and 0.724, respectively, which showed a high positive correlation. In water samples, the MPs detected in rivers were higher than those in the main waterway. However, in sediment samples, the MPs detected in the main waterway were higher than those in the rivers. It is believed that MPs are carried toward shore by ocean tide. The size of most of the sediment MPs was 20-49 µm, representing 64.1% of the entire population. The plastics detected in this study were polyethylene terephthalate (PET), polypropylene (PP), and polyethylene (PE), which originate from synthetic fibers, scrubs, and packing material. MP pollution by non-point pollution sources was investigated, with the abundance of MPs increasing by 2 to 3 times between the dry and wet seasons in water and sediment, respectively. It was determined that the inflow of MPs into rivers could have been due to non-point source pollutants from household items, roads, plants, and soil around the water sources.

Highly Selective FRET-Aided Single-Molecule Counting of MicroRNAs Labeled by Splinted Ligation.

MicroRNAs (miRNAs) are short non-coding RNAs that play an important role in regulating gene expression. Since miRNAs are abnormally expressed in various cancers, they are considered to be promising biomarkers for early cancer diagnosis. However, the short length and strong sequence similarity among miRNAs make their reliable quantification very challenging. We developed a highly selective amplification-free miRNA detection method based on Förster resonance energy transfer (FRET)-aided single-molecule counting. miRNAs were selectively labeled with FRET probes using splinted ligation. When imaged with a single-molecule FRET setup, the miRNA molecules were accurately identified by the probe's FRET. miRNA concentrations were estimated from the count of molecules. The high sensitivity of the method in finding sparse molecules enabled us to achieve a limit of detection of 31-56 amol for miR-125b, miR-100, and miR-99a. Single nucleotide mismatch could be discriminated with a very high target-to-mismatch ratio. The method accurately measured the high expression of miR-125b in gastric cancer cells, which agreed well with previous reports. The high sensitivity and accuracy of this technique demonstrated its clinical potential as a robust miRNA detection method.

Genetically Modified Ferritin Nanoparticles with Bone-Targeting Peptides for Bone Imaging.

Bone homeostasis plays a major role in supporting and protecting various organs as well as a body structure by maintaining the balance of activities of the osteoblasts and osteoclasts. Unbalanced differentiation and functions of these cells result in various skeletal diseases, such as osteoporosis, osteopetrosis, and Paget's disease. Although various synthetic nanomaterials have been developed for bone imaging and therapy through the chemical conjugation, they are associated with serious drawbacks, including heterogeneity and random orientation, in turn resulting in low efficiency. Here, we report the synthesis of bone-targeting ferritin nanoparticles for bone imaging. Ferritin, which is a globular protein composed of 24 subunits, was employed as a carrier molecule. Bone-targeting peptides that have been reported to specifically bind to osteoblast and hydroxyapatite were genetically fused to the N-terminus of the heavy subunit of human ferritin in such a way that the peptides faced outwards. Ferritin nanoparticles with fused bone-targeting peptides were also conjugated with fluorescent dyes to assess their binding ability using osteoblast imaging and a hydroxyapatite binding assay; the results showed their specific binding with osteoblasts and hydroxyapatite. Using in vivo analysis, a specific fluorescent signal from the lower limb was observed, demonstrating a highly selective affinity of the modified nanoparticles for the bone tissue. These promising results indicate a specific binding ability of the nanoscale targeting system to the bone tissue, which might potentially be used for bone disease therapy in future clinical applications.

Multi-Odor Discrimination by Rat Sniffing for Potential Monitoring of Lung Cancer and Diabetes.

The discrimination learning of multiple odors, in which multi-odor can be associated with different responses, is important for responding quickly and accurately to changes in the external environment. However, very few studies have been done on multi-odor discrimination by animal sniffing. Herein, we report a novel multi-odor discrimination system by detection rats based on the combination of 2-Choice and Go/No-Go (GNG) tasks into a single paradigm, in which the Go response of GNG was replaced by 2-Choice, for detection of toluene and acetone, which are odor indicators of lung cancer and diabetes, respectively. Three of six trained rats reached performance criterion, in 12 consecutive successful tests within a given set or over 12 sets with a success rate of over 90%. Through a total of 1300 tests, the trained animals (N = 3) showed multi-odor sensing performance with 88% accuracy, 87% sensitivity and 90% specificity. In addition, a dependence of behavior response time on odor concentrations under given concentration conditions was observed, suggesting that the system could be used for quantitative measurements. Furthermore, the animals' multi-odor sensing performance has lasted for 45 days, indicating long-term stability of the learned multi-odor discrimination. These findings demonstrate that multi-odor discrimination can be achieved by rat sniffing, potentially providing insight into the rapid, accurate and cost-effective multi-odor monitoring in the lung cancer and diabetes.

Olfactory Detection of Toluene by Detection Rats for Potential Screening of Lung Cancer.

Early detection is critical to successfully eradicating a variety of cancers, so the development of a new cancer primary screening system is essential. Herein, we report an animal nose sensor system for the potential primary screening of lung cancer. To establish this, we developed an odor discrimination training device based on operant conditioning paradigms for detection of toluene, an odor indicator component of lung cancer. The rats (N = 15) were trained to jump onto a floating ledge in response to toluene-spiked breath samples. Twelve rats among 15 trained rats reached performance criterion in 12 consecutive successful tests within a given set, or over 12 sets, with a success rate of over 90%. Through a total of 1934 tests, the trained rats (N = 3) showed excellent performance for toluene detection with 82% accuracy, 83% sensitivity, 81% specificity, 80% positive predictive value (PPV) and 83% negative predictive value (NPV). The animals also acquired considerable performance for odor discrimination even in rigorous tests, validating odor specificity. Since environmental and long-term stability are important factors that can influence the sensing results, the performance of the trained rats was studied under specified temperature (20, 25, and 30 °C) and humidity (30%, 45%, and 60% RH) conditions, and monitored over a period of 45 days. At given conditions of temperature and humidity, the animal sensors showed an average accuracy within a deviation range of ±10%, indicating the excellent environmental stability of the detection rats. Surprisingly, the trained rats did not differ in retention of last odor discrimination when tested 45 days after training, denoting that the rats' memory for trained odor is still available over a long period of time. When taken together, these results indicate that our odor discrimination training system can be useful for non-invasive breath testing and potential primary screening of lung cancer.

An enhanced ascorbate peroxidase 2/antibody-binding domain fusion protein (APEX2-ABD) as a recombinant target-specific signal amplifier.

A recombinant target-specific signal amplifier was constructed by genetically fusing the enhanced ascorbate peroxidase 2 (APEX2) and an antibody-binding domain (ABD). The fusion protein APEX2-ABD possessed the peroxidase activity and the antibody-binding capability simultaneously and replaced the conventional HRP-conjugated secondary antibodies in a TSA assay for amplifying fluorescence signals.

Fisetin, a dietary flavonoid, induces apoptosis of cancer cells by inhibiting HSF1 activity through blocking its binding to the hsp70 promoter.

Heat shock factor 1 (HSF1) is a transcription factor for heat shock proteins (HSPs) expression that enhances the survival of cancer cells exposed to various stresses. HSF1 knockout suppresses carcinogen-induced cancer induction in mice. Therefore, HSF1 is a promising therapeutic and chemopreventive target. We performed cell-based screening with a natural compound collection and identified fisetin, a dietary flavonoid, as a HSF1 inhibitor. Fisetin abolished heat shock-induced luciferase activity with an IC50 of 14 µM in HCT-116 cancer cells. The treatment of HCT-116 with fisetin inhibited proliferation with a GI50 of 23 µM. When the cells were exposed to heat shock in the presence of fisetin, the induction of HSF1 target proteins, such as HSP70, HSP27 and BAG3 (Bcl-2-associated athanogene domain 3), were inhibited. HSP70/BAG3 complexes protect cancer cells from apoptosis by stabilizing anti-apoptotic Bcl-2 family proteins. The downregulation of HSP70/BAG3 by fisetin significantly reduced the amounts of Bcl-2, Bcl-xL and Mcl-1 proteins, subsequently inducing apoptotic cell death. Chromatin immunoprecipitation assays showed that fisetin inhibited HSF1 activity by blocking the binding of HSF1 to the hsp70 promoter. Intraperitoneal treatment of nude mice with fisetin at 30mg/kg resulted in a 35.7% (P < 0.001) inhibition of tumor growth.

Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system.

Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8 mg · g(-1) leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system.

Anaerobic degradation of amino acids generated from the hydrolysis of sewage sludge.

The anaerobic degradation of each amino acid that could be generated through the hydrolysis of sewage sludge was evaluated. Stickland reaction as an intermediate reaction between two kinds of amino acids was restricted in order to evaluate each amino acid. Changes in the chemical oxygen demand (COD), T-N, NH4(+)-N, biogas, and CH4 were analysed for the anaerobic digestion process. The initial nitrogen concentration of all amino acids is adjusted as 1000 mg/L. The degradation rate of the amino acids was determined based on the ammonia form of nitrogen, which is generated by the deamination of amino acids. Among all amino acids, such as alpha-alanine, beta-alanine, lysine, arginine, glycine, histidine, cysteine, methionine, and leucine, deamination rates of cysteine, leucine, and methionine were just 61.55%, 54.59%, and 46.61%, respectively, and they had low removal rates of organic matter and showed very low methane production rates of 13.55, 71.04, and 80.77 mL CH4/g CODin, respectively. Especially for cysteine, the methane content was maintained at approximately 7% during the experiment. If wastewater contains high levels of cysteine, leucine, and methionine and Stickland reaction is not prepared, these amino acids may reduce the efficiency of the anaerobic digestion.

Signal amplification by magnetic force on polydiacetylene supramolecules for detection of prostate cancer.

A method in which a permanent magnet is introduced onto polydiacetylene (PDA) vesicle chips is introduced for enhancement of the fluorescence of PDA vesicles. This strategy can be applied to general antibody-based PDA vesicle chips to detect clinically important biomarkers for disease diagnosis.

A new method for non-labeling attomolar detection of diseases based on an individual gold nanorod immunosensor.

Herein, we present the use of a single gold nanorod sensor for detection of diseases on an antibody-functionalized surface, based on antibody-antigen interaction and the localized surface plasmon resonance (LSPR) λ(max) shifts of the resonant Rayleigh light scattering spectra. By replacing the cetyltrimethylammonium bromide (CTAB), a tightly packed self-assembled monolayer of HS(CH(2))(11)(OCH(2)CH(2))(6)OCH(2)COOH(OEG(6)) has been successfully formed on the gold nanorod surface prior to the LSPR sensing, leading to the successful fabrication of individual gold nanorod immunosensors. Using prostate specific antigen (PSA) as a protein biomarker, the lowest concentration experimentally detected was as low as 111 aM, corresponding to a 2.79 nm LSPR λ(max) shift. These results indicate that the detection platform is very sensitive and outperforms detection limits of commercial tests for PSA so far. Correlatively, its detection limit can be equally compared to the assays based on DNA biobarcodes. This study shows that a gold nanorod has been used as a single nanobiosensor to detect antigens for the first time; and the detection method based on the resonant Rayleigh scattering spectrum of individual gold nanorods enables a simple, label-free detection with ultrahigh sensitivity.

Combination of cysteine- and oligomerization domain-mediated protein immobilization on a surface plasmon resonance (SPR) gold chip surface.

Here we report an effective method for protein immobilization on a surface plasmon resonance (SPR) gold chip, describing the combination of cysteine- and oligomerization domain-mediated immobilization of enhanced green fluorescent protein (EGFP) as a model protein for the purpose of orientation-controlled surface density packing. In order to facilitate the oligomerization of EGFP, the dimeric and trimeric constructs derived from GCN4- leucine zipper domain were chosen for multimeric EGFP assembly. For orientation-controlled immobilization of the protein, EGFP modified with cysteine residues showing excellent orientation on a gold chip was used as a starting protein, as previously reported in our earlier study (Anal. Chem., 2007, 79, 2680-2687). Constructs of EGFP with oligomerization domains were genetically engineered, and corresponding fusion proteins were purified, applied to a gold chip, and then analyzed under SPR. The immobilized EGFP density on a gold chip increased according to the states of protein oligomerization, as dimeric and trimeric EGFPs displayed better adsorption capability than monomeric and dimeric forms, respectively. Fluorescence measurement corroborated the SPR results. Taken together, our findings indicated that the combination of cysteine- and oligomerization domain-mediated immobilization of protein could be used in SPR biosensor applications, allowing for an excellent orientation and high surface density simultaneously.

Design, synthesis and characterization of vanadia-doped iron-oxide pillared montmorillonite clay for the selective catalytic oxidation of H2S.

A series of vanadia-doped iron-oxide-pillared clays (V/Fe-PILCs) with various amounts of vanadia were prepared and their performance for the selective catalytic oxidation of H(2)S was investigated. V/Fe-PILCs were characterized using X-ray diffraction (XRD), surface area- and pore volume measurements, chemical analysis, Fourier-transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and temperature-programmed reduction by H(2) (H(2)-TPR). V/Fe-PILCs showed better catalytic performance than Fe-PILC without any significant SO(2) emissions. The H(2)S conversion over V/Fe-PILCs increased with increasing vanadia content up to 7 wt.%. However, it decreased at higher vanadia loading due to the decrease in surface area and the formation of the crystalline V(2)O(5) phase. The presence of water vapor in the reactant mixture resulted in a decrease of H(2)S conversion.

Detection of mutant p53 using field-effect transistor biosensor.

We assessed the abilities of wild p53 and mutant p53 proteins to interact with the consensus DNA-binding sequence using a MOSFET biosensor. This is the first report in which mutant p53 has been detected on the basis of DNA-protein interaction using a FET-type biosensor. In an effort to evaluate the performance of this protocol, we constructed the core domain of wild p53 and mutant p53 (R248W), which is DNA-binding-defective. After the immobilization of the cognate DNA to the sensing layer, wild p53 and mutant p53 were applied to the DNA-coated gate surface, and subsequently analyzed using a semiconductor analyzer. As a consequence, a significant up-shift in drain current was noted in response to wild p53, but not mutant p53, thereby indicating that sequence-specific DNA-protein interactions could be successfully monitored using a field-effect-based biosensor. These data also corresponded to the results obtained using surface plasmon resonance (SPR) measurements. Taken together, our results show that a FET-type biosensor might be promising for the monitoring of mutant p53 on the basis of its DNA-binding activity, providing us with very valuable insights into the monitoring for diseases, particularly those associated with DNA-protein binding events.

In vivo effects of NbSiR silencing on chloroplast development in Nicotiana benthamiana.

Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and thylakoid membrane development.

Large-scale expression in Escherichia coli and efficient purification of precursor and active caspase-7 by introduction of thrombin cleavage sites.

Caspases are a family of cysteine proteases that have critical roles in the apoptotic pathway. Caspase-7 is a well-known apoptotic effector that cleaves a variety of cellular substrates, and is known to be an important target in the treatment of many diseases. For efficient research, large amounts of the protein are required. However, it has been difficult to obtain sufficient quantities of either the precursor or active caspase-7 from Escherichia coli strain. In the present study, we constructed thrombin-activatable caspase-7 precursors by changing the auto-activation sites of the caspase-7 precursor into sequences susceptible to thrombin cleavage. These engineered precursors were highly expressed as soluble proteins in E. coli, and were easily purified by affinity chromatography (to levels of 10-15 mg per liter of E. coli culture), and were then readily activated by treatment with thrombin. In vitro cleavage assays and kinetic analyses revealed that the engineered active caspase-7 proteins had characteristics similar to those of wild-type caspase-7. This novel method is valuable for obtaining both precursor and active caspase-7, thereby contributing to the development of caspase-7-specific drugs to treat various diseases, including cancer and neurodegenerative conditions.

Monitoring of cleavage preference for caspase-3 using recombinant protein substrates.

The apoptotic caspases have been classified in accordance with their substrate specificities, as the optimal tetrapeptide recognition motifs for a variety of caspases have been determined via positional scanning substrate combinatorial library technology. Here, we focused on two proteolytic recognition motifs, DEVD and IETD, owing to their extensive use in cell death assay. Although DEVE and IETD have been generally considered to be selective for caspase-3 and -8, respectively, the proteolytic cleavage of these substrates does not display absolute specificity for a particular caspase. Thus, we attempted to monitor the cleavage preference for caspase-3, particularly using the recombinant protein substrates. For this aim, the chimeric GST:DEVD:EGFP and GST:IETD:EGFP proteins were genetically constructed by linking GST and EGFP with the linkers harboring DEVD and IETD. To our best knowledge, this work constitutes the first application for the monitoring of cleavage preference employing the recombinant protein substrates that simultaneously allow for mass and fluorescence analyses. Consequently, GST: IETD:EGFP was cleaved partially in response to caspase-3, whereas GST:DEVD:EGFP was completely proteolyzed, indicating that GST:DEVD:EGFP is a better substrate than GST:IETD:EGFP for caspase-3. Collectively, using these chimeric protein substrates, we have successfully evaluated the feasibility of the recombinant protein substrate for applicability to the monitoring of cleavage preference for caspase-3.

Comparative proteomic analysis of mouse melanoma cell line B16, a metastatic descendant B16F10, and B16 overexpressing the metastasis-associated tyrosine phosphatase PRL-3.

Metastasis is a complex, multistep process by which a cancer cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary cancer. A deeper understanding of the molecular events underlying metastasis will provide information that will be useful for the development of new diagnostic and therapeutic strategies. The B16 and B16F10 mouse melanoma cell lines are widely used as model system for studying many aspects of cancer biology including metastasis. Compared with B16, which has a low metastatic potential, the highly metastatic cell line B16F10 displayed a higher metastatic ability along with higher expression levels of the metastasis-associated phosphatase of regenerating liver-3 (PRL-3). B16 cells transfected with PRL-3 cDNA (B16-PRL3) had metastatic abilities comparable to those of Bl16F10 cells. To study the molecular mechanisms that underlie metastasis, the proteomes of the B16, B16F10, and B16-PRL3 cell lines were compared using two-dimensional differential in-gel electrophoresis. Proteins that varied significantly in levels between these cell lines were selected and identified using mass spectrometry. Interestingly, many proteins, especially those present in membrane fractions, were similarly up- or downregulated in both the Bl16F10 and B16-PRL3 cells lines compared to B16 cell lines. The list of similarly regulated proteins included heat shock protein 70, fascin-1, septin-6, ATP synthase beta subunit, and bone morphogenic protein receptor type IB. These proteins may play a causal role in PRL-3-mediated metastasis. These investigations open an avenue for the further characterization of the molecular mechanisms that underlie metastasis.

Novel application of surface plasmon resonance biosensor chips for measurement of advanced glycation end products in serum of Zucker diabetic fatty rats.

Advanced glycation end products (AGEs) have been implicated in diabetic complications. To measure AGEs, especially N(epsilon)-(carboxymethyl)lysine (CML), in sera from Zucker diabetic fatty rats (ZDF) and Zucker lean rats (ZL), we used a novel method of protein chip and surface plasmon resonance imaging (SPRI). Serum samples were obtained from male ZDF and ZL rats at 20 weeks of age. Antibodies to AGEs or CML were immobilized on a gold surface, which was modified by cysteine-tagged, protein-G constructs. The gold chip upon which the serum was spotted was optically coupled with a prism coupler. The reflected images from the gold chip were obtained using a charge-coupled device (CCD) camera. The direct analysis of the glycated proteins and products using SPRI showed that AGEs and CML levels were elevated in ZDF serum, compared with ZL serum. The lowest detection limit of AGEs was 10 ng/ml, with a working range covering the physiological range. These results indicate that the protein chip and SPRI system is very suitable for the measurement of glycated proteins and end products in serum samples. This system offers high sensitivity without any fluorescent or other labeling of the components and saves a substantial amount of time, resources, and labor. Our results suggest that SPRI systems can be used as a tool to diagnose diabetic complications.