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Ring-shaped replicative helicases are hexameric and play a key role in cellular DNA replication. Despite their importance, our understanding of the unwinding mechanism of replicative helicases is far from perfect. Bovine papillomavirus E1 is one of the best-known model systems for replicative helicases. E1 is a multifunctional initiator that senses and melts the viral origin and unwinds DNA. Here, we study the unwinding mechanism of E1 at the single-molecule level using magnetic tweezers. The result reveals that E1 as a single hexamer is a poorly processive helicase with a low unwinding rate. Tension on the DNA strands impedes unwinding, indicating that the helicase interacts strongly with both DNA strands at the junction. While investigating the interaction at a high force (26–30 pN), we discovered that E1 encircles dsDNA. By comparing with the E1 construct without a DNA binding domain, we propose two possible encircling modes of E1 during active unwinding.

Superparamagnetic iron oxide nanoparticles are widely used as nanoprobes for magnetic resonance imaging (MRI). In this study, a novel type of cross-linked magnetic nanoparticle was developed in an effort to improve the structural stability of amphiphilic polymer-coated iron oxide nanoparticles. Iron oxide nanocrystals were coated with a cross-linkable amphiphilic graft copolymer, poly(succinimide) grafted with folate-conjugated polyethylene glycol (PEG) and alkyl chains. The tumor-specific targeting ligand, folate, was included to target and detect cancer cells. The hydrophobic portions of the amphiphilic copolymer on the surfaces of the nanoparticles were cross-linked via an aminolysis reaction between the succinimide units and a bifunctional primary amine. The folate-conjugated cross-linked magnetic nanoparticles (F-CLMNPs) were 40 nm in diameter and displayed a low cytotoxicity, even at relatively high concentrations. The F-CLMNPs exhibited highly efficient intracellular uptake into KB cells, which overexpress the folate receptor, as determined by fluorescence microscopy, flow cytometry, and Prussian blue staining. In vivo MR images of a mouse bearing a KB cell tumor displayed a 75% drop in the T2 signal in the tumor tissues within 3 hours. These results indicated that the F-CLMNPs accumulated at the tumor site and were highly effective for tumor detection using in vivo MRI techniques.

Rapid developments in the field of nanoparticles during the last decades have led to its increased application in drug delivery approaches. The advantages of nano-based drug delivery systems include improved therapeutic efficacy of drugs and the reduction of side effects. A plethora of different nanoparticles have been explored as drug carriers for the effective treatment of cancers. Of the various organic/inorganic nanoparticles, polymeric nanoparticles have fulfilled an integral role in the advancement of drug delivery systems by virtue of the ease to incorporate and modify targeting moieties in combination with controlled drug release over prolonged periods. Furthermore, polymeric nanoparticles facilitate theranostic treatment by the incorporation of imaging agents in addition to therapeutics. Recently, stimuli-responsive polymeric nanoparticles emerged as smart drug carriers, in which drug release is affected by physical and/or chemical structural changes induced by a specific stimulus (e.g. pH, temperature, and specific enzymes). The use of these nanocomposites reduces premature drug release and maintains effective drug levels at the pathological target. This review aims to highlight advances in stimuli-triggered drug delivery approaches using polymeric nanoparticles with a focus on pH-sensitive drug- and theranostic delivery systems.

Nanocomposites are widely used to obtain an accurate diagnosis of, and to provide effective therapy for, a number of diseases, because they can be easily formulated by introducing therapeutic agents (e.g., drugs and genes) and imaging agents (e.g., magnetic nanocrystals). Furthermore, nanocomposites can be developed as all-in-one systems, which enable cancer diagnosis and therapy, as well as the simultaneous monitoring of drug behavior. In this protocol, we describe the synthesis of four pyrenyl-based polymers (pyrenyl polyethylene glycol (Py-PEG), pyrenyl dextran (Py-DEX), pyrenyl hyaluronan (Py-HA) and pyrenyl-conjugated heterofunctional PEG (pyrenyl PEG)) and their subsequent use in the preparation of multifunctional nanocomposites for different applications including multimodal imaging, targeted cancer detection and pH-sensitive drug delivery. Notably, these nanocomposites can be used to simultaneously perform multiple tasks--for example, delivering magnetic particles for early cancer detection by MRI, efficient cataloging of patient groups for personalized therapy and real-time monitoring of disease progress. Starting from the synthesis of pyrenyl-based polymers, this protocol can be completed in ∼15 d.

It is highly important to identify influenza viruses rapidly and accurately for the prevention of future pandemic outbreaks. We developed an easy and sensitive influenza virus detection method using surface-enhanced Raman scattering (SERS) antibody probes. The SERS antibody probes can be prepared easily by mixing Au nanoparticles, gold binding peptide-protein G, and antibodies without complicated chemical or biological reactions. They also retain the optimal conformation for the antibody's interaction with Influenza A/CA/07/2009 (pH1N1), allowing us to detect pH1N1 sensitively and selectively. This method provides a detection limit of 4.1 × 103 TCID per mL and is highly selective for pH1N1. We anticipate that this method can be useful in a wide range of immunoassays.

Here, we demonstrate a simple route for preparing of C 60 nano-islands on a flat surface such as a silicon wafer by using spin-coating method. The size of C 60 nano-islands was controlled by varying C 60 concentrations and rotating speeds. Moreover, the molecular structure of the nano-islands changed depending on the annealing temperature. The homogenous formation of C 60 nano-islands was revealed by scanning electron microscopy and atomic force microscopy. A structural change occurred during annealing, and the Raman spectra indicated that this change was dependent on the annealing temperature.

The toxicity of nano-graphene oxide (NGO) on development and angiogenesis was evaluated using zebrafish embryos as in vivo model system. Microinjection of NGO resulted in gross morphological defects in a dose-dependent manner partly due to the induction of apoptosis, whereas coating NGO derivatives with polyethylene glycol (PEG), a biocompatible polymer significantly attenuated its toxicity. NGO also caused abnormal branching and mispatterning of developing trunk blood vessels monitored by endothelial cell-specific fluorescent transgenic zebrafish, presumably via Vascular Endothelial Growth Factor and Notch pathways, key signaling pathways for normal angiogenic process. Interestingly, Alexa568 conjugated, PEG-coated NGO (NGO-A568) still caused angiogenic defects to the similar degree as NGO did, suggesting differential toxic effects of nanomaterials on different developmental processes. Confocal microscopy imaging of NGO- and NGO-A568-injected embryos visualized its live distribution throughout the body including the cranial vasculature. The broader utilization of embryonic zebrafish combined with in vivo , real-time, high-resolution imaging is warranted for assessing the properties of nanomaterials.

Rapid, specific and sensitive detection of pathogenic bacteria is crucial for public health and safety. Bacillus cereus is harmful as it causes foodborne illness and a number of systemic and local infections. We report a novel phage endolysin cell wall-binding domain (CBD) for B. cereus and the development of a highly specific and sensitive surface plasmon resonance (SPR)-based B. cereus detection method using the CBD. The newly discovered CBD from endolysin of PBC1, a B. cereus-specific bacteriophage, provides high specificity and binding capacity to B. cereus. By using the CBD-modified SPR chips, B. cereus can be detected at the range of 10(5)-10(8) CFU/ml. More importantly, the detection limit can be improved to 10(2) CFU/ml by using a subtractive inhibition assay based on the pre-incubation of B. cereus and CBDs, removal of CBD-bound B. cereus, and SPR detection of the unbound CBDs. The present study suggests that the small and genetically engineered CBDs can be promising biological probes for B. cereus. We anticipate that the CBD-based SPR-sensing methods will be useful for the sensitive, selective, and rapid detection of B. cereus.

We developed the photo-crosslinkable hydrogel-based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo-crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 μm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular-shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel-based 3D microfluidic device, showing that 53-75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo-crosslinkable hydrogel-based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications.

Copper is indispensable in most aerobic organisms although it is toxic if unregulated as illustrated in many neurodegenerative diseases. To elucidate the mechanisms underlying copper release from cells, a membrane-targeting reporter which can compete with extracellular copper-binding molecules is highly desirable. However, engineering a reporter protein to provide both high sensitivity and selectivity for copper(II) has been challenging, likely due to a lack of proper copper(II)-chelating strategies within proteins. Here, we report a new genetically encoded fluorescent copper(II) reporter by employing a copper-binding tripeptide derived from human serum albumin (HSA), which is one of the major copper-binding proteins in extracellular environments. Optimized insertion of the tripeptide into the green fluorescent protein leads to rapid fluorescence quenching (up to >85% change) upon copper-binding, while other metal ions have no effect. Furthermore, the high binding affinity of the reporter enables reliable copper detection even in the presence of competing biomolecules such as HSA and amyloid beta peptides. We also demonstrate that our reporter proteins can be used to visualize dynamic copper fluctuations on living HeLa cell surfaces.

We report on the targeted imaging of breast cancer using self-assembled levan nanoparticles. Indocyanine green (ICG) was encapsulated in levan nanoparticles via self-assembly. Levan-ICG nanoparticles were found to be successfully accumulated in breast cancer via specific interaction between fructose moieties in levan and overexpressed glucose transporter 5 in breast cancer cells.

In this paper, we report for the first time that graphene oxide (GO) can interact with mutagenic DNA but not intact DNA. After UV-irradiated fluorophore-linked DNA containing thymine repeats was mixed with GO, a decrease in fluorescence was observed in a time-dependent manner. In contrast, no fluorescence change was observed with intact DNA, indicating that UV irradiation of DNA resulted in the formation of mutagenic bases. Because GO is known to act as a fluorescence quencher, the decreased fluorescence implies adsorption of the UV-irradiated DNA onto GO. It appears that the decreased fluorescence might result from the greater accessibility of hydrophobic methyl groups and phenyl rings of thymine dimers to GO and from deformed DNA structures with less effective charge shielding under salt-containing conditions. Using this affinity of GO for mutagenic DNA, we could detect UV-irradiated DNA at concentrations as low as 100 pM. We were also able to analyze the ability of phototoxic drugs to catalyze the formation of mutagens under UV irradiation with GO. Because our method is highly sensitive and feasible and does not require the pretreatment of DNA, we propose that it could accelerate the screening of potential phototoxic drug candidates that would be able to sensitize mutagenic dsDNA.

As various fullerene derivatives have been developed, it is necessary to explore their photophysical properties for potential use in photoelectronics and medicine. Here, we address the photophysical properties of newly synthesized water-soluble fullerene-based nanoparticles and polyhydroxylated fullerene as a representative water-soluble fullerene derivative. They show broad emission band arising from a wide-range of excitation energies. It is attributed to the optical transitions from disorder-induced states, which decay in the nanosecond time range. We determine the kinetic properties of the singlet oxygen ((1)O2) luminescence generated by the fullerene nanoparticles and polyhydroxylated fullerene to consider the potential as photodynamic agents. Triplet state decay of the nanoparticles was longer than (1)O2 lifetime in water. Singlet oxygen quantum yield of a series of the fullerene nanoparticles is comparably higher ranging from 0.15 to 0.2 than that of polyhydroxylated fullerene, which is about 0.06.

Gold nanoparticle-conjugated fluorescent hydroxyapatite (AuFHAp) was developed as a smart nanoprobe for measuring alkaline phosphatase (ALP) activity. AuFHAp showed NIR fluorescence due to the hydrolysis of its phosphate groups by ALP. In addition, gold nanoparticles help reduce the nonspecific signal by absorbing nonspecific fluorescence. Through in vitro tests, we confirmed that the AuFHAp probe was capable of detecting ALP levels related to osteoblast activity in living cells with high fluorescence intensity.

We have developed a novel scanometric antibody probe for rapid, sensitive, and naked-eye-visible immunoassays. Using this probe, we clearly demonstrated the successful scanometric detection and identification of influenza A viruses on a microarray. In addition, the sensitivity of the scanometric immunoassay was comparable to that of the fluorescence-based method.

Levan coated cerium oxide nanoparticles (LCNPs) with the enhanced antioxidant activity were successfully synthesized and characterized. Levan and their derivatives are attractive for biomedical applications attributable to their antioxidant, anti-inflammation and anti-tumor properties. LCNPs were synthesized using the one-pot and green synthesis system with levan. For production of nanoparticles, levan plays a role as a stabilizing and reducing agent. Fourier transform infrared spectroscopy (FT-IR) analysis showed that LCNPs successfully synthesized. The morphology and size of nanoparticles were confirmed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). LCNPs have good water solubility and stability. The conjugation of levan with cerium oxide nanoparticles improved antioxidant activity. Moreover the level of ROS was reduced after treatment of LCNPs to H2O2 stimulated NIH3T3 cells. These results demonstrate that the LCNPs are useful for applying of treatment of ROS induced diseases.

A peptide-based molecular beacon (PEP-MB) was prepared for the simple, rapid, and specific detection of H1N1 viruses using a fluorescence resonance energy transfer (FRET) system. The PEP-MB exhibited minimal fluorescence in its "closed" hairpin structure. However, in the presence of H1N1 viruses, the specific recognition of the hemagglutinin (HA) protein of H1 strains by the PEP-MB causes the beacon to assume an "open" structure that emits strong fluorescence. The PEP-MB could detect H1N1 viruses within 15 min or even 5 min and can exhibit strong fluorescence even at low viral concentrations, with a detection limit of 4 copies.

We developed the photo-crosslinkable hydrogel-based 3D microfluidic device to culture neural stem cells (NSCs) and tumors. The photo-crosslinkable gelatin methacrylate (GelMA) polymer was used as a physical barrier in the microfluidic device and collagen type I gel was employed to culture NSCs in a 3D manner. We demonstrated that the pore size was inversely proportional to concentrations of GelMA hydrogels, showing the pore sizes of 5 and 25 w/v% GelMA hydrogels were 34 and 4 μm, respectively. It also revealed that the morphology of pores in 5 w/v% GelMA hydrogels was elliptical shape, whereas we observed circular-shaped pores in 25 w/v% GelMA hydrogels. To culture NSCs and tumors in the 3D microfluidic device, we investigated the molecular diffusion properties across GelMA hydrogels, indicating that 25 w/v% GelMA hydrogels inhibited the molecular diffusion for 6 days in the 3D microfluidic device. In contrast, the chemicals were diffused in 5 w/v% GelMA hydrogels. Finally, we cultured NSCs and tumors in the hydrogel-based 3D microfluidic device, showing that 53-75% NSCs differentiated into neurons, while tumors were cultured in the collagen gels. Therefore, this photo-crosslinkable hydrogel-based 3D microfluidic culture device could be a potentially powerful tool for regenerative tissue engineering applications.

As our global society has been more complex and connected closely, infectious diseases could be easily and fast propagated to everywhere. And it makes early diagnostic detection be necessary with various ways based on optical, electrical and electrochemical methods. Real-time PCR (Polymerase Chain Reaction) has been powerful detection technology to recognize the amplified target DNA fragments that would be essential to diagnostic assays, but it needs optical labels and apparatus to detect the amplification of DNA. A variety of electric/electrochemical methods have been applied for label-free detection. Impedance assay is attractive tool for biomolecule real-time sensing, however, most of impedance studies are based on the immobilization of biomolecules on the electrode surface. That ascribes to additional steps and makes impossible to reuse electrode chip and biological samples. In this study, we aimed to develop the electrical impedance detection not optical method measuring the bulk impedance of PCR product solution by nanogap electrode. Electrical impedimetric nanogap devices could be very useful for detecting slight changes of amplifying DNA fragments in PCR cycling due to double layer capacitance and charge transfer resistance between two opposing nanogap electrodes. The electrode chip was fabricated using gold thin film by easy photolithography designed the nanogapped Si/SiO2 substrate by evaporation of oxide materials. Applying electric impedance measuring in nanogap chip avoiding the use of optical labels and immobilization on the electrode surface would be very impact detection system on DNA real-time screening.

We present a nanoembossed nanoshell with a new internal location for the formation of strong electromagnetic fields. The internally nanoembossed gold nanoshell (AuNS) is fabricated by electrostatically assembling smaller silica nanoparticles (∼15.7 nm) around the silica core (∼123.6 nm) followed by growing gold nanoseeds on the core in a wet process. FDTD calculations reveal the creation of a strong electromagnetic field (|E/Ein|max = 55 at 633 nm) at sharp edges formed by the contact between the nanoembosses and the silica core. The field formation is supported by measuring the SERS signal of Ru(bpy) encapsulated in the nanoembossing silica nanoparticles. SERS signals as strong as the corresponding fluorescence are obtained. The Raman enhancement factor (EF) is estimated to be up to 10(10) at 633 nm excitation, in addition to a comparable EF at 785 nm laser excitation. The SERS intensity of the nanoembossed nanoshell layer is sufficiently high compared to the outer or the core of the nanoshell. Finally, we fabricate all-in-one nanoparticles with all the three places where the reporter dyes are loaded and acquire the highest SERS intensity to potentially enable bio-medical applications of the nanoembossed AuNS as a sensitive and reliable labeling particle.

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEP(C230X-/-) mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEP(C230X-/-) mice displayed reduced hyperlocomotion in response to intrastriatal injection of the μ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.