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Abstract: The aim of this study was the development of a bifunctional protein crosslinker-based surface plasmon resonance (SPR) biosensor for rapid detection of aflatoxin B1 (AFB1), a potent carcinogen. A fusion protein was obtained by genetically fusing gold binding protein (GBP) that binds strongly to gold surfaces to protein G (ProG) that interacts with the Fc portion of antibodies. It was used as a bifunctional crosslinker for rapid self-oriented immobilization of antibodies on gold substrates without any chemical treatment. SPR analyses demonstrated the binding of the GBP-ProG crosslinker to the gold surface was superior to that of an only ProG via currently used self-assembled monolayers of alkanethiol due to the GBP property. As a result, anti-AFB1 antibodies were 36% more immobilized on the GBP-ProG layer than the ProG layer. When the GBP-ProG crosslinker-based SPR chips were fabricated with the best density (100 μg/mL) of anti-AFB1 antibodies, they could detect AFB1 as low as 1 μg/mL in both buffer and corn extracts and selectively detect it with negligible SPR responses in control toxins (zearalenone and ochratoxin A). These results mean the GBP-ProG is more useful than the thiolated chemical linkers for development of gold substrate-based immunosensors, and this GBP-ProG crosslinker-based immunosensor could detect small molecules effectively. [Copyright &y& Elsevier]

Abstract: We demonstrated in vitro small ubiquitin-like modifier (SUMO)-mediated modification (SUMOylation) of RanGTPase activating protein-1 (RanGAP1) by using bioluminescence resonance energy transfer (BRET) for studying protein interactions. Renilla luciferase (Rluc) was fused to SUMO, and RanGAP1, the binding partner of SUMO, was fused to enhanced yellow fluorescence protein (EYFP). Upon binding of SUMO and RanGAP1, BRET was observed between EYFP (donor) and Rluc (acceptor) in the presence of E1 (Aos1/Uba2) and E2 (Ubc9) enzymes, whereas mutation (K524A) of RanGAP1 at its SUMO binding site prevented significant energy transfer. Comparing BRET and fluorescence resonance energy transfer (FRET) efficiencies using this in vitro model system, we observed that BRET efficiency was 3-fold higher than FRET efficiency, due to the lower background signal intensity of EYFP in the BRET system. Consequently, BRET system is expected to be useful for in vitro analysis of SUMOylation as well as studying other protein interactions. [Copyright &y& Elsevier]

In this study, we examined the impact of environmental perturbation on the movement of the toxic bloom-forming alga Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara [syn. H. carterae (Hulburt) F.J.R. Taylor] between vegetative and resting cell phases of the life history. Resting state induction, in batch culture, was most effective when vegetative cells were subjected to low temperature (10° C) and darkness for extended time periods. Heterosigma cells in stasis had neither a cell wall nor scales but were surrounded by a calyx, most probably of polysaccharide composition. The resting cell was completely immobile, although both flagella remained attached. Heterosigma resting cells did not require a maturation period before successful activation to the vegetative state could occur. Cell division and motility were impacted sequentially during both the induction and activation phases of resting cell development. Our data show that Heterosigma had an obligate light requirement for resting cell activation. In replete medium, very low light fluences of 5 μmol photons·m- 2 ·s- 1 were as effective as 60 μmol photons·m- 2 ·s- 1 in the initiation of activation. Such sensitivity to extremely low light might give Heterosigma a competitive advantage for bloom formation in nature. Reduced nitrate levels significantly shortened the temporal transition of vegetative cells into the resting cell phase of the life history. Additionally, when resting cells induced in nitrate-limited medium were activated under nitrate-replete condition, the efficiency of the activation response was directly correlated to light availability. Both vegetative and resting cells maintained a haploid DNA complement. Rapid amplified polymorphic DNA (RAPD) analysis demonstrated variation in genetic identity among axenic Heterosigma strains. Strain identity influenced success in... [ABSTRACT FROM AUTHOR]

In this study, we examined the impact of environmental perturbation on the movement of the toxic bloom-forming alga Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara [syn. H. carterae (Hulburt) F.J.R. Taylor] between vegetative and resting cell phases of the life history. Resting state induction, in batch culture, was most effective when vegetative cells were subjected to low temperature (10° C) and darkness for extended time periods. Heterosigma cells in stasis had neither a cell wall nor scales but were surrounded by a calyx, most probably of polysaccharide composition. The resting cell was completely immobile, although both flagella remained attached. Heterosigma resting cells did not require a maturation period before successful activation to the vegetative state could occur. Cell division and motility were impacted sequentially during both the induction and activation phases of resting cell development. Our data show that Heterosigma had an obligate light requirement for resting cell activation. In replete medium, very low light fluences of 5 μmol photons·m- 2 ·s- 1 were as effective as 60 μmol photons·m- 2 ·s- 1 in the initiation of activation. Such sensitivity to extremely low light might give Heterosigma a competitive advantage for bloom formation in nature. Reduced nitrate levels significantly shortened the temporal transition of vegetative cells into the resting cell phase of the life history. Additionally, when resting cells induced in nitrate-limited medium were activated under nitrate-replete condition, the efficiency of the activation response was directly correlated to light availability. Both vegetative and resting cells maintained a haploid DNA complement. Rapid amplified polymorphic DNA (RAPD) analysis demonstrated variation in genetic identity among axenic Heterosigma strains. Strain identity influenced success in... [ABSTRACT FROM AUTHOR]

Aptamers are single-stranded DNA or RNA molecules that can be identified through an iterative in vitro selection–amplification process. Among them, fluorogenic aptamers in response to small molecules have been of great interest in biosensing and bioimaging due to their rapid fluorescence turn-on signals with high target specificity and low background noise. In this review, we report recent advances in fluorogenic aptasensors and their applications to in vitro diagnosis and cellular imaging. These aptasensors modulated by small molecules have been implemented in different modalities that include duplex or molecular beacon-type aptasensors, aptazymes, and fluorogen-activating aptamer reporters. We highlight the working principles, target molecules, modifications, and performance characteristics of fluorogenic aptasensors, and discuss their potential roles in the field of biosensor and bioimaging with future directions and challenges. [ABSTRACT FROM AUTHOR]

In this paper, the fault current limiting (FCL) characteristics of a flux-coupled type superconducting fault current limiter (SFCL) with parallel connection between two windings in a DC system were analyzed. The flux-coupled type SFCL was composed of two coils connected in parallel and a superconducting element (SE), which was connected in series with the secondary coil. The flux-coupled type SFCL works in DC systems similar to those in AC systems. Before a fault occurs, the respective magnetic fluxes generated by the two coils connected in parallel offset each other, maintaining the voltage induced in the two coils at zero. In case of a fault, however, resistance is generated in the SE, preventing the magnetic fluxes generated by the two coils from offsetting each other. Thus, some voltage is induced in the two coils, and this starts to limit the fault current. DC short circuit tests were conducted, and the test results confirmed that the flux-coupled type SFCL with the two parallel connected coils was effective in limiting the fault current in a DC system. Additionally, the effect of the wiring direction of the two coils on the SFCL's FCL performance and operating current, limiting impedance, and instantaneous power load was further analyzed, and as a result, the performance conditions of the SFCL in a DC system were determined. [ABSTRACT FROM AUTHOR]

Abstract: Interaction between streptavidin and biotin on poly(amidoamine) (PAMAM) dendrimer-activated surfaces and on self-assembled monolayers (SAMs) was quantitatively studied by using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface protein density was systematically varied as a function of protein concentration and independently quantified using the ellipsometry technique. Principal component analysis (PCA) and principal component regression (PCR) were used to identify a correlation between the intensities of the secondary ion peaks and the surface protein densities. From the ToF-SIMS and ellipsometry results, a good linear correlation of protein density was found. Our study shows that surface protein densities are higher on dendrimer-activated surfaces than on SAMs surfaces due to the spherical property of the dendrimer, and that these surface protein densities can be easily quantified with high sensitivity in a label-free manner by ToF-SIMS. [Copyright &y& Elsevier]

After the publication of this work [1] errors were found in Figs. 1a and 5d. [ABSTRACT FROM AUTHOR]

Glucagon, a peptide hormone clinically used to treat acute hypoglycemia in diabetes patients, is readily degenerated by undergoing fibrillation under pharmaceutical conditions. Since glucagon is employed as a typical model system for structural investigation of amyloid fibril formation of proteins, production of recombinant glucagon is in demand to facilitate mutagenesis and isotope labeling for nuclear magnetic resonance (NMR) studies. In this study, we established a method to produce recombinant glucagon in Escherichia coli. Recombinant plasmids were constructed to express a maltose-binding protein-fused glucagon, which was cleaved by factor-Xa protease to yield glucagon peptide without any N-terminal extra residues. The final product was clearly identified and characterized by immunoblotting, mass spectrometry, circular dichroism spectroscopy, and backbone NMR assignments of the [13C/15N] isotope-enriched sample. Cellular activity of the recombinant glucagon in hepatocytes was confirmed by monitoring characteristic gene expressions. Site-directed mutagenesis was successfully performed using our recombinant production system. Our bacterial production system and the recombinant glucagon not only provide an economical route of glucagon manufacturing, but also enable NMR-based structural investigation of glucagon fibrillation. [Display omitted] • Bacterial production of a recombinant glucagon was established using Escherichia coli. • MBP used as a fusion tag was cleaved by factor-Xa to yield isolated glucagon. • Final product was validated by mass spectrometry and activity assay in hepatocytes. • Mutagenesis and isotope labeling were successful using the production system. • Backbone NMR assignments were completed by heteronuclear multidimensional NMR. [ABSTRACT FROM AUTHOR]

To resolve the problem of target specificity and light transmission to deep-seated tissues in photodynamic therapy (PDT), we report a cancer cell-targeted photosensitizer using photoprotein-conjugated upconversion nanoparticles (UCNPs) with high target specificity and efficient light transmission to deep tissues. Core-shell UCNPs with low internal energy back transfer were conjugated with recombinant proteins that consists of a photosensitizer (KillerRed; KR) and a cancer cell-targeted lead peptide (LP). Under near infrared (NIR)-irradiating condition, the UCNP-KR-LP generated superoxide anion radicals as reactive oxygen species via NIR-to-green light conversion and exhibited excellent specificity to target cancer cells through receptor-mediated cell adhesion. Consequently, this photosensitizing process facilitated rapid cell death in cancer cell lines (MCF-7, MDA-MB-231, and U-87MG) overexpressing integrin beta 1 (ITGB1) receptors but not in a cell line (SK-BR-3) with reduced ITGB1 expression and a non-invasive normal breast cell line (MCF-10A). In contrast to green light irradiation, NIR light irradiation exhibited significant PDT efficacy in cancer cells located beneath porcine skin tissues up to a depth of 10 mm, as well as in vivo tumor xenograft mouse models. This finding suggests that the designed nanocomposite is useful for sensing and targeting various deep-seated tumors. [ABSTRACT FROM AUTHOR]

Introduction: Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein with putative functions in cell proliferation, angiogenesis and differentiation. Expression of ECM1 in several types of carcinoma suggests that it may promote tumor development. In this study, we investigated the role of ECM1 in oncogenic cell signaling in breast cancer, and potential mechanisms for its effects.Methods: In order to find out the functional role of ECM1, we used the recombinant human ECM1 and viral transduction systems which stably regulated the expression level of ECM1. We examined the effect of ECM1 on cell proliferation and cell signaling in vitro and in vivo. Moreover, tissues and sera of patients with breast cancer were used to confirm the effect of ECM1.Results: ECM1 protein was increased in trastuzumab-resistant (TR) cells, in association with trastuzumab resistance and cell proliferation. Through physical interaction with epidermal growth factor receptor (EGFR), ECM1 potentiated the phosphorylation of EGFR and extracellular signal-regulated kinase upon EGF treatment. Moreover, ECM1-induced galectin-3 cleavage through upregulation of matrix metalloproteinase 9 not only improved mucin 1 expression, but also increased EGFR and human epidermal growth factor receptor 3 protein stability as a secondary signaling.Conclusions: ECM1 has important roles in both cancer development and trastuzumab resistance in breast cancer through activation of EGFR signaling. [ABSTRACT FROM AUTHOR]

Despite the simplicity and usability of gold nanoparticle (AuNP)-based colorimetry in a large panel of bioassays, this technique still suffers from a poor detection limit. To overcome this issue, we report an improved AuNP-based colorimetric assay in combination with simple centrifugation and silver enhancement. As a proof-of-concept, two types of enzymes (i.e., protein phosphatase and protease) were constructed based on the self-assembly of thiol-stabilized AuNPs in the presence of peptides and metal ions. When the AuNP solutions were subjected to a short cycle of centrifugation and silver enhancement, the signal-to-background ratio (SBR) was distinctly improved by a factor of 5–10 in both enzyme activity assays, as compared to that of AuNP-based colorimetry. The detection limit achieved by using the silver enhancement was determined to be improved by a factor of 1.0–3.4. Furthermore, grayscale images of the silver enhancement allowed for a rapid and simple enzyme assay that did not require measuring the absorbance. Due to its ability to improve the detection senstivity in a facile way, we anticipate that this approach will be suitable for use in many AuNP colorimetric assays. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the spatial and energetic distributions of electrons trapped within a SiO2/HfO2 dual layer gate stack (EOT = 1 nm) of fully processed high-k/metal gate nFETs were investigated by discharging-sweep mode amplitude charge pumping (DSACP). DSACP enables the separate energy profiling of the traps in the SiO2 and HfO2 layers of a SiO2/HfO2 gate stack. The electrical measurement of the spatial/energetic trap profiles with DSACP is based on the electron de-trapping mechanism, which allows scanning to be performed below the conduction band of Si in terms of both the depth and energy. The results show that shallower traps appear in the HfO2 layer with increasing discharging time and a significant correlation exists between the density of the shallow traps and positive bias temperature instability (PBTI) characteristics. [Copyright &y& Elsevier]

Despite the therapeutic potential of recombinant proteins, their cell permeabilities and stabilities remain significant challenges. Here we demonstrate that cyclized recombinant proteins can be used as universal cargos for permeable and stable delivery into cells and polydiacetylene liposomes. Utilizing a split intein-mediated process, cyclized model fluorescent proteins containing short tetraarginine (R 4) and hexahistidine (H 6) tags were generated without compromising their native protein functions. Strikingly, as compared to linear R 4 /H 6 -tagged proteins, the cyclized counterparts have substantially increased permeabilities in both cancer cells and synthetic liposomes, as well as higher resistances to enzymatic degradation in cancer cells. These properties are likely a consequence of structural constraints imposed on the proteins in the presence of short functional peptides. Additionally, photodynamic therapy by cyclized photoprotein-loaded liposomes in cancer cells was significantly improved in comparison to that by their non-cyclized counterparts. These findings suggest that our strategy will be universally applicable to intercellular delivery of proteins and therapeutics. • Cyclization of proteins with tags enhances membrane permeability and stability. • Functions of cyclized proteins are effective in both cells and PCDA liposomes. • This strategy allows liposome-mediated cytosolic delivery of proteins. • Cyclized photoproteins enable photodynamic therapeutic effect on cancer cells. • This strategy serves as a universal cargo platform for protein delivery. [ABSTRACT FROM AUTHOR]

Cetuximab is a murine-human chimeric monoclonal antibody (mAb) that is clinically used to treat epidermal growth factor receptor (EGFR)-positive cancers. As antibody fragment engineering has emerged as an economic alternative to mAb drugs via bacterial production, we have previously generated FM318, a recombinant Fab adopted from cetuximab. Here, in an effort to facilitate industrial development, we searched for useful mutations that could increase its production yields. Amino acid substitutions were selected to resemble the humanized sequence to avoid unexpectedly raising immunogenic problems by the mutations. As a result, FM329, which accommodates L3Q/L4 M mutations in the light chain and S15G/Q16G in the heavy chain Fd, showed a high production yield in a fed-batch operation, reaching approximately 3.5 times FM318 productivity, with its structure and EGFR-binding affinity being maintained. Additionally, for a potential application to antibody-drug conjugates, a cytotoxic agent, DM1 was successfully linked to FM329 with an average drug-to-antibody ratio of 1.4. The conjugate showed dramatically increased anticancer activity with retention of EGFR-binding affinity. Collectively, we suggest that the partially humanized recombinant cetuximab Fab, FM329, and its DM1 conjugate would serve as promising platforms to develop an economic alternative to cetuximab and/or an improved drug candidate for a potent anti-cancer therapy. [ABSTRACT FROM AUTHOR]

An insufficient sensitivity of aptamer-integrated colorimetric gold nanoparticles (AuNPs) is a common challenge in the detection of environmental chemicals. We report the use of a modified aptamer/AuNP-based sensor in conjunction with a fluorescing single-stranded DNA aptamer for the sensitive detection of bisphenol A (BPA), which is a major endocrine disruptor (EDC). While an anti-BPA single-stranded DNA aptamer was bound with BPA, a weak fluorescence signal was observed upon the addition of SYBR Green-I (SG-I), which is specific to a duplex region of the free aptamer. This reaction was accompanied by a subsequent aggregation of unmodified AuNPs (citrate-stabilized AuNPs) at high salt concentrations, leading to a color change from reddish to purple. In contrast, the absence of BPA elicited a high fluorescence signal from SG-I and produced no color change in the AuNP solution even upon the addition of salt ions. Unlike aptamers that are labeled at their ends with fluorophores, our strategy gave rise to neither a loss of target-binding affinity nor a perturbation of the AuNP colorimetry. Thus, a quantitative analysis with a broad dynamic range was achieved by combining the fluorescent and colorimetric measurements at BPA concentrations ranging over four orders of magnitude. Our approach also yielded a greater detection sensitivity (as low as 9 pg·mL −1 ) than classical AuNP colorimetry or other aptamer-combined methods Moreover, this method enabled the detection of BPA in a small fraction extracted from thermal paper with a high specificity among EDCs. We anticipate that this approach will facilitate further advances in the design of traditional AuNP colorimetric sensors using different aptamers for chemical sensing in the environmental and clinical fields. [ABSTRACT FROM AUTHOR]

Despite the major role of cancer antigen 125 (CA125) in cancer progression, its structural diversity makes antibody-based immunoassay of this protein difficult. Here we report the selection of an anti-CA125 ssDNA aptamer and its application to sensitive detection of CA125 when used together with a CA125-specific antibody immobilized on a three-dimensional network of carbon nanotubes (3DN-CNTs). With exceptional stability and easy modifications over antibodies, the selected ssDNA aptamer (rCAA-8) showed high binding affinity (166 nM) for recombinant CA125, which enabled CA125-specific imaging in ovarian cancer cells (OVCAR-3). Furthermore, when compared to other fluorescent assays based on graphene oxide or flat surface and traditional enzyme-linked immunosorbent assay, a chip-based assay using a 3DN-CNT surface and anti-CA125 antibody-aptamer pair resulted in higher sensitivity and broader dynamic range as a function of CA125 concentration, due to high target specificity and high surface loading density. Our on-chip aptamer-based assay will facilitate sensitive and specific monitoring of CA125 in the biological and clinical fields. [ABSTRACT FROM AUTHOR]

We report the development of amplification-free surface-enhanced Raman scattering (SERS)-based DNA assays for the rapid and highly sensitive detection of prostate cancer antigen 3 (PCA3) mimic DNA. This technique does not require any DNA amplification process using thermo-cycles in conventional polymerase chain reaction (PCR) due to its highly sensitive detection capability. Herein, the PCA3 mimic DNA, composed of 45 nucleotide sequences, was sandwiched between two probe DNA-immobilized particles (ASO 735 -conjugated detection HGNs and ASO 683 -immobilized capture magnetic beads) by hybridization reactions. Its quantitative analysis was performed by monitoring the characteristic Raman peak intensity of sandwich DNA complexes. The limit of detection (LOD) is estimated to be 2.7 fM, which is about four orders of magnitude more sensitive than that of conventional PCR. This SERS-based DNA assay technique is expected to be a potentially useful tool for early disease diagnosis. [ABSTRACT FROM AUTHOR]

Although cyclic AMP receptor protein ( CRP) has long served as a typical example of effector-mediated protein allostery, mechanistic details into its regulation have been controversial due to discrepancy between the known crystal structure and NMR structure of apo- CRP. Here, we report that the recombinant protein corresponding to its C-terminal DNA-binding domain ( CDD) forms a dimer. This result, together with structural information obtained in the present NMR study, is consistent with the previous crystal structure and validates its relevance also in solution. Therefore, our findings suggest that dissociation of the CDD may be critically involved in cAMP-induced allosteric activation of CRP. [ABSTRACT FROM AUTHOR]

Despite therapeutic benefits of singlet oxygen (1O 2), the development of reliable 1O 2 detection methods is challenging due to its short lifespan and fast reactivity. Herein, we report a photodynamic color sensor for visualizing photoinduced 1O 2 using a 10,12-pentacosadiynoic acid (PCDA) vesicle and a mini singlet oxygen generator (miniSOG). The miniSOG produced 1O 2 by photoactivation, which subsequently reacted with the PCDA vesicle, resulting in a rapid color change from colorless monomeric PCDA to blue-colored polydiacetylene (PDA) vesicles (from yellow to green in the miniSOG-mixed solutions) through electron delocalization-mediated polymerization. Therefore, the color change from PCDA to PDA enabled the rapid and simple detection of photoinduced 1O 2 and its scavengers on a microplate. Notably, the PCDA vesicles exhibited better sensitivity and stability for photoinduced 1O 2 than classical chromogenic reagents. Along with the unique chromatic property and structural versatility of PCDA, we suggest that our approach is highly suitable for visualizing 1O 2 in a rapid and simple way. [Display omitted] • Rapid determination of 1O 2 is highly demanded for its therapeutic applications. • A photodynamic color sensor for 1O 2 was developed using diacetylene vesicles. • Colorless PCDA vesicles turned into blue PDA vesicles by photoinduced 1O 2. • This platform is useful for visualizing 1O 2 in a rapid and simple way. [ABSTRACT FROM AUTHOR]

Activity-based monitoring of cell-secreted proteases has gained significant interest due to the implication of these substances in diverse cellular functions. Here, we demonstrated a cell-based method of monitoring protease activity using fluorescent cell-permeable peptides. The activatable peptide consists of anionic (EEEE), cleavable, and cationic sequences (RRRR) that enable intracellular delivery by matrix metalloproteinase-2 (MMP2), which is secreted by living cancer cells. Compared to HT-29 cells (MMP2-negative), HT-1080 cells (MMP2-positive) showed a strong fluorescence response to the short fluorescent peptide via cell-secreted protease activation. Our approach is expected to find applications for the rapid visualization of protease activity in living cells. [ABSTRACT FROM AUTHOR]

Acteoside, an active phenylethanoid glycoside, has been used traditionally as an anti-inflammatory agent. The molecular mechanism by which acteoside reduces inflammation was investigated in lipopolysaccharide (LPS)-induced Raw264.7 cells and in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. In vitro, acteoside inhibits high mobility group box 1 (HMGB1) release and iNOS/NO production and induces heme oxygenase-1 (HO-1) expression in a concentration-dependent manner, while HO-1 siRNA antagonizes the inhibition of HMGB1 and NO. The effect of acteoside is inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and Nfr2 siRNA, indicating that acteoside induces HO-1 via p38 MAPK and NF-E2-related factor 2 (Nrf2). In vivo, acteoside increases survival and decreases serum and lung HMGB1 levels in CLP-induced sepsis. Overall, these results that acteoside reduces HMGB1 release and may be beneficial for the treatment of sepsis. [ABSTRACT FROM AUTHOR]

Abstract: Among proteases, matrix metalloproteinases (MMPs) have been of significant interest because they are considered as one of the promising biomarkers in association with cancer metastasis, inflammation and other degenerative diseases. Many attempts based on the optical sensing have been made to analyze the activity of MMPs, but most of them require an expensive fluorescence readout and a labor-intensive process. To circumvent this issue, we demonstrated a simple colorimetric detection of protease activity by using carboxy gold nanoparticles (AuNPs) and histidine-containing peptides via metal-affinity coordination. Due to their higher surface-to-volume ratio, the nanometer size of AuNPs enables the surface ligands to function like a chelator, providing greater affinity with metal ions, even in the absence of chelators. With no additional modification by multidentate ligands, the carboxy AuNPs were easily aggregated and changed in color (from reddish-brown to violet) after adding peptide substrates with hexahistidine at both ends and metal ions, whereas the presence of proteases in solution prevented NP aggregation by cleaving the peptides, thereby retaining the original color of the AuNPs. When the extinction ratio (E 520/E 700) of the AuNP solution was measured as a function of matrix metalloproteinase concentration in a single reaction, there was good linearity from as low as 3nM to 52nM. This approach is anticipated to be useful in designing other diagnostic nanosensors. [Copyright &y& Elsevier]

Abstract: We report the novel activity-based detection of antifreeze protein (AFP), also known as ice-binding protein (IBP), using freeze-labile gold nanoparticles (AuNPs) in order to overcome labor-intensive and low throughput issues of the current method based on thermal hysteresis (TH). Upon the addition of either CnAFP from the Antarctic diatom Chaetoceros neogracile or LeIBP from the Arctic yeast Leucosporidium sp. to mercaptosuccinic acid-capped AuNP, the self-assembly of AuNPs was highly inhibited after a freezing/thawing cycle, leading to no color change in the AuNP solution. As a result, the aggregation parameter (E 520/E 650) of AuNP presented the rapid detection of both the concentration-dependent activity and stability of two AFPs with high sensitivity, where the detection range was 100-fold lower than that of the TH-based method. We suggest that our newly developed method is very suitable for simple and high-throughput measurement of AFP activity. [Copyright &y& Elsevier]

We report a gold nanoparticle (AuNP)-based fluorescence quenching system via metal coordination for the simple assay of protease activity. Carboxy AuNPs (5 nm in core diameter) functioned as both quenchers and metal chelators without requiring further modification with multidentate ligands; therefore, they were strongly associated with the hexahistidine regions of dye-tethered peptides in the presence of Ni(II) ions, leading to notable fluorescence quenching over the varying molar ratios of dye to AuNP. Upon the addition of matrix metalloproteinase-7 (MMP-7), the fluorescent intensity was efficiently recovered in one-pot mixture especially at 10:1-100:1 molar ratios of dye to AuNP. Consequently, the dequenching degree was dependent on the MMP-7 concentration in a hyperbolic manner, ranging from as low as 10 to 1,000 ng mL. In this regard, we anticipate that the developed system will give us a general way to construct nanoparticle-dye conjugates and will find applications in the analyses of many other proteases mediating significant biological processes with low background and high sensitivity. [ABSTRACT FROM AUTHOR]

To resolve time-consuming and imperceptible monitoring problems in the traditional systematic evolution of ligands by exponential enrichment (SELEX), we report gold nanoparticle-assisted SELEX (GNP-SELEX) as a visual, proofreading, and self-monitoring platform and its application to small molecule-binding single-stranded DNA (ssDNA) aptasensors. Through the colorimetric changes between rounds, GNP-SELEX enabled the rapid determination of target-specific aptamer library enrichment with neither target modification nor extra monitoring process. We identified ssDNA aptamers with high selectivity and binding affinity by targeting two small molecules (brassinolide; BL and bisphenol A; BPA) as a model. The rational design of selected aptamers by 3D molecular simulation increased their ability to detect BL or BPA in real samples as bioreceptors. These results suggest that GNP-SELEX is useful as a self-monitoring platform to discover ssDNA aptamers as well as to develop aptasensors for diverse targets in a rapid and simple way. • GNP-SELEX as a colorimetric SELEX platform was demonstrated. • GNP-SELEX enabled visual self-monitoring in and between SELEX rounds. • We discovered novel ssDNA aptamers to brassinolide and bisphenol A. • This platform facilitated the development of aptasensors against two small molecules. [ABSTRACT FROM AUTHOR]

• We report an ICP-coupled electrokinetic method for the detection of LMW thiols. • The interaction between the redox regulator and DNA operator sensed LMW thiols. • The protein/DNA complex was rapidly separated by LMW thiols in microfluidics. • This platform is useful for monitoring rapid redox changes in LMW thiols. Despite the diagnostic potential of low-molecular-weight (LMW) thiols in human diseases, their rapid and simple detection is challenging due to their reversible redox changes. Herein, we report a rapid electrokinetic detection of LMW thiols using the interaction between Bacillus subtilis -derived organic hydroperoxide resistance regulatory protein (OhrR BS) and its operator DNA element in ion concentration polarization (ICP)-coupled microfluidic multiple channels. The dimeric OhrR BS was tightly bound to the dye-labeled dsDNA element with high binding affinity (K D ≈ 4 nM) under the reduced conditions. The presence of organic hydroperoxide (OHP) and LMW thiol (X–SH) caused rapid oxidation of the reduced Cys residue (OhrR BS –SH) of the protein into the sulfenic acid form (OhrR BS –SOH) by OHP and subsequently into the disulfide form (OhrR BS –S–S–X) by LMW thiol via S-thiolation, leading to a rapid release of OhrR BS from the fluorescent dsDNA element. Based on this principle, the fluorescence zones of the dsDNA-protein complex in response to LMW thiols were rapidly separated in the microfluidic channels by electrokinetic mobility. Owing to the ability of ICP-coupled microfluidics to concentrate charged samples, this electrokinetic method enabled the rapid determination (<35 min) and improved sensitivity (>2 μM) of LMW thiols, depending on their molecular weights and concentrations. Additionally, this strategy enabled the simultaneous detection of free and total LMW thiols in mouse serum. These results suggest that the ICP-coupled microfluidic platform in combination with the OhrR BS –DNA complex will be useful for monitoring rapid redox changes of LMW thiols in real samples. [ABSTRACT FROM AUTHOR]

Anthocyanins play critical roles in protecting plant tissues against diverse stresses. The complicated regulatory networks induced by various environmental factors modulate the homeostatic level of anthocyanins. Here, we show that anthocyanin accumulation is induced by brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana) shoots and shed light on the underlying regulatory mechanism. We observed that anthocyanin levels are altered considerably in BR-related mutants, and BRs induce anthocyanin accumulation by upregulating the expression of anthocyanin biosynthetic genes. Our genetic analysis indicated that BRASSINAZOLE RESISTANT 1 (BZR1) and PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) are essential for BR-induced anthocyanin accumulation. The BR-responsive transcription factor BZR1 directly binds to the PAP1 promoter, regulating its expression. In addition, we found that intense anthocyanin accumulation caused by the pap1-D-dominant mutation is significantly reduced in BR mutants, implying that BR activity is required for PAP1 function after PAP1 transcription. Moreover, we demonstrated that BZR1 physically interacts with PAP1 to cooperatively regulate the expression of PAP1-target genes, such as TRANSPARENT TESTA 8, DIHYDROFLAVONOL 4-REDUCTASE, and LEUKOANTHOCYANIDIN DIOXYGENASE. Our findings indicate that BZR1 functions as an integral component of the PAP1-containing transcription factor complex, contributing to increased anthocyanin biosynthesis. Notably, we also show that functional interaction of BZR1 with PAP1 is required for anthocyanin accumulation induced by low nitrogen stress. Taken together, our results demonstrate that BR-regulated BZR1 promotes anthocyanin biosynthesis through cooperative interaction with PAP1 of the MBW complex., Competing Interests: Conflict of interest statement. The authors declare no confilct of interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Purpose: To unravel the mechanism regulating the phosphorylation of glycogen synthase kinase 3 (GSK3) and the correlation between the inhibitory phosphorylation of GSK3α and sperm motility in human., Materials and Methods: The phosphorylation and priming phosphorylated substrate-specific kinase activity of GSK3 were examined in human spermatozoa with various motility conditions., Results: In human spermatozoa, GSK3α/β was localized in the head, midpiece, and principal piece of tail and p-GSK3α(Ser21) was enriched in the midpiece. The ratio of p-GSK3α(Ser21)/GSK3α was positively coupled with normal sperm motility criteria of World Health Organization. In high-motility spermatozoa, p-GSK3α(Ser21) phosphotyrosine (p-Tyr) proteins but p-GSK3α(Tyr279) markedly increased together with decreased kinase activity of GSK3 after incubation in Ca 2+ containing medium. In high-motility spermatozoa, p-GSK3α(Ser21) levels were negatively coupled with kinase activity of GSK3, and which was deregulated in low-motility spermatozoa. In high-motility spermatozoa, 6-bromo-indirubin-3'-oxime, an inhibitor of kinase activity of GSK3 increased p-GSK3α(Ser21) and p-Tyr proteins. p-GSK3α(Ser21) and p-Tyr protein levels were decreased by inhibition of PKA and Akt. Calyculin A, a protein phosphatase-1/2A inhibitor, markedly increased the p-GSK3α(Ser21) and p-Tyr proteins, and significantly increased the motility of low-motility human spermatozoa., Conclusions: Down regulation of kinase activity of GSK3α by inhibitory phosphorylation was positively coupled with human sperm motility, and which was regulated by Ca 2+ , PKA, Akt, and PP1. Small-molecule inhibitors of GSK3 and PP1 can be considered to potentiate human sperm motility., Competing Interests: The authors have nothing to disclose., (Copyright © 2024 Korean Society for Sexual Medicine and Andrology.)

Owing to the strong absorption of water in the near-infrared (NIR) region near 1.0 μm, this wavelength is considered unsuitable as an imaging and analytical signal in biological environments. However, 1.0 μm NIR can be converted into heat and used as a local water-molecular heating strategy for the photothermal therapy of biological tissues. Herein, we describe a Nd-Yb co-doped nanomaterial (water-heating nanoparticles (NPs)) as strong 1.0 μm emissive NPs to target the absorption band of water. Furthermore, introducing Tm ions into the water-heating NPs improve the NIR lifetime, enabling the development of a NIR imaging-guided water-heating probe (water-heating NIR NPs). In the glioblastoma multiforme male mouse model, tumor-targeted water-heating NIR NPs reduce the tumor volume by 78.9% in the presence of high-resolution intracranial NIR long-lifetime imaging. Hence, water-heating NIR NPs can be used as a promising nanomaterial for imaging and photothermal ablation in deep-tissue-bearing tumor therapy., (© 2023. The Author(s).)

Photodynamic therapy (PDT) has been considered a noninvasive and cost-effective modality for tumor treatment. However, the complexity of tumor microenvironments poses challenges to the implementation of traditional PDT. Here, we review recent advances in PDT to resolve the current problems. Major breakthroughs in PDTs are enabling significant progress in molecular medicine and are interconnected with innovative strategies based on smart bio/nanomaterials or therapeutic insights. We focus on newly developed PDT strategies designed by tailoring photosensitive reactive oxygen species generation, which include the use of proteinaceous photosensitizers, self-illumination, or oxygen-independent approaches. While these updated PDT platforms are expected to enable major advances in cancer treatment, addressing future challenges related to biosafety and target specificity is discussed throughout as a necessary goal to expand the usefulness of PDT.

Despite the potential of photodynamic therapy (PDT), its comprehensive use in cancer treatment has not been achieved because of the nondegradable risks of photosensitizing drugs and limits of light penetration and instrumentation. Here, we present bioluminescence (BL)-induced proteinaceous PDT (BLiP-PDT), through the combination of luciferase and a reactive oxygen species (ROS)-generating protein (Luc-RGP), which is self-luminescent and degradable. After exposure to coelenterazine- h as a substrate for luciferase without external light irradiation, Luc-RGP fused with a small lead peptide-induced breast cancer cell death through the generation of BL-sensitive ROS in the plasma membrane. Even with extremely low light energy, BLiP-PDT exhibited targeted effects in primary breast cancer cells from patients and in in vivo tumor xenograft mouse models. These findings suggest that BLiP-PDT is immediately useful as a promising theranostic approach against various cancers., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Despite the diverse roles of cell-secreted proteases in the extracellular matrix (ECM), classical methods to analyze protease activity have not been explored at the cell culture site. Here, we report a stable, matrix-sticky, and protease-sensitive extracellular reporter that comprises a collagen-binding protein and a Förster resonance energy transfer (FRET) coupler of an enhanced green fluorescent protein and a small dye molecule. The extracellular FRET reporter via split intein-mediated protein trans-splicing is able to adhere to collagen matrices, leading to fluorescence changes by matrix metalloproteinase-2 (MMP2) activity during living cell culture without impeding cell viability. When a proMMP2 mutant (Y581A) with altered protease secretion and activity was transfected into cancer cells, the reporter revealed a dramatic reduction in MMP2 activity in both two- and three-dimensional culture systems, compared with cells transfected with wild-type proMMP2. Our reporter is immediately amenable to monitor protease activity in diverse ECM-resident cells as well as to study protease-related extracellular signaling and tissue remodeling.

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

: Glucose biosensors have an important place in the diagnosis of diabetes as well as in various food and biotechnological processes. Recent advances in nanomaterials have directly improved enzymatic glucose biosensors owing to their distinguished structural and physiochemical properties. Here, we review the recent developments in electrochemical and fluorescent glucose biosensors based on nanomaterials. New technologies that combine nanomaterials with glucose-sensing enzymes will result in promising glucose biosensors with high specificity and sensitivity.

A secondary mutation (T790M) in epidermal growth factor receptor (EGFR) is a hallmark of acquired resistance to EGFR inhibitors used to treat non-small-cell lung cancer (NSCLC). Therefore, identifying the T790M mutation is crucial to guide treatment decisions. Given that DNA sequencing methods are time-consuming and insensitive, we developed and investigated the feasibility of using molecular beacons for the detection of the T790M mutation in EGFR. A molecular beacon complementary to the region of the secondary EGFR mutation (T790M) was designed and used in NSCLC samples bearing drug-sensitive and -resistant EGFR mutations. For a rapid and simple assay, we attempted to use the molecular beacon with real-time PCR and in situ fluorescence imaging. The ability of the designed molecular beacon to specifically detect the T790M mutation of EGFR was tested for samples from two patients with drug resistance and compared with conventional DNA sequencing methods. The molecular beacon successfully detected the T790M mutation in patient samples with drug resistance. The sensitivity of the molecular beacon, which detected as little as 2% of genomic DNA from the drug-resistant cells (H1975), was much higher than direct sequencing. Furthermore, in situ fluorescence imaging with the molecular beacon gave rise to a distinguishable signal for the T790M mutation in drug-resistant cells. The molecular beacon-based approach enabled rapid and sensitive detection of the EGFR mutation (T790M) in NSCLC with in situ fluorescence imaging, which can be directed to determine various treatment options in patients with cancer.

We describe a chip-based method to detect protein glycosylation based on the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs). Our assay system relies on modulations in the energy transfer between the nanoparticles on a surface. The photoluminescence (PL) of lectin-coated QDs (energy donor) immobilized on a glass slide is quenched by carbohydrate-coated AuNPs (energy acceptor), and the presence of the glycoprotein causes the increase of the PL of QDs. As a proof-of-concept, Concanavalin A-coated QDs (ConA-QDs) and dextran-coated AuNPs (Dex-AuNPs) were used to detect the mannosylated proteins. As a result, the PL intensity of QDs was found to be linearly correlated with the concentration and the number of glycan moiety of the glycoprotein. We anticipated that our simple assay system will find applications for the analysis of glycoproteins with high selectivity and sensitivity in a high-throughput manner.

A label-free, activity-based assay of matrix metalloproteinase (MMP) and its inhibition was demonstrated on peptide-conjugated gold nanoparticles (AuNPs) with nonbiofouling poly(oligo(ethylene glycol) methacrylate) (pOEGMA) films using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Following surface-initiated atom-transfer radical polymerization of OEGMA on a Si/SiO2 substrate, the MMP activity was determined by analyzing the cleaved peptide fragments using TOF-SIMS on the peptide-conjugated AuNPs. The use of nonbiofouling pOEGMA films in conjunction with AuNPs synergistically enhanced the sensitivity of assays for MMP activity and its inhibition in human serum. The detection sensitivity of MMP-7 in serum was as low as 20 ng mL(-1) (1 pmol mL(-1)), and the half-maximal inhibitory concentration (IC50) of minocycline, which is a MMP-7 inhibitor, was estimated to be 450 nM. It is anticipated that the developed system will be broadly useful for conducting activity-based assays of serum proteases, as well as for screening of their inhibitors, with high sensitivity in a high-throughput manner.

Rapid and sensitive assay of proteases and their inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. We developed a multiplexed assay system of proteases and their inhibition by measuring the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) on a glass slide. In this system, while the photoluminescence (PL) of donor QDs immobilized on a surface was quenched due to the presence of AuNPs (energy acceptor) in close proximity, the protease activity caused modulation in the efficiency of the energy transfer between the acceptor and donor, thus enabling the protease assay. In comparison to the QD-dye system, the conjugate of the QD-AuNP gave rise to higher energy transfer efficiency, resulting in quantitative assay of proteases with more sensitivity. When matrix metalloproteinase, caspase, and thrombin were tested, a multiplexed assay was successfully achieved since the AuNP could be used as a common energy acceptor in conjunction with QDs having different colors. Our system is anticipated to find applications in the diagnosis of protease-related diseases and screening of potential drugs with high sensitivity in a high-throughput way.

We demonstrate that protein kinase can be assayed with high sensitivity on peptide-conjugated gold nanoparticles (AuNPs). Phosphorylation of peptides on the AuNP-monolayers was detected by using an anti-phosphotyrosine antibody (alpha-pY) and Cy3-labeled secondary antibody (Cy3-alpha-mIgG) as a probing molecule. When compared to conventional self-assembled monolayers (SAMs), spherical and three-dimensional geometry of AuNPs led to high surface density of peptide substrate and easy accessibility to enzyme, and consequently the resulting AuNP monolayers gave rise to improved detection sensitivity. Blocking of peptide-conjugated AuNPs with a poly(ethylene glycol) (PEG) also contributed to a higher signal-to-background ratio in kinase and its inhibition assays. The use of AuNPs as the platform surface will enable highly sensitive detection of protein kinases in a high-throughput manner.

We demonstrate the effects of protein orientation and trehalose on a quantitative analysis of surface-immobilized proteins by using time-of-flight secondary ion mass spectrometry (TOF-SIMS). As our model protein, streptavidin (SA) was quantitatively immobilized on a solid surface at different configurations by random or oriented immobilization and subsequently treated with trehalose. The resulting surface was analyzed by using TOF-SIMS and surface plasmon resonance (SPR) spectroscopy, where the secondary ion spectra from SA were compared with the surface density of the protein. In the case of oriented immobilization, the ion peak intensities measured by TOF-SIMS were correlated well with the SPR data, regardless of the presence of trehalose. Alternatively, trehalose significantly increased correlation between TOF-SIMS and SPR data for the randomly immobilized SA. It is likely that a trehalose-treated surface is less vulnerable to denaturation, thus leading to a reliable quantification of surface-immobilized proteins by TOF-SIMS. Our results show that TOF-SIMS can be used for understanding biophysical states such as orientation and denaturation of surface-immobilized proteins as well as for quantifying proteins within the field of biosensors and biochips.