Advanced Bioluminescence Reporter with Engineered Luciferase via Sequence-Guided Mutagenesis.

luciferase (Luc) is the preeminent secreted luciferase widely used in cell-based reporter assays. By employing sequence-guided mutagenesis informed by alignments of diverse copepod luciferase sequences, we identified key amino acids that significantly enhance bioluminescence (BL) intensity. Among the mutated proteins expressed in bacteria, five individual mutations (M60L, K88Q, F89Y, I90L, or S103T) independently increased BL intensity by 1.

Transcription factors BZR1 and PAP1 cooperate to promote anthocyanin biosynthesis in Arabidopsis shoots.

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.

Regulation of Phosphorylation of Glycogen Synthase Kinase 3α and the Correlation with Sperm Motility in Human.

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.

Cyclized proteins with tags as permeable and stable cargos for delivery into cells and liposomes.

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) and hexahistidine (H) tags were generated without compromising their native protein functions.

Photosensitizing deep-seated cancer cells with photoprotein-conjugated upconversion nanoparticles.

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.

A local water molecular-heating strategy for near-infrared long-lifetime imaging-guided photothermal therapy of glioblastoma.

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.

Activatable Peptides for Rapid and Simple Visualization of Protease Activity Secreted in Living Cells.

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.

Gold nanoparticle-assisted SELEX as a visual monitoring platform for the development of small molecule-binding DNA aptasensors.

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.

Tailoring photosensitive ROS for advanced photodynamic therapy.

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.

Self-luminescent photodynamic therapy using breast cancer targeted proteins.

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- 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.

Collagen-Immobilized Extracellular FRET Reporter for Visualizing Protease Activity Secreted by Living Cells.

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.

Microbial Redox Regulator-Enabled Pulldown for Rapid Analysis of Plasma Low-Molecular-Weight Biothiols.

Although low-molecular-weight (LMW) biothiols function as a disease indicator in plasma, rapidly and effectively analyzing them remains challenging in the extracellular oxidative environment due to technical difficulties. Here, we report a newly designed, affinity pulldown platform using a -derived organic hydroperoxide resistance regulatory (OhrR) protein and its operator dsDNA for rapid and cost-effective analyses of plasma LMW biothiols. In the presence of organic hydroperoxide, LMW biothiols triggered the rapid dissociation of FAM-labeled dsDNA from FLAG-tagged OhrR via S-thiolation of OhrR on anti-FLAG antibody-coated beads, which led to a strong increase of fluorescence intensity in the supernatant after pulldown.

Rapid and sensitive determination of bisphenol A using aptamer and split DNAzyme.

Despite the increasing concern regarding bisphenol A (BPA) as an endocrine disrupting chemical (EDC) upon environmental or human exposure, development of simple method for BPA detection has been hampered, due to the lack of a stable bioreceptor and signal generator. Here, we report a nucleic acid-based rapid and sensitive method for BPA detection, which constitutes a ssDNA aptamer and ssDNAzyme. When the peroxidase-like DNAzyme sequence was split into two parts (one incorporated into the anti-BPA aptamer as a target recognition element and the other into the complementary sequence as a bait), the presence of BPA hindered the association of the split DNA sequence, leading to a reduced signal in the DNAzyme-triggered chemiluminescence (CL).

Correction to: ECM1 regulates cell proliferation and trastuzumab resistance through activation of EGF-signaling.

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

Identification of the Thioredoxin-Like 2 Autoantibody as a Specific Biomarker for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has a higher risk of death within 5 years of being diagnosed than the other forms of breast cancer. It is the second leading cause of death due to cancer among women. Currently, however, no diagnostic blood-based biomarker exists to identify the early stages of TNBC.

Facile Determination of Sodium Ion and Osmolarity in Artificial Tears by Sequential DNAzymes.

Despite high relevance of tear osmolarity and eye abnormality, numerous methods for detecting tear osmolarity rely upon expensive osmometers. We report a reliable method for simply determining sodium ion-based osmolarity in artificial tears using sequential DNAzymes. When sodium ion-specific DNAzyme and peroxidase-like DNAzyme were used as a sensing and detecting probe, respectively, the concentration of Na⁺ in artificial tears could be measured by absorbance or fluorescence intensity, which was highly correlated with osmolarity over the diagnostic range (² > 0.

C-terminal dimerization of apo-cyclic AMP receptor protein validated in solution.

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.

On-Chip Peptide Mass Spectrometry Imaging for Protein Kinase Inhibitor Screening.

Protein kinases are enzymes that are important targets for drug discovery because of their involvement in regulating the essential cellular processes. For this reason, the changes in protein kinase activity induced by each drug candidate (the inhibitor in this case) need to be accurately determined. Here, an on-chip secondary ion mass spectrometry (SIMS) imaging technique of the peptides was developed for determining protein kinase activity and inhibitor screening without a matrix.

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

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).

Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis.

Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides.

Nanoparticles for Use in Enzyme Assays.

Nanoparticles (NPs) have created new ways to enhance the performance of classical biosensors in analytical sciences. NPs with unprecedented physiochemical properties can serve both as excellent carriers of bioreceptors and as signal enhancers, leading to improved assay platforms with high sensitivity and selectivity. Because enzymes play central roles in many cellular functions, specific and precise assays of their functions are of great significance in medical science and biotechnology.

Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity.

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification.

Creating Anti-icing Surfaces via the Direct Immobilization of Antifreeze Proteins on Aluminum.

Cryoprotectants such as antifreeze proteins (AFPs) and sugar molecules may provide a solution for icing problems. These anti-icing substances protect cells and tissues from freezing by inhibiting ice formation. In this study, we developed a method for coating an industrial metal material (aluminum, Al) with AFP from the Antarctic marine diatom, Chaetoceros neogracile (Cn-AFP), to prevent or delay ice formation.