Genetically encoded biosensors for the detection of rapamycin: toward the screening of agonists and antagonists.

Biosensors are valuable tools for the rapid screening of biological targets with high sensitivity and specificity. It is important to screen biological events in their native context for pharmacological and toxicological applications. However, in vitro biosensors often require purified probes and targets for screening, thus providing limited information on the biological activities of targets in their native environment. To address this issue, we developed a cell-based sensing system that could detect a biologically active small molecule, rapamycin (Rapa). We designed a reporter system based on fluorescence translocation by signal peptide reconstitution. Herein, signal peptides are activated by conditional protein splicing without the need for refolding into a functional tertiary structure, thus eliminating false positives and negatives due to mere binding or misfolding. The developed biosensor demonstrated excellent sensitivity with a limit of detection of 0.1 nM, and it was able to screen the agonist and antagonist of Rapa. The developed cell-based sensing system could contribute to improving the screening system aimed to identify the natural mimetics of Rapa and potential drug candidates.

Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules.

Live-cell-based biosensors have emerged as a useful tool for biotechnology and chemical biology. Genetically encoded sensor cells often use bimolecular fluorescence complementation or fluorescence resonance energy transfer to build a reporter unit that suffers from nonspecific signal activation at high concentrations. Here, we designed genetically encoded sensor cells that can report the presence of biologically active molecules via fluorescence-translocation based on split intein-mediated conditional protein trans-splicing (PTS) and conditional protein trans-cleavage (PTC) reactions. In this work, the target molecules or the external stimuli activated intein-mediated reactions, which resulted in activation of the fluorophore-conjugated signal peptide. This approach fully valued the bond-making and bond-breaking features of intein-mediated reactions in sensor construction and thus eliminated the interference of false-positive signals resulting from the mere binding of fragmented reporters. We could also avoid the necessity of designing split reporters to refold into active structures upon reconstitution. These live-cell-based sensors were able to detect biologically active signaling molecules, such as Ca2+ and cortisol, as well as relevant biological stimuli, such as histamine-induced Ca2+ stimuli and the glucocorticoid receptor agonist, dexamethasone. These live-cell-based sensing systems hold large potential for applications such as drug screening and toxicology studies, which require functional information about targets.

Detection of Metallothionein in Javanese Medaka (Oryzias javanicus), Using a scFv-Immobilized Protein Chip.

Environmental pollution by various industrial chemicals and biological agents poses serious risks to human health. Especially, marine contamination by potentially toxic elements (PTEs) has become a global concern in recent years. Many efforts have been undertaken to monitor the PTE contamination of the aquatic environment. However, there are few approaches available to assess the PTE exposure of aquatic organisms. In this research, we developed a strategy to evaluate the heavy metal exposure of marine organisms, by measuring the expression levels of metallothionein protein derived from Oryzias javanicus (OjaMT). OjaMT is a biomarker of heavy metal exposure because the expression level increases upon heavy metal exposure. The developed assay is based on a real-time, label-free surface plasmon resonance (SPR) measurement. Anti-OjaMT antibody and anti-OjaMT single-chain fragment of variable region (scFv) were used as detection probes. Two types of SPR sensor chips were fabricated, by immobilizing antibody or Cys3-tagged scFv (scFv-Cys3) in a controlled orientation and were tested for in situ label-free OjaMT detection. Compared to the antibody-presenting sensor chips, the scFv-presenting sensor chips showed improved performance, displaying enhanced sensitivity and enabling semi-quantitative detection. The portable SPR system combined with scFv-immobilized sensor chips is expected to provide an excellent point-of-care testing system that can monitor target biomarkers in real time.

Inhibitory peptides derived from Hepatitis C virus NS5A for reducing clinical symptoms of dengue virus infection.

Lethal Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) caused by Dengue virus (DENV) infection necessitate the development of effective treatments. Peptides derived from the N-terminal amphipathic α-helix of hepatitis C virus (HCV) NS5A exhibit antiviral activity by disrupting liposomes with high curvatures, such as virus envelopes. This study engineered five peptides from HCV genotype 3a NS5A N-terminal α-helix and screened them for neutralizing efficacy against three DENV serotypes. Two peptides, 3a 3/20 and DS-05, showed superior therapeutic efficacy against DENV and were further evaluated in treating DHF/DSS induced by mouse-adapted DENV infection. Administration of 3a 3/20 and DS-05 post-infection significantly improved mortality and weight loss associated with DHF/DSS in AG6 mice. These peptides reduced viral load in internal organs and viremia to levels comparable with the positive control drug, JNJ-A07, a DENV NS3-NS4B inhibitor. Additionally, they attenuated the cytokine storm in the blood and expression of inflammatory cytokines in internal organ tissues, ameliorating liver and kidney dysfunction after DENV infection. Histopathological analysis revealed significant suppression of damages in internal organs. These findings suggest that the 3a 3/20 and DS-05 peptides improve clinical symptoms of DHF/DSS induced by DENV infection, indicating their potential for clinical application.

Frontal Alpha Asymmetry Correlates with Suicidal Behavior in Major Depressive Disorder.

OBJECTIVE: Based on the constant associations made between major depressive disorder (MDD) and alpha asymmetry, and MDD and suicide, this study aimed to examine the relationship between frontal alpha asymmetry and suicide in MDD patients. METHODS: Sixty-six MDD patients, of whom fifteen were male and fifty-one were female, were recruited. Independent groups were created based on the median score of frontal alpha asymmetry: the left dominant (LD) group and the right dominant (RD) group. The alpha band (8‒12 Hz) and its sub-bands (i.e., low alpha band: 8‒10 Hz; high alpha band: 10‒12 Hz) were of interest. Source level alpha asymmetry was calculated as well. RESULTS: Suicidal behavior was positively correlated with the asymmetry indices of the low alpha band and the alpha band in the LD group and that of the high alpha band in the RD group. Source level analysis revealed positive correlations between suicidal behavior and the asymmetry index of the low alpha band in the LD group. CONCLUSION: Frontal alpha asymmetry, especially that of the low alpha band, might reflect the cognitive deficits associated with suicidal behaviors in MDD patients.

Molecular origin of AuNPs-induced cytotoxicity and mechanistic study.

Gold nanoparticles (AuNPs) with diverse physicochemical properties are reported to affect biological systems differently, but the relationship between the physicochemical properties of AuNPs and their biological effects is not clearly understood. Here, we aimed to elucidate the molecular origins of AuNP-induced cytotoxicity and their mechanisms, focusing on the surface charge and structural properties of modified AuNPs. We prepared a library of well-tailored AuNPs exhibiting various functional groups and surface charges. Through this work, we revealed that the direction or the magnitude of surface charge is not an exclusive factor that determines the cytotoxicity of AuNPs. We, instead, suggested that toxic AuNPs share a common structural characteristics of a hydrophobic moiety neighbouring the positive charge, which can induce lytic interaction with plasma membrane. Mechanistic study showed that the toxic AuNPs interfered with the formation of cytoskeletal structure to slow cell migration, inhibited DNA replication and caused DNA damage via oxidative stress to hinder cell proliferation. Gene expression analysis showed that the toxic AuNPs down-regulated genes associated with cell cycle processes. We discovered structural characteristics that define the cytotoxic AuNPs and suggested the mechanisms of their cytotoxicity. These findings will help us to understand and to predict the biological effects of modified AuNPs based on their physicochemical properties.

Allergen Microarrays for In Vitro Diagnostics of Allergies: Comparison with ImmunoCAP and AdvanSure.

BACKGROUND: In vitro detection of the allergen-specific IgE antibody (sIgE) is a useful tool for the diagnosis and treatment of allergies. Although multiple simultaneous allergen tests offer simple and low-cost screening methods, these platforms also have limitations with respect to multiplexibility and analytical performance. As an alternative assay platform, we developed and validated a microarray using allergen extracts that we termed "GOLD" chip. METHODS: Serum samples of 150 allergic rhinitis patients were used in the study, and the diagnostic performance of the microarray was compared with that of AdvanSure (LG Life Sciences, Daejun, Korea) and ImmunoCAP (Phadia, Uppsala, Sweden). Standard IgE samples were used for the quantitative measurement of sIgEs. RESULTS: The microarray-based assay showed excellent performance in the quantitative measurement of sIgEs, demonstrating a linear correlation within the range of sIgE concentrations tested. The limit of detection (LOD) was lower than 0.35 IU/mL, which is the current standard for the LOD cut-off. The assay also provided highly reproducible sets of data. The total agreement percentage of positive and negative calls was 92.2% compared with ImmunoCAP. Moreover, an outstanding correlation was observed between the microarray and the ImmunoCAP results, with Cohen's kappa and Pearson correlation coefficient values of 0.80 and 0.79, respectively. CONCLUSIONS: The microarray-based in vitro diagnostic platform offers a sensitive, reproducible, and highly quantitative method to detect sIgEs. The results showed strong correlations with that of ImmunoCAP. These results suggest that the new allergen microarray can serve as a useful alternative to current screening platforms, ultimately becoming a first-line screening method.

Inhibitory Effects of Novel SphK2 Inhibitors on Migration of Cancer Cells.

BACKGROUND: Cell migration is an essential process for survival and differentiation of mammalian cells. Numerous diseases are induced or influenced by inappropriate regulation of cell migration, which plays a key role in cancer cell metastasis. In fact, very few anti-metastasis drugs are available on the market. SphKs are enzymes that convert sphingosine to sphingosine-1-phosphate (S1P) and are known to control various cellular functions, including migration of cells. In human, SphK2 is known to promote apoptosis, suppresses cell growth, and controls cell migration; in addition, the specific ablation of SphK2 activity was reported to inhibit cancer cell metastasis. OBJECTIVE: The previously identified SG12 and SG14 are synthetic analogs of sphingoid and can specifically inhibit the functions of SphK2. We investigated the effects of the SphK2 specific inhibitors on the migratory behavior of cells. METHOD: We investigated how SG12 and SG14 affect cell migration by monitoring both cumulative and individual cell migration behavior using HeLa cells. RESULTS: SG12 and SG14 mutually showed stronger inhibitory effects with less cytotoxicity compared with a general SphK inhibitor, N,N-dimethylsphingosine (DMS). The mechanistic aspects of specific SphK2 inhibition were studied by examining actin filamentation and the expression levels of motility-related genes. CONCLUSION: The data revealed that SG12 and SG14 resemble DMS in decreasing overall cell motility, but differ in that they differentially affect motility parameters and motility-related signal transduction pathways and therefore actin polymerization, which are not altered by DMS. Our findings show that SphK2 inhibitors are putative candidates for anti-metastatic drugs.

Curcumin extends life span, improves health span, and modulates the expression of age-associated aging genes in Drosophila melanogaster.

BACKGROUND: Curcumin, an extract from the rhizome of the plant Curcuma longa (turmeric), has been widely used as a spice and herbal medicine in Asia. It has been suggested to have many biological activities, such as antioxidative, antiinflammatory, anticancer, chemopreventive, and antineurodegenerative properties. We evaluated the impact of curcumin on life span, fecundity, feeding rate, oxidative stress, locomotion, and gene expression in two different wild-type Drosophila melanogaster strains, Canton-S and Ives, under two different experimental conditions. RESULTS: We report that curcumin extended the life span of two different strains of D. melanogaster, an effect that was accompanied by protection against oxidative stress, improvement in locomotion, and chemopreventive effects. Life span extension was gender and genotype specific. Curcumin also modulated the expression of several aging-related genes, including mth, thor, InR, and JNK. CONCLUSIONS: The observed positive effects of curcumin on life span and health span in two different D. melanogaster strains demonstrate a potential applicability of curcumin treatment in mammals. The ability of curcumin to mitigate the expression levels of age-associated genes in young flies suggests that the action of curcumin on these genes is a cause, rather than an effect, of its life span-extending effects.