Functional Screening System Research Articles

Conversion of dehydrocoelenterazine to coelenterazine in mammalian cells: Identification using cultured cells stably expressing coelenterazine-utilizing luciferase

Dehydrocoelenterazine (dCTZ) is a dehydrogenated form of coelenterazine (CTZ), which is well-known as the luciferin responsible for the bioluminescence reaction in marine organisms. In this report, we demonstrate for the first time that dCTZ is readily reduced to CTZ in mammalian cells. Using an FDSS®/μCell functional drug screening system, the conversion of dCTZ to CTZ in cells was identified through the luciferin (CTZ)-luciferase reaction in Chinese hamster ovary K1 (CHO–K1) cell lines, which stably expressed CTZ-utilizing luciferases of Renilla luciferase (RLase) or QL-nanoKAZ (a mutant of the 19 kDa protein of Oplophorus luciferase). After loading dCTZ into CHO–K1 cells expressing RLase or QL-nanoKAZ, the luminescence from both cells was detected within 10 s and continued for over 30 min. Thus, dCTZ permeates mammalian cells and is immediately converted to CTZ. This suggests that dCTZ could potentially be used as a substitute for CTZ in in vivo assays of the CTZ-dependent luminescence systems.

A Functional Hydrogel Bead-Based High-Throughput Screening System for Mammalian Cells with Enhanced Secretion of Therapeutic Antibodies.

Droplet-based high-throughput screening systems are an emerging technology that provides a quick test to screen millions of cells with distinctive characteristics. Biopharmaceuticals, specifically therapeutic proteins, are produced by culturing cells that secrete heterologous recombinant proteins with different populations and expression levels; therefore, a technology to discriminate cells that produce more target proteins is needed. Here, we present a droplet-based microfluidic strategy for encapsulating, screening, and selecting target cells with redox-responsive hydrogel beads (HBs). As a proof-of-concept study, we demonstrate the enrichment of hybridoma cells with enhanced capability of antibody secretion using horseradish peroxidase (HRP)-catalyzed hydrogelation of tetra-thiolate poly(ethylene glycol); hybridoma cells were encapsulated in disulfide-bonded HBs. Recombinant protein G or protein M with a C-terminal cysteine residue was installed in the HBs via disulfide bonding to capture antibodies secreted from the cells. HBs were fluorescently stained by adding the protein L-HRP conjugate using a tyramide signal amplification system. HBs were then separated by fluorescence-activated droplet sorting and degraded by reducing the disulfide bonds to recover the target cells. Finally, we succeeded in the selection of hybridoma cells with enhanced antibody secretion, indicating the potential of this system in the therapeutic protein production.

Rapid Identification of High-Temperature Responsive Genes Using Large-Scale Yeast Functional Screening System in Potato.

As the third largest global food crop, potato plays an important role in ensuring food security. However, it is particularly sensitive to high temperatures, which seriously inhibits its growth and development, thereby reducing yield and quality and severely limiting its planting area. Therefore, rapid, and high-throughput screening for high-temperature response genes is highly significant for analyzing potato high-temperature tolerance molecular mechanisms and cultivating new high-temperature-tolerant potato varieties. We screened genes that respond to high temperature by constructing a potato cDNA yeast library. After high-temperature treatment at 39 °C, the yeast library was subjected to high-throughput sequencing, and a total of 1931 heat resistance candidate genes were screened. Through GO and KEGG analysis, we found they were mainly enriched in "photosynthesis" and "response to stimuli" pathways. Subsequently, 12 randomly selected genes were validated under high temperature, drought, and salt stress using qRT-PCR. All genes were responsive to high temperature, and most were also induced by drought and salt stress. Among them, five genes ectopically expressed in yeast enhance yeast's tolerance to high temperatures. We provide numerous candidate genes for potato response to high temperature stress, laying the foundation for subsequent analysis of the molecular mechanism of potato response to high temperature.

Evaluation of FMS Scores of Competitive CrossFit Athletes by Gender

Functional Movement Screening System (FMS) is a testing and assessment protocol used to evaluate the body movement and movement capacity of athletes or physically active individuals. Analysis of FMS scores by gender can provide important information to personalize training programs, identify weak points, and improve the movement quality of athletes. Therefore, the aim of this study was to evaluate the FMS scores of competitive CrossFit athletes according to gender. A total of 22 athletes, 14 males with a mean age of 26.79±5.16 years and 8 females with a mean age of 32.38±3.74 years participated in the study. Participants were administered the FMS™ test consisting of seven tasks (per the FMS™ manual). In the analysis of the data, after the normality distribution was made, the Man-Whitney U test was performed for the data that did not show the normal distribution in an independent two-group comparison. A statistically significant difference was found in the right(p=0.04) and left (p=0.04) shoulder mobility score, shoulder mobility final score (p=0.01), and active straight leg raise score-right (p=0.02) measurements of the athletes according to gender. As a result, females were found to have higher 'shoulder mobility' and 'active straight leg raising' scores than males on a movement basis. In addition, the total FMS scores were found to be above average for both genders.

A systematic identification of cold tolerance genes in peanut using yeast functional screening system

Peanut (Arachis hypogaea L.) is a thermophilic crop, and low temperature leads to a significant reduction in annual yields. Despite a few cold tolerant germplasms or cultivars have been discovered and developed, molecular mechanisms governing peanut cold tolerance is poorly understood. Identification of keys genes involved in cold tolerance is the first step to address the underlying mechanism. In this study, we isolated and characterized 157 genes with potentials to confer cold tolerance in peanut by using a yeast functional screening system. GO (Gene ontology) and KEGG (Kyoto encyclopedia of genes and genomes) enrichment analysis of these genes revealed that ribosome and photosynthesis proteins might play essential roles in peanut cold response. Transcriptome results indicated that 60 cold tolerance candidate genes were significantly induced or depressed by low temperature. qRT-PCR analysis demonstrated that several candidate genes could be also regulated by salt or drought stress. Individual overexpression of two UDP-glycosyltransferases (AhUGT2 and AhUGT268) in transgenic yeast cells could enhance their tolerance to multiple abiotic stress. In conclusion, this study advances our understanding of the mechanisms associated with the cold stress responses in peanut, and offers valuable gene resources for genetic improvement of abiotic stress tolerance in crops.

EFEITO DO TREINAMENTO FUNCIONAL SOBRE A QUALIDADE DE ACERTO DOS JOGADORES DE BADMINTON

ABSTRACT Introduction: The importance of physical training in badminton is reflected in the quality of technical and tactical movements in the field, improving the athlete the physical strength enough as support and the level of physical fitness necessary for their sports performance, reducing sports injuries and, mainly, increasing their hitting rates. Objective: Study the effect of functional training on the hit rate in badminton players. Methods: Bibliographic and experimental research methods were used through the ITN badminton technical level test and the functional movement screening system (FMS). The effect of functional training was compared and analyzed considering baseline return quality and functional movement quality of 12 young badminton players. Results: Compared to the pre-test, the mean baseline reaching depth and accuracy statistical score increased significantly after the 8 weeks of functional training (P<0.05); the control group baseline depth and accuracy test score improved slightly but not significantly (P>0.05). Conclusion: Young badminton players who add functional training to physical training demonstrate more relevant impacts on their hitting rates. These exercises have also demonstrated that they can strengthen muscles and increase shoulder, hip, and knee flexibility. Level of evidence II; Therapeutic studies - investigating treatment outcomes.

Functional mining of novel terpene synthases from metagenomes

BackgroundTerpenes are one of the most diverse and abundant classes of natural biomolecules, collectively enabling a variety of therapeutic, energy, and cosmetic applications. Recent genomics investigations have predicted a large untapped reservoir of bacterial terpene synthases residing in the genomes of uncultivated organisms living in the soil, indicating a vast array of putative terpenoids waiting to be discovered.ResultsWe aimed to develop a high-throughput functional metagenomic screening system for identifying novel terpene synthases from bacterial metagenomes by relieving the toxicity of terpene biosynthesis precursors to the Escherichia coli host. The precursor toxicity was achieved using an inducible operon encoding the prenyl pyrophosphate synthetic pathway and supplementation of the mevalonate precursor. Host strain and screening procedures were finely optimized to minimize false positives arising from spontaneous mutations, which avoid the precursor toxicity. Our functional metagenomic screening of human fecal metagenomes yielded a novel β-farnesene synthase, which does not show amino acid sequence similarity to known β-farnesene synthases. Engineered S. cerevisiae expressing the screened β-farnesene synthase produced 120 mg/L β-farnesene from glucose (2.86 mg/g glucose) with a productivity of 0.721 g/L∙h.ConclusionsA unique functional metagenomic screening procedure was established for screening terpene synthases from metagenomic libraries. This research proves the potential of functional metagenomics as a sequence-independent avenue for isolating targeted enzymes from uncultivated organisms in various environmental habitats.

Engineering a U-box of E3 ligase E4B through yeast surface display-based functional screening generates a variant with enhanced ubiquitin ligase activity

Ubiquitination is the covalent attachment of ubiquitin (Ub) to substrate proteins and regulates several cellular processes, including protein degradation. Ub ligases (E3s) bring a Ub-conjugated enzyme E2 (E2–Ub) and the target protein closer to enable ubiquitination. In this study, we engineered a U-box domain of human U-box-type E3 E4B (E4BU) to enhance its function as a Ub ligase by accelerating the rate of Ub transfer directly from Ub-loaded human E2 UbcH5b (E2(UbcH5b)–Ub) to the proximal substrate. We developed a functional screening system for the E4BU library using a yeast surface display system combined with fluorescence-activated cell sorting (FACS) to isolate functionally improved variants. This phenotypic screening system yielded an E4BU variant, E4BU(#8), which exhibited an approximately 4-fold greater Ub ligase activity rate in the yeast displayed form than that of the E4BU wild-type. When E4BU(#8) was fused to a green fluorescent protein (GFP)-specific nanobody, the fusion protein polyubiquitinated GFP in proportion to the concentration and incubation time, with an approximately 3-fold faster Ub ligase activity rate than the previously isolated E4BU(NT) variant. Importantly, the engineered E4BU(#8) retained endogenous Lys48-linked polyubiquitination activity, which is essential for substrate degradation by the 26S proteasome. Our results indicated that E4BU(#8), which binds to and allosterically stimulates E2(UbcH5b)–Ub to enhance Ub transferase activity to a substrate, may be valuable in designing biological molecules for targeted protein degradation.

Identifying Acute Cardiac Hazard in Early Drug Discovery Using a Calcium Transient High-Throughput Assay in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Introduction: Early identification of cardiac risk is essential for reducing late-stage attrition in drug development. We adapted the previously published cardiac hazard risk-scoring system using a calcium transient assay in human stem cell-derived CMs for the identification of cardiac risks recorded from the new hiPSC-CM line and investigated its predictivity and translational value based on the screening of a large number of reference and proprietary compounds. Methods: Evaluation of 55 reference drugs provided the translation of various pharmacological effects into a single hazard label (no, low, high, or very high hazard) using a Ca2+-sensitive fluorescent dye assay recorded by -by FDSS/µCell Functional Drug Screening System (Hamamatsu on hiPSC-CM line (FCDI iCell Cardiomyocytes2). Results: Application of the adapted hazard scoring system in the Ca2+ transient assay, using a second hiPS-CM line, provided comparable scoring results and predictivity of hazard, to the previously published scoring approach, with different pharmacological drug classes, as well as screening new chemical entities (NCE’s) using a single hazard label from four different scoring levels (no, low, high, or very high hazard). The scoring system results also showed minimal variability across three different lots of hiPSC-CMs, indicating good reproducibility of the cell line. The predictivity values (sensitivity and specificity) for drug-induced acute cardiac risk for QT-interval prolongation and Torsade de pointes (TdPs) were >95% and statistical modeling confirmed the prediction of proarrhythmic risk. The outcomes of the NCEs also showed consistency with findings in other well-established in vitro and in vivo cardiac risk assays. Conclusion: Evaluation of a large list of reference compounds and internal NCEs has confirmed the applicability of the adaptations made to the previously published novel scoring system for the hiPSC-CMs. The validation also established the predictivity for drug-induced cardiac risks with good translation to other established preclinical in vitro and in vivo assays, confirming the application of this novel scoring system in different stem cell-CM lines for early cardiac hazard identification.

Targeted exon skipping of NF1 exon 17 as a therapeutic for neurofibromatosis type I

We investigated the feasibility of utilizing an exon-skipping approach as a genotype-dependent therapeutic for neurofibromatosis type 1 (NF1) by determining which NF1 exons might be skipped while maintaining neurofibromin protein expression and GTPase activating protein (GAP)-related domain (GRD) function. Initial in silico analysis predicted exons that can be skipped with minimal loss of neurofibromin function, which was confirmed by in vitro assessments utilizing an Nf1 cDNA-based functional screening system. Skipping of exons 17 or 52 fit our criteria, as minimal effects on protein expression and GRD activity were noted. Antisense phosphorodiamidate morpholino oligomers (PMOs) were utilized to skip exon 17 in human cell lines with patient-specific pathogenic variants in exon 17, c.1885G>A, and c.1929delG. PMOs restored functional neurofibromin expression. To determine the in vivo significance of exon 17 skipping, we generated a homozygous deletion of exon 17 in a novel mouse model. Mice were viable and exhibited a normal lifespan. Initial studies did not reveal the presence of tumor development; however, altered nesting behavior and systemic lymphoid hyperplasia was noted in peripheral lymphoid organs. Alterations in T and B cell frequencies in the thymus and spleen were identified. Hence, exon skipping should be further investigated as a therapeutic approach for NF1 patients with pathogenic variants in exon 17, as homozygous deletion of exon 17 is consistent with at least partial function of neurofibromin.

UBQLN4 is an ATM substrate that stabilizes the anti-apoptotic proteins BCL2A1 and BCL2L10 in mesothelioma.

ATM serine/threonine kinase (ATM; previously known as ataxia‐telangiectasia mutated) plays a critical role in maintaining genomic stability and regulates multiple downstream pathways, such as DNA repair, cell cycle arrest, and apoptosis. As a serine/threonine kinase, ATM has an array of downstream phosphorylation substrates, including checkpoint effector checkpoint kinase 2 (CHK2). ATM inhibits cell cycle progression by phosphorylating and activating CHK2, which plays an important role in the formation and development of tumors and participates in DNA repair responses after double‐stranded DNA breaks. In this study, we used a recently developed mammalian functional genetic screening system to explore a series of ATM substrates and their role in DNA damage to enhance our understanding of the DNA damage response. Ubiquilin 4 (UBQLN4), which belongs to the ubiquilin family characterized by its ubiquitin‐like (UBL) and ubiquitin‐associated (UBA) domains, was identified as a new substrate for ATM. UBQLN4 is involved in various intracellular processes, such as autophagosome maturation, p21 regulation, and motor axon morphogenesis. However, the biological function of UBQLN4 remains to be elucidated. In this study, we not only identified UBQLN4 as a substrate for ATM, but also found that UBQLN4 interacts with and stabilizes the anti‐apoptotic proteins Bcl‐2‐related protein A1 (BCL2A1) and Bcl‐2‐like protein 10 (BCL2L10) and prevents mesothelioma cell apoptosis in response to DNA damage. These findings expand our understanding of the role of UBQLN4 in mesothelioma and provide new insights into potential mesothelioma treatments targeting substrates for ATM.

Development of an Automatic Functional Movement Screening System with Inertial Measurement Unit Sensors

Background: In this study, an automatic scoring system for the functional movement screen (FMS) was developed. Methods: Thirty healthy adults fitted with full-body inertial measurement unit sensors completed six FMS exercises. The system recorded kinematics data, and a professional athletic trainer graded each participant. To reduce the number of input variables for the predictive model, ordinal logistic regression was used for subset feature selection. The ensemble learning algorithm AdaBoost.M1 was used to construct classifiers. Accuracy and F score were used for classification model evaluation. The consistency between automatic and manual scoring was assessed using a weighted kappa statistic. Results: When all the features were used, the predict model presented moderate to high accuracy, with kappa values between fair to very good agreement. After feature selection, model accuracy decreased about 10%, with kappa values between poor to moderate agreement. Conclusions: The results indicate that higher prediction accuracy was achieved using the full feature set compared with using the reduced feature set.

Identification of citrus immune regulators involved in defence against Huanglongbing using a new functional screening system.

SummaryHuanglongbing (HLB) is the most devastating citrus disease in the world. Almost all commercial citrus varieties are susceptible to the causal bacterium, Candidatus Liberibacter asiaticus (CLas), which is transmitted by the Asian citrus psyllid (ACP). Currently, there are no effective management strategies to control HLB. HLB‐tolerant traits have been reported in some citrus relatives and citrus hybrids, which offer a direct pathway for discovering natural defence regulators to combat HLB. Through comparative analysis of small RNA profiles and target gene expression between an HLB‐tolerant citrus hybrid (Poncirus trifoliata × Citrus reticulata) and a susceptible citrus variety, we identified a panel of candidate defence regulators for HLB‐tolerance. These regulators display similar expression patterns in another HLB‐tolerant citrus relative, with a distinct genetic and geographic background, the Sydney hybrid (Microcitrus virgata). Because the functional validation of candidate regulators in tree crops is always challenging, we developed a novel rapid functional screening method, using a C. Liberibacter solanacearum (CLso)/potato psyllid/Nicotiana benthamiana interaction system to mimic the natural transmission and infection circuit of the HLB complex. When combined with efficient virus‐induced gene silencing in N. benthamiana, this innovative and cost‐effective screening method allows for rapid identification and functional characterization of regulators involved in plant immune responses against HLB, such as the positive regulator BRCA1‐Associated Protein, and the negative regulator Vascular Associated Death Protein.

Abstract 1402: Preclinical evaluation of a novel senescence-associated miRNA miR-3140-3p for malignant pleural mesothelioma

Abstract Cellular senescence acts as a natural barrier against tumor progression Our previous study showed that selective induction of senescence using miR-22, one of senescence-associated microRNAs (SA-miRNAs), suppressed tumor progression (JCB 2011) In this study, we developed functional screening system using miRNA library to identify a potent SA-miRNA that suppress malignant tumor such as anti-cancer drug resistant and cancer stem cells. We focused on microRNA inducing the senescent phenotype in normal human fibroblast cell After transfection of 2028 miRNA mimics library in normal human fibroblast TIG-3, we evaluate the senescence phenotypes such as cell growth arrest and size enlargement using high content analyzer, and found that 349 miRNAs induced cellular senescence More importantly, we identified miR-3140-3p as potent SA-miRNA that significantly suppressed tumor cell proliferation in several types of cancer cells including cancer stem cells and anti-cancer drug resistant cells. We focused on malignant pleural mesothelioma (MPM) which is an aggressive tumor developed from the mesothelial lining cells of the pleura MPM is caused by direct asbestos exposure and is an incurable tumor type for the lack of effective treatment In addition, as Japan is one of the world's highest importers and users of asbestos, the cases of mesothelioma are expected to increase in the near future Therefore, we investigated the possibility of whether SA-miRNAs showed tumor suppressive effects against MPM cell lines Moreover, we tried to develop a new treatment or medicine for the MPM patients using SA-miRNA In vitro and in vivo analysis revealed that several SA-miRNAs drastically suppressed the tumor cell proliferation Based on these findings, the modulation of SA-miRNA is a promising approach to improve the treatment of MPM patients In addition, we performed and completed pre-clinical trials and design first-in-human study in Japan. Citation Format: Yuki Yamamoto, Ayaka Nishiura, Kimiyoshi Yano, Noriaki Matsuda, Ryou-u Takahashi, Yasuhiro Tsutani, Morihito Okada, Hidetoshi Tahara. Preclinical evaluation of a novel senescence-associated miRNA miR-3140-3p for malignant pleural mesothelioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1402.

Identification of novel compounds against Tat-mediated human immunodeficiency virus-1 transcription by high-throughput functional screening assay

Trans-activator (Tat)-mediated human immunodeficiency virus type 1 (HIV-1) transcription is essential for the replication of HIV-1 and is considered a potent therapeutic target for HIV-1 inhibition. In this study, the Library of Pharmacologically Active Compounds (LOPAC1280) was screened using our dual-reporter screening system for repositioning as Tat-inhibitory compounds. Consequently, two compounds were found to be potent, with low cytotoxicity. Of these two compounds, Roscovitine (CYC202) is already known to be a Tat inhibitor, while gemcitabine has been newly identified as an inhibitor of Tat-mediated transcription linked to viral production and replication. In an additional screening using the ribonucleoside analogues of gemcitabine, two analogues (2′-C-methylcytidine and 3-deazauridine) showed a specific Tat-inhibitory effect linked to their anti-HIV-1 activity. Interestingly, these compounds did not affect Tat protein directly, while the mechanism underlying their inhibition of Tat-mediated transcription was linked to pyrimidine biosynthesis, rather than to alteration of the dNTP pool, influenced by the inhibition of ribonucleotide reductase.Taken together, the proposed functional screening system is a useful tool for the identification of inhibitors of Tat-mediated HIV-1 transcription from among a large number of compounds, and the inhibitory effect of HIV-1 transcription by gemcitabine and its analogues may suggest a strategy for developing a new class of therapeutic anti-HIV drugs.

Two-Tier Screening Platform for Directed Evolution of Aminoacyl-tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency.

Genetic code expansion through amber stop codon suppression provides a powerful tool for introducing non-proteinogenic functionalities into proteins for a broad range of applications. However, ribosomal incorporation of noncanonical amino acids (ncAAs) by means of engineered aminoacyl-tRNA synthetases (aaRSs) often proceeds with significantly reduced efficiency compared to sense codon translation. Here, we report the implementation of a versatile platform for the development of engineered aaRSs with enhanced efficiency in mediating ncAA incorporation by amber stop codon suppression. This system integrates a white/blue colony screen with a plate-based colorimetric assay, thereby combining high-throughput capabilities with reliable and quantitative measurement of aaRS-dependent ncAA incorporation efficiency. This two-tier functional screening system was successfully applied to obtain a pyrrolysyl-tRNA synthetase (PylRS) variant (CrtK-RS(4.1)) with significantly improved efficiency (+250-370 %) for mediating the incorporation of Nϵ -crotonyl-lysine and other lysine analogues of relevance for the study of protein post-translational modifications into a target protein. Interestingly, the beneficial mutations accumulated by CrtK-RS(4.1) were found to localize within the noncatalytic N-terminal domain of the enzyme and could be transferred to another PylRS variant, improving the ability of the variant to incorporate its corresponding ncAA substrate. This work introduces an efficient platform for the improvement of aaRSs that could be readily extended to other members of this enzyme family and/or other target ncAAs.

New tumor suppressor microRNAs target glypican-3 in human liver cancer.

Glypican-3 (GPC3) is an oncogene, frequently upregulated in liver malignancies such as hepatocellular carcinoma (HCC) and hepatoblastoma and constitutes a potential molecular target for therapy in liver cancer. Using a functional screening system, we identified 10 new microRNAs controlling GPC3 expression in malignant liver cells, five of them e.g. miR-4510, miR-203a-3p, miR-548aa, miR-376b-3p and miR-548v reduce GPC3 expression. These 5 microRNAs were significantly downregulated in tumoral compared to non-tumoral liver and inhibited tumor cell proliferation. Interestingly, miR-4510 inversely correlated with GPC3 mRNA and protein in HCC samples. This microRNA also induced apoptosis of hepatoma cells and blocked tumor growth in vivo in the chick chorioallantoic membrane model. We further show that the tumor suppressive effect of miR-4510 is mediated through direct targeting of GPC3 mRNA and inactivation of Wnt/β-catenin transcriptional activity and signaling pathway. Moreover, miR-4510 up-regulated the expression of several tumor suppressor genes while reducing the expression of other pro-oncogenes. In summary, we uncovered several new microRNAs targeting the oncogenic functions of GPC3. We provided strong molecular, cellular and in vivo evidences for the tumor suppressive activities of miR-4510 bringing to the fore the potential value of this microRNA in HCC therapy.

Screening of randomly mutagenized glucagon-like peptide-1 library by using an integrated yeast-mammalian assay system

Glucagon-like peptide-1 (GLP1) is a 30-amino acid peptide hormone activating the GLP1 receptor (GLP1R), a class B G-protein coupled receptor (GPCR), and is considered to be effective for treating diabetes and other metabolic diseases. Phage display is the first innovative technology in order to prepare and screen a large polypeptide library including GLP1R agonists, but this methodology is not as effective in discovering functional peptides such as activators for GPCRs. Here, we report a novel functional screening system for GPCR-acting peptides, which integrates a yeast peptide secretion system into a biological detection system with GPCR-producing mammalian cells. Using this screening system, we found attractive GLP1R agonists with several substitutions from a random mutant GLP1 library which was secreted by yeast, Saccharomyces cerevisiae. This system established here not only enables peptides to be analyzed in the soluble form but also needs no chemical synthesis, purification, and condensation of peptides of interests, and therefore, can be widely applied to the discovery of novel bioactive peptides acting on GPCRs.

Carboxylic Acid Fullerene (C60) Derivatives Attenuated Neuroinflammatory Responses by Modulating Mitochondrial Dynamics.

Fullerene (C60) derivatives, a unique class of compounds with potent antioxidant properties, have been reported to exert a wide variety of biological activities including neuroprotective properties. Mitochondrial dynamics are an important constituent of cellular quality control and function, and an imbalance of the dynamics eventually leads to mitochondria disruption and cell dysfunctions. This study aimed to assess the effects of carboxylic acid C60 derivatives (C60–COOH) on mitochondrial dynamics and elucidate its associated mechanisms in lipopolysaccharide (LPS)-stimulated BV-2 microglial cell model. Using a cell-based functional screening system labeled with DsRed2-mito in BV-2 cells, we showed that LPS stimulation led to excessive mitochondrial fission, increased mitochondrial localization of dynamin-related protein 1 (Drp1), both of which were markedly suppressed by C60–COOH pretreatment. LPS-induced mitochondria reactive oxygen species (ROS) generation and collapse of mitochondrial membrane potential (ΔΨm) were also significantly inhibited by C60–COOH. Moreover, we also found that C60–COOH pretreatment resulted in the attenuation of LPS-mediated activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling, as well as the production of pro-inflammatory mediators. Taken together, these findings demonstrated that carboxylic acid C60 derivatives may exert neuroprotective effects through regulating mitochondrial dynamics and functions in microglial cells, thus providing novel insights into the mechanisms of the neuroprotective properties of carboxylic acid C60 derivatives.

Functional screening system for yeast-secreted peptides acting on G-protein coupled receptors.

We established a novel functional screening system for peptides acting on G-protein coupled receptors (GPCRs). Peptides are a promising drug scaffold because of their intermediate molecular size between that of therapeutic small molecules and antibodies. They also offer potential advantages of targeting not only membrane proteins but also intracellular protein–protein interactions. Phage display technology has been used for exploring novel peptides acting on GPCRs, but it is unclear whether the identified peptides functionally modulate targets because the technology selects peptides based on binding ability but not functional activity to targets. In a novel screening system that we established, yeast cells were utilized as a peptide producer while mammalian cells stably producing the receptor for glucagon-like peptide 1 (GLP1R) were used as a biosensor for receptor activation. Three kinds of GLP1R agonists secreted by yeasts were successfully detected for their functional activities without any purification and condensation of those peptides. By applying the functional screening system, we were able to identify GLP1R agonist-secreting yeasts based on GLP1R activation from the cell mixture containing a number of background yeasts that produced non-active control peptides. Further applications of this system would include not only activity evaluation of bioactive peptides without chemical synthesis but also discovery of novel peptides activating druggable GPCRs.