•Helical-structured Ag microcoils can be used as a SERS substrate.•Surface-enhanced Raman scattering (SERS) effect in Ag microcoils is anisotropic.•SERS effect is dominated by geometry of Ag ...microcoils.•Sharp corners or bends act as hot spots in Ag microcoils.
Ag nanostructure-based surface-enhanced Raman scattering (SERS) substrates tend to be oxidized and corroded by sulfides owing to their large specific surface area, resulting in severe deterioration. Contrastingly, micrometer-sized Ag structures are more chemically stable. Nevertheless, no reports exist on the utilization of Ag microstructures as SERS substrates, as they lack hot spots. Herein, for the first time, SERS is observed in helical Ag structures with coil diameters and pitches of the micrometer order. The geometry-dominated anisotropic SERS effect is investigated by controlling the shape of Ag microcoils. A new model is proposed to elucidate the mechanism of hot spot formation in Ag microcoils. The results of this study should aid efforts to develop improved SERS substrates.
The differentiation of pancreatic endocrine cells from human pluripotent stem cells has been thoroughly investigated for their application in cell therapy against diabetes. Although non-endocrine ...cells are inevitable contaminating by-products of the differentiation process, a comprehensive profile of such cells is lacking. Therefore, we characterized non-endocrine cells in iPSC-derived pancreatic islet cells (iPIC) using single-cell transcriptomic analysis. We found that non-endocrine cells consist of (1) heterogeneous proliferating cells, and (2) cells with not only pancreatic traits but also liver or intestinal traits marked by FGB or AGR2. Non-endocrine cells specifically expressed FGFR2, PLK1, and LDHB. We demonstrated that inhibition of pathways involving these genes selectively reduced the number of non-endocrine cells in the differentiation process. These findings provide useful insights into cell purification approaches and contribute to the improvement of the mass production of endocrine cells for stem cell-derived cell therapy for diabetes.
Selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875), an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by ...activating FFAR1 expressed in pancreatic β cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca(2+) influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand γ-linolenic acid (γ-LA). Augmentation of glucose-induced insulin secretion by fasiglifam, γ-LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA) levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs in vivo. Point mutations of FFAR1 differentially affected Ca(2+) influx activities of fasiglifam and γ-LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.
Various types of antipsychotics have been developed for the treatment of schizophrenia since the accidental discovery of the antipsychotic activity of chlorpromazine. Although all clinically ...effective antipsychotic agents have common properties to interact with the dopamine D2 receptor (D2R) activation, their precise mechanisms of action remain elusive. Antipsychotics are well known to induce transcriptional changes of immediate early genes (IEGs), raising the possibility that gene expressions play an essential role to improve psychiatric symptoms. Here, we report that while different classes of antipsychotics have complex pharmacological profiles against D2R, they share common transcriptome fingerprint (TFP) profile of IEGs in the murine brain in vivo by quantitative real-time PCR (qPCR). Our data showed that various types of antipsychotics with a profound interaction of D2R including haloperidol (antagonist), olanzapine (antagonist), and aripiprazole (partial agonist) all share common spatial TFPs closely homologous to those of D2R antagonist sulpiride, and elicited greater transcriptional responses in the striatum than in the nucleus accumbens. Meanwhile, D2R agonist quinpirole and propsychotic NMDA antagonists such as MK-801 and phencyclidine (PCP) exhibited the contrasting TFP profiles. Clozapine and propsychotic drug methamphetamine (MAP) displayed peculiar TFPs that reflect their unique pharmacological property. Our results suggest that transcriptional responses are conserved across various types of antipsychotics clinically effective in positive symptoms of schizophrenia and also show that temporal and spatial TFPs may reflect the pharmacological features of the drugs. Thus, we propose that a TFP approach is beneficial to evaluate novel drug candidates for antipsychotic development.
Purinergic P2Y
receptors, G-protein coupled receptors that primarily couple with Gα
-proteins, are activated equipotently by adenosine-5'-triphosphate (ATP) and uridine-5'-triphosphate. Evidence ...suggests that P2Y
agonists make potential drug candidates for the treatment of cardiovascular diseases. However, selective non-nucleotide, small-molecule P2Y
agonists have yet to be developed. In this report, we discuss Compound 89, a novel non-nucleotide allosteric P2Y
agonist that was active in signal transduction and gene induction, and in our in vitro cardiac hypertrophy model. Compound 89 exhibited selective P2Y
agonistic activity and potentiated responses to the endogenous agonist ATP, while exhibiting no agonistic activities for four other Gα
-coupled human P2Y (hP2Y) receptors and one representative Gα
-coupled hP2Y
receptor. Its P2Y
agonistic effect on mouse P2Y
receptors suggested non-species-specific activity. Compound 89 acted as a pure positive allosteric modulator in a Ca
mobilization assay of neonatal rat cardiomyocytes; it potentiated ATP-induced expression of genes in the nuclear receptor 4A family (negative regulators of hypertrophic stimuli in cardiomyocytes). Additionally, Compound 89 attenuated isoproterenol-induced cardiac hypertrophy, presumably through dose-dependent interaction with pericellular ATP. These results indicate that Compound 89 is potentially efficacious against cardiomyocytes and therefore a good proof-of-concept tool for elucidating the therapeutic potential of P2Y
activation in various cardiovascular diseases.
Many drugs of abuse and most neuropharmacological agents regulate G protein-coupled receptors (GPCRs) in the central nervous system (CNS)_ENREF_1. The striatum, in which dopamine D1 and D2 receptors ...are enriched, is strongly innervated by the ventral tegmental area (VTA), which is the origin of dopaminergic cell bodies of the mesocorticolimbic dopamine system_ENREF_3 and plays a central role in the development of psychiatric disorders_ENREF_4. Here we report the comprehensive and anatomical transcript profiling of 322 non-odorant GPCRs in mouse tissue by quantitative real-time PCR (qPCR), leading to the identification of neurotherapeutic receptors exclusively expressed in the CNS, especially in the striatum. Among them, GPR6, GPR52, and GPR88, known as orphan GPCRs, were shown to co-localize either with a D2 receptor alone or with both D1 and D2 receptors in neurons of the basal ganglia. Intriguingly, we found that GPR52 was well conserved among vertebrates, is Gs-coupled and responsive to the antipsychotic drug, reserpine. We used three types of transgenic (Tg) mice employing a Cre-lox system under the control of the GPR52 promoter, namely, GPR52-LacZ Tg, human GPR52 (hGPR52) Tg, and hGPR52-GFP Tg mice. Detailed histological investigation suggests that GPR52 may modulate dopaminergic and glutamatergic transmission in neuronal circuits responsible for cognitive function and emotion. In support of our prediction, GPR52 knockout and transgenic mice exhibited psychosis-related and antipsychotic-like behaviors, respectively. Therefore, we propose that GPR52 has the potential of being a therapeutic psychiatric receptor. This approach may help identify potential therapeutic targets for CNS diseases.
Transplantation of differentiated cells from human-induced pluripotent stem cells (hiPSCs) holds great promise for clinical treatments. Eliminating the risk factor of malignant cell transformation is ...essential for ensuring the safety of such cells. This study was aimed at assessing and mitigating mutagenicity that may arise during the cell culture process in the protocol of pancreatic islet cell (iPIC) differentiation from hiPSCs.
We evaluated the mutagenicity of differentiation factors used for hiPSC-derived pancreatic islet-like cells (iPICs). We employed Ames mutagenicity assay, flow cytometry analysis, immunostaining, time-resolved fluorescence resonance energy transfer-based (TR-FRET) cell-free dose-response assays, single-cell RNA-sequencing and in vivo efficacy study.
We observed a mutagenic effect of activin receptor-like kinase 5 inhibitor II (ALK5iII). ALK5iII is a widely used β-cell inducer but no other tested ALK5 inhibitors induced β-cells. We obtained kinase inhibition profiles and found that only ALK5iII inhibited cyclin-dependent kinases 8 and 19 (CDK8/19) among all ALK5 inhibitors tested. Consistently, CDK8/19 inhibitors efficiently induced β-cells in the absence of ALK5iII. A combination treatment with non-mutagenic ALK5 inhibitor SB431542 and CDK8/19 inhibitor senexin B afforded generation of iPICs with in vitro cellular composition and in vivo efficacy comparable to those observed with ALK5iII.
Our findings suggest a new risk mitigation approach for cell therapy and advance our understanding of the β-cell differentiation mechanism.
Mechanisms controlling development, growth, and metabolism are coordinated in response to changes in environmental conditions, enhancing the likelihood of survival to reproductive maturity. Much ...remains to be learned about the molecular basis underlying environmental influences on these processes. C. elegans larvae enter a developmentally dormant state called L1 diapause when hatched into nutrient-poor conditions. The nematode pten homologue daf-18 is essential for maintenance of survival and germline stem cell quiescence during this period (Fukuyama et al., 2006; Sigmond et al., 2008), but the details of the signaling network(s) in which it functions remain to be elucidated. Here, we report that animals lacking both aak-1 and aak-2, which encode the two catalytic α subunits of AMP-activated protein kinase (AMPK), show reduced viability and failure to maintain mitotic quiescence in germline stem cells during L1 diapause. Furthermore, failure to arrest germline proliferation has a long term consequence; aak double mutants that have experienced L1 diapause develop into sterile adults when returned to food, whereas their continuously fed siblings are fertile. Both aak and daf-18 appear to maintain germline quiescence by inhibiting activity of the common downstream target, TORC1 (TOR Complex 1). In contrast, rescue of the lethality phenotype indicates that aak-2 acts not only in the intestine, as does daf-18, but also in neurons, likely promoting survival by preventing energy deprivation during L1 diapause. These results not only provide evidence that AMPK contributes to survival during L1 diapause in a manner distinct from that by which it controls dauer diapause, but they also suggest that AMPK suppresses TORC1 activity to maintain stem cell quiescence.
Differentiation of pancreatic endocrine cells from human pluripotent stem cells (PSCs) has been thoroughly investigated for application in cell therapy against diabetes. In the context of induced ...pancreatic endocrine cell implantation, previous studies have reported graft enlargement resulting from off-target pancreatic lineage cells. However, there is currently no documented evidence of proliferative off-target cells beyond the pancreatic lineage in existing studies. Here, we show that the implantation of seven-stage induced PSC-derived pancreatic islet cells (s7-iPICs) leads to the emergence of unexpected off-target cells with proliferative capacity via in vivo maturation. These cells display characteristics of both mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs), termed proliferative MSC- and SMC-like cells (PMSCs). The frequency of PMSC emergence was found to be high when 10
s7-iPICs were used. Given that clinical applications involve the use of a greater number of induced cells than 10
, it is challenging to ensure the safety of clinical applications unless PMSCs are adequately addressed. Accordingly, we developed a detection system and removal methods for PMSCs. To detect PMSCs without implantation, we implemented a 4-wk-extended culture system and demonstrated that putative PMSCs could be reduced by compound treatment, particularly with the taxane docetaxel. When docetaxel-treated s7-iPICs were implanted, the PMSCs were no longer observed. This study provides useful insights into the identification and resolution of safety issues, which are particularly important in the field of cell-based medicine using PSCs.
Purinergic P2Y
2
receptors, G-protein coupled receptors that primarily couple with Gα
q/11
-proteins, are activated equipotently by adenosine-5′-triphosphate (ATP) and uridine-5′-triphosphate. ...Evidence suggests that P2Y
2
agonists make potential drug candidates for the treatment of cardiovascular diseases. However, selective non-nucleotide, small-molecule P2Y
2
agonists have yet to be developed. In this report, we discuss Compound 89, a novel non-nucleotide allosteric P2Y
2
agonist that was active in signal transduction and gene induction, and in our
in vitro
cardiac hypertrophy model. Compound 89 exhibited selective P2Y
2
agonistic activity and potentiated responses to the endogenous agonist ATP, while exhibiting no agonistic activities for four other Gα
q/11
-coupled human P2Y (hP2Y) receptors and one representative Gα
i/o
-coupled hP2Y
12
receptor. Its P2Y
2
agonistic effect on mouse P2Y
2
receptors suggested non-species-specific activity. Compound 89 acted as a pure positive allosteric modulator in a Ca
2+
mobilization assay of neonatal rat cardiomyocytes; it potentiated ATP-induced expression of genes in the nuclear receptor 4A family (negative regulators of hypertrophic stimuli in cardiomyocytes). Additionally, Compound 89 attenuated isoproterenol-induced cardiac hypertrophy, presumably through dose-dependent interaction with pericellular ATP. These results indicate that Compound 89 is potentially efficacious against cardiomyocytes and therefore a good proof-of-concept tool for elucidating the therapeutic potential of P2Y
2
activation in various cardiovascular diseases.
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