Radical Caging Strategy for Cholinergic Optopharmacology.

Photo-caged methodologies have been indispensable for elucidating the functional mechanisms of pharmacologically active molecules at the cellular level. A photo-triggered removable unit enables control of the photo-induced expression of pharmacologically active molecular function, resulting in a rapid increase in the concentration of the bioactive compound near the target cell. However, caging the target bioactive compound generally requires specific heteroatom-based functional groups, limiting the types of molecular structures that can be caged. We have developed an unprecedented methodology for caging/uncaging on carbon atoms using a unit with a photo-cleavable carbon-boron bond. The caging/uncaging process requires installation of the CH2-B group on the nitrogen atom that formally assembles an N-methyl group protected with a photoremovable unit. N-Methylation proceeds by photoirradiation via carbon-centered radical generation. Using this radical caging strategy to cage previously uncageable bioactive molecules, we have photocaged molecules with no general labeling sites, including acetylcholine, an endogenous neurotransmitter. Caged acetylcholine provides an unconventional tool for optopharmacology to clarify neuronal mechanisms on the basis of photo-regulating acetylcholine localization. We demonstrated the utility of this probe by monitoring uncaging in HEK cells expressing a biosensor to detect ACh on the cell surface, as well as Ca2+ imaging in Drosophila brain cells (ex vivo).

Squaraine probes for the bimodal staining of lipid droplets and endoplasmic reticulum imaging in live cells.

Lipid droplets (LDs) have emerged as a hot target for cancer therapeutics in recent years owing to findings that have shown them to be key organelles involved in maintaining cellular stability and regulating inter-organelle communication through molecular trafficking. LDs emerge from the endoplasmic reticulum (ER) as a form of cellular homeostasis control. We herein report the study of a library of asymmetric squaraines as superior fluorescence probes to track and image LDs in their native state and environment within cancer cells. The probes are highly selective towards LDs and displayed prominent bright fluorescence with just 1 µM probe concentration. They also possess bimodal LD and ER staining capability via the simple diffusion of small lipophilic molecules. The probes almost instantly stained LDs, while the ER staining rate is dependent on the probe's lipophilicity and the incubation duration. These "on-demand" organelle-selective probes are highly desirable tools for revealing the role of LDs in governing many cellular processes, especially in malignant cells.

Heat-sterilized Bifidobacterium breve prevents depression-like behavior and interleukin-1ß expression in mice exposed to chronic social defeat stress.

Major depressive disorder (MDD) is a common and serious psychiatric disease that involves brain inflammation. Bifidobacterium breve is commonly used as a probiotic and was shown to improve colitis and allergic diseases by suppressing the inflammatory response. Heat-sterilized B. breve has beneficial effects on inflammation. We hypothesize, therefore, that this probiotic might reduce depression symptoms. We tested this is a mouse model of social defeat stress. C57BL/6J mice exposed to chronic social defeat stress (CSDS) for five consecutive days developed a mild depression-like behavior characterized by a social interaction impairment. CSDS also altered the gut microbiota composition, such as increased abundance of Bacilli, Bacteroidia, Mollicutes, and Verrucomicrobiae classes and decreased Erysipelotrichi class. The prophylactic effect of heat-sterilized B. breve as a functional food ingredient was evaluated on the depression-like behavior in mice. The supplementation started two weeks before and lasted two weeks after the last exposure to CSDS. Two weeks after CSDS, the mice showed deficits in social interaction and increased levels of inflammatory cytokines, including interleukin-1ß (IL-1ß) in the prefrontal cortex (PFC) and hippocampus (HIP). Heat-sterilized B. breve supplementation significantly prevented social interaction impairment, suppressed IL-1ß increase in the PFC and HIP, and modulated the alteration of the gut microbiota composition induced by CSDS. These findings suggest that heat-sterilized B. breve prevents depression-like behavior and IL-1ß expression induced by CSDS through modulation of the gut microbiota composition in mice. Therefore, heat-sterilized B. breve used as an ingredient of functional food might prevent MDD.

Epo-C12 inhibits peroxiredoxin 1 peroxidase activity.

Epo-C12 is a synthetic derivative of epolactaene, isolated from Penicillium sp. BM 1689-P. Epo-C12 induces apoptosis in human acute lymphoblastoid leukemia BALL-1 cells. In our previous studies, seven proteins that bind to Epo-C12 were identified by a combination of pull-down experiments using biotinylated Epo-C12 (Bio-Epo-C12) and mass spectrometry. In the present study, the effect of Epo-C12 on peroxiredoxin 1 (Prx 1), one of the proteins that binds to Epo-C12, was investigated. Epo-C12 inhibited Prx 1 peroxidase activity. However, it did not suppress its chaperone activity. Binding experiments between Bio-Epo-C12 and point-mutated Prx 1s suggest that Epo-C12 binds to Cys52 and Cys83 in Prx 1. The present study revealed that Prx 1 is one of the target proteins through which Epo-C12 exerts an apoptotic effect in BALL-1 cells.

Sonic hedgehog enhances calcium oscillations in hippocampal astrocytes.

Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute time scale, indicating a noncanonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a Gi coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP-degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of Gq/11-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, Gi mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.

An affinity change model to elucidate the rotation mechanism of V1-ATPase.

V-ATPases are ubiquitous proton-transporting ATPases of eukaryotic and prokaryotic membranes that utilize energy from ATP hydrolysis. The hydrophilic catalytic part called V1-ATPase is composed of a ring-shaped hexametric A3B3 complex and a central DF shaft. We previously proposed a rotation mechanism of the Enterococcus hirae V1-ATPase based on the crystal structures of the V1 and A3B3 complexes. However, the driving force that induces the conformational changes of A3B3 and rotation of the DF shaft remains unclear. In this study, we investigated the binding affinity changes between subunits of V1-ATPase by surface plasmon resonance analysis. The binding of ATP to subunit A was found to considerably increase the affinity between the A and B subunits, and thereby ATP binding contributes to forming the A1B1 tight conformation. Furthermore, the DF shaft bound to the reconstituted A1B1 complex with high affinity, suggesting that the tight A1B1 complex is a major binding unit of the shaft in the A3B3 ring complex. Based on these results, we propose that rotation of the V1-ATPase is driven by affinity changes between each subunit via thermal fluctuations.

Rotation mechanism of Enterococcus hirae V1-ATPase based on asymmetric crystal structures.

In various cellular membrane systems, vacuolar ATPases (V-ATPases) function as proton pumps, which are involved in many processes such as bone resorption and cancer metastasis, and these membrane proteins represent attractive drug targets for osteoporosis and cancer. The hydrophilic V(1) portion is known as a rotary motor, in which a central axis DF complex rotates inside a hexagonally arranged catalytic A(3)B(3) complex using ATP hydrolysis energy, but the molecular mechanism is not well defined owing to a lack of high-resolution structural information. We previously reported on the in vitro expression, purification and reconstitution of Enterococcus hirae V(1)-ATPase from the A(3)B(3) and DF complexes. Here we report the asymmetric structures of the nucleotide-free (2.8 Å) and nucleotide-bound (3.4 Å) A(3)B(3) complex that demonstrate conformational changes induced by nucleotide binding, suggesting a binding order in the right-handed rotational orientation in a cooperative manner. The crystal structures of the nucleotide-free (2.2 Å) and nucleotide-bound (2.7 Å) V(1)-ATPase are also reported. The more tightly packed nucleotide-binding site seems to be induced by DF binding, and ATP hydrolysis seems to be stimulated by the approach of a conserved arginine residue. To our knowledge, these asymmetric structures represent the first high-resolution view of the rotational mechanism of V(1)-ATPase.

RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells.

Adenosine triphosphate (ATP) provides energy for the regulation of multiple cellular processes in living organisms. Capturing the spatiotemporal dynamics of ATP in single cells is fundamental to our understanding of the mechanisms underlying cellular energy metabolism. However, it has remained challenging to visualize the dynamics of ATP in and between distinct intracellular organelles and its interplay with other signaling molecules. Using single fluorescent proteins, multicolor ATP indicators were developed, enabling the simultaneous visualization of subcellular ATP dynamics in the cytoplasm and mitochondria of cells derived from mammals, plants, and worms. Furthermore, in combination with additional fluorescent indicators, the dynamic interplay of ATP, cAMP, and Ca2+ could be visualized in activated brown adipocyte. This set of indicator tools will facilitate future research into energy metabolism.

Dopamine D1 Receptor-Mediated Transmission Maintains Information Flow Through the Cortico-Striato-Entopeduncular Direct Pathway to Release Movements.

In the basal ganglia (BG), dopamine plays a pivotal role in motor control, and dopamine deficiency results in severe motor dysfunctions as seen in Parkinson's disease. According to the well-accepted model of the BG, dopamine activates striatal direct pathway neurons that directly project to the output nuclei of the BG through D1 receptors (D1Rs), whereas dopamine inhibits striatal indirect pathway neurons that project to the external pallidum (GPe) through D2 receptors. To clarify the exact role of dopaminergic transmission via D1Rs in vivo, we developed novel D1R knockdown mice in which D1Rs can be conditionally and reversibly regulated. Suppression of D1R expression by doxycycline treatment decreased spontaneous motor activity and impaired motor ability in the mice. Neuronal activity in the entopeduncular nucleus (EPN), one of the output nuclei of the rodent BG, was recorded in awake conditions to examine the mechanism of motor deficits. Cortically evoked inhibition in the EPN mediated by the cortico-striato-EPN direct pathway was mostly lost during suppression of D1R expression, whereas spontaneous firing rates and patterns remained unchanged. On the other hand, GPe activity changed little. These results suggest that D1R-mediated dopaminergic transmission maintains the information flow through the direct pathway to appropriately release motor actions.

Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum.

Ca2+ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca2+ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N3-fura-2) to a specific site of subcellular compartments to realize focal Ca2+ sensing. Using scAVD (single-chain avidin)-biotin interaction and a copper-free click reaction system, we linked N3-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca2+ sensors conjugated with four N3-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N3-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N3-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N3-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca2+ concentration, ER-localized N3-fura-2 successfully sensed the Ca2+ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N3-fura-2 to subcellular compartments and the ability of sensing focal Ca2+ level changes with the specifically targeted Ca2+ sensors.

Autocrine pathways involving S100A8 and/or S100A9 that are postulated to regulate the immunological functions of macrophages in rats.

The development of ulcerative colitis (UC) is closely associated with abnormally functioning macrophages. Rat S100A8 (r-S100A8) and r-S100A9 (S100 proteins) is abundantly expressed in immune cells of myeloid origin, macrophages; however, it remains unclear why r-S100A9 is dominantly expressed in the macrophages of UC rats (UCR). The purpose of this study was to verify the immunological roles of S100 proteins in UCR. We observed the distribution of S100 protein-positive macrophages in the large colons of UCR using a fluorescent immunological staining method, so that S100 protein-positive macrophages were restricted to the rectal tissues of the UCR, and that the mRNA levels of r-S100A8 and r-S100A9 were up-regulated by stimulation with recombinant rat S100A8 (rr-S100A8) alone and rr-S100A9 alone, respectively. When the changes in the mRNA levels of r-S100A8 and r-S100A9 in macrophages were examined in in vitro study by PCR and real-time PCR, the mRNA levels of anti-inflammatory and inflammatory cytokines increased selectively after stimulation with rr-S100A8 alone and rr-S100A9 alone, respectively. These results suggest that autocrine signal transduction pathways involving S100 proteins regulate the immunological functions of macrophages to maintain homeostasis in the gastrointestinal tract. This may be depended on expression balance of S100 proteins in macrophages. It is strongly suggested that in UCR the immune functions of macrophages are regulated in a complex manner by r-S100A8 and/or r-S100A9 through undefined autocrine pathways on the cells.

Micro-thermography in millimeter-scale animals by using orally-dosed fluorescent nanoparticle thermosensors.

We propose an instant micro-thermography method using a fluorescent-nanoparticle thermosensor capable of reporting temperature as the fluorescence intensity ratio of the temperature-sensitive dye to the reference. We demonstrate "temperature mapping" inside a fruit fly larva that was orally dosed with nanoparticle thermosensors.

Crystal structure of the central axis DF complex of the prokaryotic V-ATPase.

V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of the central axis D subunit and a membrane c ring that are connected by F and d subunits. Here we determined the crystal structure of the DF complex of the prokaryotic V-ATPase of Enterococcus hirae at 2.0-Å resolution. The structure of the D subunit comprised a long left-handed coiled coil with a unique short ß-hairpin region that is effective in stimulating the ATPase activity of V(1)-ATPase by twofold. The F subunit is bound to the middle portion of the D subunit. The C-terminal helix of the F subunit, which was believed to function as a regulatory region by extending into the catalytic A(3)B(3) complex, contributes to tight binding to the D subunit by forming a three-helix bundle. Both D and F subunits are necessary to bind the d subunit that links to the c ring. From these findings, we modeled the entire rotor complex (DFdc ring) of V-ATPase.

Design strategy for blends of biodegradable polyester and thermoplastic starch based on a molecular dynamics study of the phase-separated interface.

Molecular insight into the phase-separated interface formed when biodegradable polyesters and thermoplastic starch (TPS) are melt-blended is valuable for the design of composites. In this study, eight different interfaces combining four major biodegradable polyesters (PLA, PBS, PHB and PBAT) and two TPSs [unmodified TPS (nTPS) and citrate-modified TPS (cTPS)] were investigated by using molecular dynamics (MD) simulations. According to the MD simulation results, PBS, PHB and PBAT diffuse readily into the TPS and form compatible interfaces, whereas PLA is less compatible with the TPS. The results of tensile simulations show that PBS and PBAT adhere well to TPS; in particular, PBS/cTPS and PBAT/cTPS exhibit high interfacial-fracture energy (G). Both PLA and PHB blended with TPS exhibit low G because PLA is less compatible with TPS and PHB and TPS have low electrostatic interaction. The reason for the high G of PBS/cTPS and PBAT/cTPS is thought to be a combination of three factors: (i) formation of a deep compatible interface, (ii) suppression of void growth by electrostatic interactions and (iii) absorption of strain energy by a change in the conformation of the molecular chains. These three interfacial adhesion mechanisms should be considered when designing biodegradable polyester/TPS blends with good mechanical properties.

Effects of Heat-Killed Lacticaseibacillus paracasei MCC1849 on Immune Parameters in Healthy Adults-A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study.

Previous clinical studies have shown that heat-killed Lacticaseibacillus paracasei MCC1849 suppresses subjective symptoms among healthy adults. However, the mechanism underlying this beneficial effect remains unclear. This clinical study aimed to investigate the effects of MCC1849 on immune functions in humans. In this randomized, double-blind, placebo-controlled, parallel-group study, 100 healthy adults were randomly divided into MCC1849 or placebo groups. Participants ingested test powder with 5 × 1010 MCC1849 cells or placebo powder for 4 weeks. Immune functions were evaluated using expression levels of CD86 and HLA-DR on dendritic cells (DCs), neutrophils, and natural killer cells. The expression levels of interferon (IFN)-α, -ß, and -γ in peripheral blood mononuclear cells incubated with Cpg2216 in vitro were quantified. Efficacy analysis was performed on participants in the per-protocol set (placebo group; n = 47, MCC1849 group; n = 49). The expression level of CD86 on pDCs and the gene expression levels of IFN-α, -ß, and -γ upon TLR9 agonist stimulation were significantly higher in the MCC1849 group at 4 weeks. No side effects were observed. This is the first report to show the positive effects of MCC1849 on human immune cells. These findings reveal one possible mechanism of how MCC1849 suppresses subjective symptoms.

Effects of Bifidobacterium longum BB536 and Bifidobacterium breve MCC1274 on Body Composition in Normal and Overweight Adults in Randomized Placebo-Controlled Study.

Visceral fat accumulation is considered to be associated with a higher risk of chronic diseases. We investigated the effects of Bifidobacterium longum subsp. longum (B. longum) BB536 and Bifidobacterium breve (B. breve) MCC1274 on body composition, including visceral fat, in a randomized, parallel-group, placebo-controlled study. Participants were between 29 and 64 years of age and had a body mass index (BMI) of greater than 23 and less than 30. One hundred participants were randomly assigned to the probiotics group or placebo group. Participants were administered probiotic capsules containing 1 × 1010 colony-forming units (CFUs) of B. longum BB536 and 5 × 109 CFU of B. breve MCC1274 or placebo capsules without bifidobacteria for 16 weeks. In the probiotics group, abdominal visceral fat area, total abdominal fat area, and serum triglyceride levels were significantly decreased compared to those in the placebo group. Additionally, the increase in BMI observed in the placebo group was significantly suppressed in the probiotics group. This study showed that B. longum BB536 and B. breve MCC1274 reduced abdominal visceral fat and total fat levels in healthy normal and overweight adults, suggesting their beneficial effects on body composition.

Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution.

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

Immunomodulatory activity of heat-killed Lacticaseibacillus paracaseiMCC1849 based on the activation of plasmacytoid dendritic cells in the peripheral blood of healthy adults.

Probiotics are widely used in food for their health benefits to the host. Inactivated probiotics also reportedly improve the intestinal environment and immune regulation. Our previous studies showed that heat-killed Lacticaseibacillus paracasei MCC1849 (hk-MCC1849) effectively induced IL-12 production in mouse spleen cells and significantly reduced cold symptoms in clinical trial subjects. To further elucidate the mechanism of host immune regulation by hk-MCC1849, human peripheral blood mononuclear cells (PBMCs) were cocultured with hk-MCC1849. The Toll-like receptor 9 ligands CpG-ODN 2216 and hk-MCC1849 and the heat-killed Lacticaseibacillus rhamnosus ATCC53103 were used as positive and negative controls, respectively. The results showed that, compared with the control, hk-MCC1849 significantly increased the expression of the plasmacytoid dendritic cell (pDC) marker CD86 (p < .0001) and the pDC marker HLA-DR (p < .001) in PBMCs. The expression levels of the IL-12p40, IFNα, IFNα1, IFNγ, and ISG15 genes were significantly increased after coculture with hk-MCC1849 (p < .05, p < .05, p < .05, p < .05, and p < .05, respectively, vs. control). Furthermore, to confirm whether hk-MCC1849 directly interacted with pDCs, DCs were enriched with PBMCs following 24 h of coculture with hk-MCC1849. Phagocytosis of fluorescently labeled hk-MCC1849 by pDCs was observed, and there were significant increases in CD86 (p < .05) and HLA-DR (p < .0001) expression in pDCs. These results suggest that hk-MCC1849 exerts a potential immunomodulatory effect on the host through the activation of peripheral pDCs.

Bifidobacterium breve M-16V regulates the autonomic nervous system via the intestinal environment: A double-blind, placebo-controlled study.

We conducted a randomized controlled trial to investigate the potential of Bifidobacterium breve M-16 V to improve mood in humans. In this evaluation, we incorporated the use of near-infrared spectroscopy (NIRS), which has been used to evaluate mood states in studies with small sample sizes. Participants were given B. breve M-16 V (20 billion cells/day) for 6 weeks, and their mood state was assessed before and after ingestion. NIRS data were collected at rest and during a mental arithmetic task (under stress). Intake of B. breve M-16 V decreased the heart rate under stress and increased levels of the GABA-like substance pipecolic acid in stool samples. In addition, B. breve M-16 V improved mood and sleep scores in participants with high anxiety levels. These results suggest that B. breve M-16 V affects the metabolites of the gut microbiota and has the potential to modulate the autonomic nervous system and to improve mood and sleep.