Naphthyridine-Based Electron Push-Pull-Type Amine-Reactive Fluorescent Probe for Sensing Amines and Proteins in Aqueous Media.

In bioengineering, fluorescent amine-reactive probes are invaluable for the detection of amine species. In particular, targeting probes for lysine, which has a free amino group in amino acids, are a valid method for protein detection. For this purpose, many fluorescent "turn-on type" probes with amine reactivity have been developed; however, they require improvements. In the typical florescence probes, BODIPY and NBD analogs have small Stokes shifts based on absorption and emission and lability in an aqueous environment, respectively. In this study, a new class of fluorescent probes, 1,8-Nap-F, based on the electron push-pull-type 1,8-naphthyridine framework, was designed and investigated as an amine-reactive probe. Generally, electron push-pull-type fluorophores exhibit a large Stokes shift at the expense of fluorescent enhancement in aqueous media; thus, there is a trade-off between possessing a large Stokes shift and intense emission. However, 1,8-Nap-F reacts with primary amines, yielding emissive amine products with a large Stokes shift (>70 nm) without fluorescence quenching and side products, even in an aqueous environment, thereby overcoming the disadvantages of electron push-pull-type fluorophores and lability in aqueous conditions. By applying the specific features of 1,8-Nap-F, we achieved selective lysine detection and fluorescence bioimaging, such as endoplasmic reticulum-selective protein labeling and organelle staining, in living cells by utilizing amine-substituted derivatives.

Acid responsiveness of emissive morpholinyl aminoquinolines and their use for cell fluorescence imaging.

Herein, we report emissive aminoquinoline derivatives (TFMAQ) containing alkylmorpholine and arylmorpholine groups and their photophysical properties, acid-responsiveness, and organelle targeting. The alkylmorpholine group is well-known to favour accumulation in lysosomes and be acid-responsive, but, counterintuitively, the TFMAQ derivatives containing ethylmorpholine groups showed limited accumulation in lysosomes and, instead, preferential accumulation in lipid droplets. The findings reported here will aid the development of organelle/tissue specific dyes for cell imaging and diagnosis.

The role of the cannabinoid system in fear memory and extinction in male and female mice.

The prevalence of post-traumatic stress disorder (PTSD) is higher in women than in men. Among both humans and mice, females exhibit higher resistance to fear extinction than males, suggesting that differences between sexes in fear-extinction processes are involved in the pathophysiology of such fear-related diseases. Sex differences in molecular mechanisms underlying fear memory and extinction are unclear. The cannabinoid (CB) system is well known to be involved in fear memory and extinction, but this involvement is based mainly on experiments using male rodents. It is not known whether there are sex differences in the role of the CB system in fear memory and extinction. To explore this possibility, we investigated the effects of pharmacological manipulations of the CB system on the retrieval and extinction of contextual fear memory in male and female mice. WIN55,212-2, a CB receptor (CBR) agonist, augmented the retrieval of fear memory in both sexes, but SR141716 (a CB1R antagonist) did not affect it in either sex. An enhancement of 2-arachidonylglycerol (2-AG, one of the two major endocannabinoids) via JZL184 (an inhibitor of the 2-AG hydrolase monoacylglycerol lipase [MAGL]), augmented the retrieval of fear memory through the activation of CB1R but not CB2R in female mice. In contrast, the enhancement of N-arachidonylethanolamine (AEA, the other major endocannabinoid) via URB597, an inhibitor of an AEA hydrolase (fatty acid amide hydrolase-1) did not show any effects on the retrieval of fear memory in either sex. WIN55,212-2, SR141716, and JZL184 inhibited fear extinction irrespective of sex. URB enhanced fear extinction in females that were in diestrus phase at the first extinction session, but not in males. These results suggest that although the role of CB1R in the retrieval and extinction of contextual fear memory is common among males and females, the effects of an increase in endocannabinoid levels on the retrieval or extinction of contextual fear memory differ between the sexes.

Characterizing clinical outcomes and factors associated with conduction gaps in VISITAG SURPOINT-guided catheter ablation for atrial fibrillation.

PURPOSE: Although usefulness of VISITAG SURPOINT (VS) on pulmonary vein isolation (PVI) in catheter ablation of atrial fibrillation has been reported, optimal VS thresholds can depend on the inter-tag distance (ITD) and vice versa. We validated the efficacy of PVI with lower target ITDs and VS values than in previous studies. METHODS: Retrospective review of consecutive patients (N = 100) with paroxysmal (n = 32) or persistent AF (n = 68) undergoing VS-guided ablation between 09/2018 and 08/2019 was conducted. All procedures were performed by two operators. Target VS values were 425 (anterior), 375 (posterior), and 325 (near the esophagus). Target ITD was 4 mm. RESULTS: Acute PVI was achieved in all cases, however, 13 residual gaps in 12 patients were observed after initial encirclement (first pass isolation: 88%). Ten gaps due to spontaneous PV reconnections (PVR) were found in nine patients (9%). These 23 gaps had similar median VS (gap-related vs non-gap: 429 vs 410, P = .4545) and power (36 vs 36W, P = .4843), higher contact force (13.8 vs 11.0g, P = .0061), and larger ITD (5.3 vs 3.7mm, P < .001) when compared to the remaining tags. Only ITDs were independently associated with gap formation in multivariate analysis. One-year Kaplan-Meier freedom from any atrial arrhythmia was 87.2%. Eight patients received repeat ablation (8.1%) and of these, 6 (75%) were free from PVR. CONCLUSION: Favorable rates of first pass isolation, acute PVR, and long-term procedure success were achieved using lower VS values than in previous reports. With a target VS value of 375-425, ITDs of 4 mm was sufficient for durable PVI.

Both IRBIT and long-IRBIT bind to and coordinately regulate Cl-/HCO3- exchanger AE2 activity through modulating the lysosomal degradation of AE2.

Anion exchanger 2 (AE2) plays crucial roles in regulating cell volume homeostasis and cell migration. We found that both IRBIT and Long-IRBIT (L-IRBIT) interact with anion exchanger 2 (AE2). The interaction occurred between the conserved AHCY-homologous domain of IRBIT/L-IRBIT and the N-terminal cytoplasmic region of AE2. Interestingly, AE2 activity was reduced in L-IRBIT KO cells, but not in IRBIT KO cells. Moreover, AE2 activity was slightly increased in IRBIT/L-IRBIT double KO cells. These changes in AE2 activity resulted from changes in the AE2 expression level of each mutant cell, and affected the regulatory volume increase and cell migration. The activity and expression level of AE2 in IRBIT/L-IRBIT double KO cells were downregulated if IRBIT, but not L-IRBIT, was expressed again in the cells, and the downregulation was cancelled by the co-expression of L-IRBIT. The mRNA levels of AE2 in each KO cell did not change, and the downregulation of AE2 in L-IRBIT KO cells was inhibited by bafilomycin A1. These results indicate that IRBIT binding facilitates the lysosomal degradation of AE2, which is inhibited by coexisting L-IRBIT, suggesting a novel regulatory mode of AE2 activity through the binding of two homologous proteins with opposing functions.

Quantum key distribution with correlated sources.

In theory, quantum key distribution (QKD) offers information-theoretic security. In practice, however, it does not due to the discrepancies between the assumptions used in the security proofs and the behavior of the real apparatuses. Recent years have witnessed a tremendous effort to fill the gap, but the treatment of correlations among pulses has remained a major elusive problem. Here, we close this gap by introducing a simple yet general method to prove the security of QKD with arbitrarily long-range pulse correlations. Our method is compatible with those security proofs that accommodate all the other typical device imperfections, thus paving the way toward achieving implementation security in QKD with arbitrary flawed devices. Moreover, we introduce a new framework for security proofs, which we call the reference technique. This framework includes existing security proofs as special cases, and it can be widely applied to a number of QKD protocols.

Sex-dependent opposite effects of a tropomyosin-related kinase B receptor (TrkB) agonist 7,8-dihydroxyflavone on cued fear extinction in mice.

Tropomyosin-related kinase B receptor (TrkB) is one of the new candidate receptors for drugs targeting psychiatric and neurodegenerative disorders. Recently, 7,8-dihydroxyflavone (7,8-DHF) has been identified as a selective TrkB agonist that crosses the blood-brain barrier after oral or intraperitoneal administration, and it enhances cued fear extinction in male rodents. However, its effects on females remain unclear. Preclinical research including both sexes is important for the development of treatment, particularly, for stress-related disorders such as post-traumatic stress disorder because such disorders are more prevalent in women. Therefore, we investigated the effects of 7,8-DHF on cued and contextual fear extinction in both male and female mice. Here we demonstrated that the administration of 7,8-DHF before each extinction session attenuated cued fear extinction in females; conversely, it enhanced cued fear extinction in males. However, administration of 7,8-DHF immediately after each extinction session did not affect cued fear extinction in either sex. Moreover, in contextual fear extinction, administration of 7,8-DHF before each extinction session did not affect fear extinction in either sex. Thus, 7,8-DHF showed sex-dependent opposite effects on cued fear extinction in mice when administered before but not immediately after each extinction session. Our results could contribute to the development of pharmacotherapy involving 7,8-DHF, particularly for stress-related disorders.

Selective synthesis of substituted amino-quinoline derivatives by C-H activation and fluorescence evaluation of their lipophilicity-responsive properties.

Push-pull type fluorescent amino-quinoline derivatives (TFMAQ) bearing phenyl aromatic groups in the 8-position (TFMAQ-8Ar series) were synthesized via palladium-catalyzed C-H activation reaction in short steps. The N-arylation or C-H activation reactions were selectively controlled with high yield by combinations of palladium and phosphine ligands. The TFMAQ-8Ar analogues exhibited fluorescent solvatochromism in non-polar and polar solvents. In non-polar solvent, the absolute fluorescence quantum yield was high, wheareas the fluorescence was almost quenched in polar solvent. The TFMAQ-8Ar derivatives also showed high fluorescence emission at solid state owing to the planar structure between the quinoline ring and phenyl ring at the 7-amino group, as demonstrated by X-ray crystal structure analysis. The fluorescence imaging of 3T3-L1 cell using TFMAQ-8Ar derivatives was performed by confocal laser microscopy. Strong and specific emissions at lipid droplets were observed owing to the accumulation of TFMAQ-8Ar derivatives. Therefore, we propose that the TFMAQ-8Ar derivatives should become a versatile fluorescence probe for the live imaging of lipid droplets.

The Phosphatidylinositol 3-Kinase p110α/PTEN Signaling Pathway Is Crucial for HIV-1 Entry.

Human immunodeficiency virus type 1 (HIV-1) drives multiple signaling pathways to facilitate its cellular entry and replication. The interaction between HIV-1 envelope (env) protein and target cell surface CD4 first activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, and the subsequent interaction between HIV-1 env glycoprotein and CCR5/CXCR4 coreceptors establishes viral fusion and entry. Four isoforms of the class-I PI3K catalytic subunits (p110α, p110ß, p110γ, and p110δ) have been identified so far, but the isoform(s) involved in the HIV-1 entry is still unknown. This study aimed to identify the PI3K isoform(s) using recently developed isoform-specific inhibitors and the roles of their negative regulators, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1), in HIV-1 infection. We found that the PI3K p110α isoform-specific inhibitor PIK-75 suppressed HIV-1 entry in HIV-1 permissive T cells, PM1 cells, and TZM-bl cells (HeLa cell-derived indicator cells that coexpress CD4, CCR5, and CXCR4) and decreased the HIV-1-induced phosphorylation of Akt. Moreover, wild-type PTEN (but neither phosphatase-deficient PTEN nor wild-type SHIP1) was a key regulator of HIV-1 entry. Cell-to-cell fusion by HIV-1 env-CD4 interaction was suppressed in the presence of PI3K p110α-specific inhibitor. These data suggest that the PI3K p110α/PTEN signaling pathway is indispensable for HIV-1 entry, including HIV-1 env-mediated cell-to-cell fusion.

Sex differences in the effects of adult short-term isolation rearing on contextual fear memory and extinction.

Fear conditioning and extinction is a useful tool for understanding the pathogenesis of fear-related disorders including post-traumatic stress disorder (PTSD) and for developing treatments for them. To investigate the role of sub-brain regions or molecular mechanisms in fear conditioning and extinction, neuroscientists have been employing an optogenetic or in vivo recording technique, in which placement of an optical fiber or an electrode into the brain region of a free-moving mouse is essential. These methods require isolation rearing (at least one week) from the brain surgery to the behavioral test. Although such short-term adult rearing has been shown not to influence fear memory and extinction in males, the effect in females remains unclear. In the present study, we investigated the effect on fear memory and fear extinction of adult isolation rearing during the one week before contextual fear conditioning in both male and female mice. This short-term adult isolation rearing increased fear responses in the contextual fear memory test in females but not in males. On the other hand, the rearing showed no effect on fear responses during fear extinction or the recall test in either sex. In summary, adult short-term isolation rearing enhanced only fear memory, and only in females.

Splicing variation of Long-IRBIT determines the target selectivity of IRBIT family proteins.

IRBIT [inositol 1,4,5-trisphosphate receptor (IP3R) binding protein released with inositol 1,4,5-trisphosphate (IP3)] is a multifunctional protein that regulates several target molecules such as ion channels, transporters, polyadenylation complex, and kinases. Through its interaction with multiple targets, IRBIT contributes to calcium signaling, electrolyte transport, mRNA processing, cell cycle, and neuronal function. However, the regulatory mechanism of IRBIT binding to particular targets is poorly understood. Long-IRBIT is an IRBIT homolog with high homology to IRBIT, except for a unique N-terminal appendage. Long-IRBIT splice variants have different N-terminal sequences and a common C-terminal region, which is involved in multimerization of IRBIT and Long-IRBIT. In this study, we characterized IRBIT and Long-IRBIT splice variants (IRBIT family). We determined that the IRBIT family exhibits different mRNA expression patterns in various tissues. The IRBIT family formed homo- and heteromultimers. In addition, N-terminal splicing of Long-IRBIT changed the protein stability and selectivity to target molecules. These results suggest that N-terminal diversity of the IRBIT family and various combinations of multimer formation contribute to the functional diversity of the IRBIT family.

Fundamental rate-loss trade-off for the quantum internet.

The quantum internet holds promise for achieving quantum communication-such as quantum teleportation and quantum key distribution (QKD)-freely between any clients all over the globe, as well as for the simulation of the evolution of quantum many-body systems. The most primitive function of the quantum internet is to provide quantum entanglement or a secret key to two points efficiently, by using intermediate nodes connected by optical channels with each other. Here we derive a fundamental rate-loss trade-off for a quantum internet protocol, by generalizing the Takeoka-Guha-Wilde bound to be applicable to any network topology. This trade-off has essentially no scaling gap with the quantum communication efficiencies of protocols known to be indispensable to long-distance quantum communication, such as intercity QKD and quantum repeaters. Our result-putting a practical but general limitation on the quantum internet-enables us to grasp the potential of the future quantum internet.

Metallomics approach to changes in element concentration during differentiation from fibroblasts into adipocytes by element array analysis.

We aimed to establish an element array analysis that involves the simultaneous detection of all elements in cells and the display of changes in element concentration before and after a cellular event. In this study, we demonstrated changes in element concentration during the differentiation of 3T3-L1 mouse fibroblasts into adipocytes. This metallomics approach yielded unique information of cellular response to physiological and toxicological events.

IRBIT regulates CaMKIIα activity and contributes to catecholamine homeostasis through tyrosine hydroxylase phosphorylation.

Inositol 1,4,5-trisphosphate receptor (IP3R) binding protein released with IP3 (IRBIT) contributes to various physiological events (electrolyte transport and fluid secretion, mRNA polyadenylation, and the maintenance of genomic integrity) through its interaction with multiple targets. However, little is known about the physiological role of IRBIT in the brain. Here we identified calcium calmodulin-dependent kinase II alpha (CaMKIIα) as an IRBIT-interacting molecule in the central nervous system. IRBIT binds to and suppresses CaMKIIα kinase activity by inhibiting the binding of calmodulin to CaMKIIα. In addition, we show that mice lacking IRBIT present with elevated catecholamine levels, increased locomotor activity, and social abnormalities. The level of tyrosine hydroxylase (TH) phosphorylation by CaMKIIα, which affects TH activity, was significantly increased in the ventral tegmental area of IRBIT-deficient mice. We concluded that IRBIT suppresses CaMKIIα activity and contributes to catecholamine homeostasis through TH phosphorylation.

Measurement-device-independent quantum key distribution for Scarani-Acin-Ribordy-Gisin 04 protocol.

The measurement-device-independent quantum key distribution (MDI QKD) was proposed to make BB84 completely free from any side-channel in detectors. Like in prepare & measure QKD, the use of other protocols in MDI setting would be advantageous in some practical situations. In this paper, we consider SARG04 protocol in MDI setting. The prepare & measure SARG04 is proven to be able to generate a key up to two-photon emission events. In MDI setting we show that the key generation is possible from the event with single or two-photon emission by a party and single-photon emission by the other party, but the two-photon emission event by both parties cannot contribute to the key generation. On the contrary to prepare & measure SARG04 protocol where the experimental setup is exactly the same as BB84, the measurement setup for SARG04 in MDI setting cannot be the same as that for BB84 since the measurement setup for BB84 in MDI setting induces too many bit errors. To overcome this problem, we propose two alternative experimental setups, and we simulate the resulting key rate. Our study highlights the requirements that MDI QKD poses on us regarding with the implementation of a variety of QKD protocols.

Irbit mediates synergy between ca(2+) and cAMP signaling pathways during epithelial transport in mice.

BACKGROUND & AIMS: The cyclic adenosine monophosphate (cAMP) and Ca(2+) signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. METHODS: We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6(-/-) mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit(-/-)) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl(-) current. Protein interactions were determined by immunoprecipitation analyses. RESULTS: Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca(2+) and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit(-/-) or Slc26a6(-/-) mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation. CONCLUSIONS: Irbit promotes synergy between the Ca(2+) and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjögren syndrome.

Cot kinase promotes Ca2+ oscillation/calcineurin-independent osteoclastogenesis by stabilizing NFATc1 protein.

Osteoclasts are multinuclear bone-resorbing cells formed by the fusion of monocyte/macrophage-lineage precursor cells. Activation of the transcription factor NFATc1 (nuclear factor of activated T cells c1) by the receptor activator of NF-κB ligand (RANKL) is critical for osteoclast differentiation. In our previous report (Y. Kuroda, C. Hisatsune, T. Nakamura, K. Matsuo, and K. Mikoshiba. Proc. Natl. Acad. Sci. U. S. A. 105:8643, 2008), we demonstrated that osteoblasts induce osteoclast differentiation via Ca(2+) oscillation/calcineurin-dependent and -independent NFATc1 activation pathways; however, the mechanism underlying the latter remained unclear. Here we show that Cot, a serine/threonine kinase also known as tumor progression locus 2 (Tpl-2), directly phosphorylates all Ca(2+)/calcineurin-regulated NFAT family members (NFATc1 through NFATc4) and increases their protein levels. Moreover, Cot activity in osteoclasts was enhanced via cell-cell interaction with osteoblasts, and Cot promoted Ca(2+) oscillation/calcineurin-independent osteoclastogenesis by increasing NFATc1 stability through phosphorylation. We propose that NFAT activation in vivo occurs via phosphorylation-induced protein stabilization, even in the absence of Ca(2+) oscillation and calcineurin activity.

Syntaxin 1B suppresses macropinocytosis and semaphorin 3A-induced growth cone collapse.

Growth cone collapse is a crucial process for repulsive axon guidance and is accompanied by a reduction in growth cone surface area. This process of reduction may be regulated by endocytosis; however, its molecular mechanism is unclear. Macropinocytosis is a clathrin-independent form of endocytosis in which large areas of plasma membrane can be engulfed. We have reported previously that macropinocytosis is induced in growth cones of chick dorsal root ganglion neurons by semaphorin 3A (Sema3A), a repulsive axon guidance cue, and that Sema3A-induced reduction in growth cone surface area and macropinocytic vacuole area were correlated, suggesting a positive role for macropinocytosis in Sema3A-induced growth cone collapse. In the present study, we found that syntaxin 1B (Syx1B), a membrane trafficking protein, is a negative regulator of macropinocytosis, and its expression is downregulated by Sema3A signaling. Macropinocytosis inhibitor ethylisopropylamiloride or Syx1B overexpression suppressed Sema3A-induced macropinocytosis and growth cone collapse. These results indicate that Syx1B couples macropinocytosis-mediated massive internalization of the plasma membrane to Sema3A-induced growth cone collapse.

IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway.

Fluid and HCO(3)(-) secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO(3)(-) in secretory glands is fueled by Na(+)/HCO(3)(-) cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl(-)/HCO(3)(-) exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP(3)) receptor-binding protein released with IP(3) (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO(3)(-) secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase-regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.

Isolation of inositol 1,4,5-trisphosphate receptor-associating proteins and selective knockdown using RNA interference.

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are IP(3)-gated Ca(2+) release channels localized on intracellular Ca(2+) stores and play a role in the generation of complex patterns of intracellular Ca(2+) signals. We show herein experimental protocols for the identification of associating proteins of IP(3)R isoforms from various cells and tissues using affinity column chromatography and for the specific knockdown of the expression of IP(3)R isoforms and their associating proteins using RNA interference. These methods will provide clues to understand the exact nature of how the signaling complex contributes to the generation of spatio-temporal patterns of intracellular Ca(2+) signals.