Development of nanobody-based POLArIS orientation probes enabled multi-color/multi-target orientation imaging in living cells.

Fluorescence polarization microscopy (FPM) can visualize the dipole orientation of fluorescent molecules and has been used for analyzing architectural dynamics of biomolecules including cytoskeletal proteins. To monitor the orientation of target molecules by FPM, target molecules need to be labeled with fluorophores in a sterically constrained manner, so that the fluorophores do not freely rotate. Recently, a versatile probe for such labeling using fluorescent proteins, POLArIS (Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest), was reported. POLArIS is a fusion protein consisting of a non-immunoglobulin-based recombinant binder Affimer and a green fluorescent protein (GFP), where the Affimer and GFP are rigidly connected to each other. POLArIS probe for molecules of interest can be developed through phage display screening of Affimer. This screening is followed by the rigid connection of fluorescent proteins to the selected Affimers. The Affimer-based POLArIS, however, cannot be used with animal immune libraries for selecting specific binder clones. In addition, multi-color FPM by POLArIS was not available due to the lack of color variations of POLArIS. In this study, we have developed new versions of POLArIS with nanobodies, which are compatible with animal immune libraries, and expanded color variations of POLArIS with cyan/green/yellow/red fluorescent proteins, enabling multi-color orientation imaging for multiple targets. Using nanobody-based POLArIS orientation probes, we performed two-color FPM of F-actin and vimentin in living cells. Furthermore, we made nanobody-based POLArIS probes that have different dipole orientations for adjusting the orientation of fluorescence polarization with respect to the target molecules. These nanobody-based POLArIS with options of colors and dipole orientations will enhance the performance of this probe for broader applications of fluorescence polarization imaging in living cells, tissues, and whole organisms.

POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

Genetically encoded orientation probes for F-actin for fluorescence polarization microscopy.

Fluorescence polarization microscopy, which can visualize both position and orientation of fluorescent molecules, is useful for analyzing architectural dynamics of proteins in vivo, especially that of cytoskeletal proteins such as actin. Fluorescent phalloidin conjugates and SiR-actin can be used as F-actin orientation probes for fluorescence polarization microscopy, but a lack of appropriate methods for their introduction to living specimens especially to tissues, embryos, and whole animals hampers their applications to image the orientation of F-actin. To solve this problem, we have developed genetically encoded F-actin orientation probes for fluorescence polarization microscopy. We rigidly connected circular permutated green fluorescent protein (GFP) to the N-terminal α-helix of actin-binding protein Lifeact or utrophin calponin homology domain (UtrCH), and normal mEGFP to the C-terminal α-helix of UtrCH. After evaluation of ensemble and single particle fluorescence polarization with the instantaneous FluoPolScope, one of the constructs turned out to be suitable for practical usage in live cell imaging. Our new, genetically encoded F-actin orientation probe, which has a similar property of an F-actin probe to conventional GFP-UtrCH, is expected to report the 3D architecture of the actin cytoskeleton with fluorescence polarization microscopy, paving the way for both the single molecular orientation imaging in cultured cells and the sub-optical resolution architectural analysis of F-actin networks analysis of F-actin in various living systems.

Semi-in situ atomic force microscopy imaging of intracellular neurofilaments under physiological conditions through the 'sandwich' method.

Neurofilaments are intermediate filament proteins specific for neurons and characterized by formation of biochemically stable, obligate heteropolymers in vivo While purified or reassembled neurofilaments have been subjected to morphological analyses by electron microscopy and atomic force microscopy, there has been a need for direct imaging of cytoplasmic genuine intermediate filaments with minimal risk of artefactualization. In this study, we applied the modified 'cells on glass sandwich' method to exteriorize intracellular neurofilaments, reducing the risk of causing artefacts through sample preparation. SW13vim(-) cells were double transduced with neurofilament medium polypeptide (NF-M) and alpha-internexin (α-inx). Cultured cells were covered with a cationized coverslip after prestabilization with tannic acid to form a sandwich and then split into two. After confirming that neurofilaments could be deposited on ventral plasma membranes exposed via unroofing, we performed atomic force microscopy imaging semi-in situ in aqueous solution. The observed thin filaments, considered to retain native structures of the neurofilaments, exhibited an approximate periodicity of 50-60 nm along their length. Their structural property appeared to reflect the morphology formed by their constituents, i.e. NF-M and α-inx. The success of semi-in situ atomic force microscopy of exposed bona fide assembled neurofilaments through separating the sandwich suggests that it can be an effective and alternative method for investigating cytoplasmic intermediate filaments under physiological conditions by atomic force microscopy.

Direct label-free measurement of the distribution of small molecular weight compound inside thick biological tissue using coherent Raman microspectroscopy.

Distributions of small molecular weight (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterise the localisation of large biomolecules, they cannot be easily applied to the cases with small molecule compounds. We used CARS spectroscopy to detect and identify a label-free small molecule compound. To facilitate detection in aqueous environment, we utilised time-resolved and phase-sensitive techniques to reduce non-resonant background generated from water. We applied this technique to detect small molecular weight compound, taurine, inside mouse cornea tissue immersed in taurine solution as an initial model experiment. We detected a Raman peak of taurine near wavenumber 1033 cm(-1) inside cornea and successfully characterised its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualise the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines.

In vivo molecular labeling of halogenated volatile anesthetics via intrinsic molecular vibrations using nonlinear Raman spectroscopy.

Halogenated volatile anesthetics are frequently used for inhaled anesthesia in clinical practice. No appropriate biological method has been available for visualizing their localization in action. Therefore, despite their frequent use, the mechanism of action of these drugs has not been fully investigated. We measured coherent anti-Stokes Raman scattering (CARS) spectra of sevoflurane and isoflurane, two of the most representative volatile anesthetics, and determined the low-frequency vibrational modes without nonresonant background disturbance. Molecular dynamics calculations predict that these modes are associated with multiple halogen atoms. Because halogen atoms rarely appear in biological compounds, the entire spectral landscape of these modes is expected to be a good marker for investigating the spatial localization of these drugs within the intracellular environment. Using live squid giant axons, we could detect the unique CARS spectra of sevoflurane for the first time in a biological setting.

Relationships between psychological distress, coping styles, and HPA axis reactivity in healthy adults.

Psychological distress and coping styles have been suggested to relate to altered function in the hypothalamic-pituitary-adrenal (HPA) axis, although there remains much to be understood about their relationships. High and low cortisol levels (or reactivity) both represent HPA axis dysfunction, with accumulated evidence suggesting that they are linked to different types of psychopathology. The dexamethasone (DEX)/corticotropin-releasing hormone (CRH) test has been extensively used to identify HPA axis abnormalities in various psychiatric conditions including mood disorders; however, the possible associations of psychological distress and coping styles with HPA axis function have not been well documented using this test. Here, we examined the relationships of HPA axis reactivity as measured by the DEX/CRH test with subjectively perceived psychological distress and coping styles, both of which were assessed with self-report questionnaires, in 121 healthy volunteers. Subjects were divided into three groups by the cortisol suppression pattern, namely the incomplete-suppressors (DST-Cortisol ≥ 5 µg/dL or DEX/CRH-Cortisol ≥ 5 µg/dL), moderate-suppressors (DST-Cortisol < 5 µg/dL and 1 µg/dL ≤ DEX/CRH -Cortisol < 5 µg/dL), and enhanced-suppressors (DST-Cortisol < 5 µg/dL and DEX/CRH-Cortisol < 1 µg/dL). The enhanced-suppressors showed significantly higher scores in obsessive-compulsive, interpersonal sensitivity and anxiety symptoms and significantly more frequent use of avoidant coping strategy, compared to the other two groups. These results point to the important role of enhanced suppression of cortisol, or blunted cortisol reactivity, in non-clinical psychopathology such as avoidant coping strategy and greater psychological distress.

Adhesamine, a new synthetic molecule, accelerates differentiation and prolongs survival of primary cultured mouse hippocampal neurons.

Attachment to the substrate is essential for both survival and differentiation of various kinds of cells, such as neurons and epithelial cells. We recently found a small synthetic molecule, adhesamine, which boosts adhesion and growth of mammalian cells. In the present study, we applied adhesamine to primary cultured hippocampal neuronal cells and compared its effects with those of PLL (poly-L-lysine), which is widely used as a substrate for cell cultures. Neurons grown on adhesamine-coated coverslips survived for up to 1 month without a feeder layer of glial cells, and had greater viability than cells grown on PLL-coated coverslips. Morphological analysis revealed that neurons cultured with adhesamine exhibited earlier differentiation, i.e. earlier axonal outgrowth and dendritic maturation with enhanced neurite branching, than neurons cultured with PLL. Synaptic formation and postsynaptic responses were evident as early as 4 days in cells cultured with adhesamine. Acceleration of differentiation is mediated by earlier activation of the signalling pathways from heparan sulfate in the extracellular matrix to both FAK (focal adhesion kinase) and MAPK (mitogen-activated protein kinase). Improved survival rates and accelerated maturation of neurons exposed to adhesamine suggest that this completely synthetic molecule may be a useful reagent for culturing neuronal cells.

Kinesin-1/Hsc70-dependent mechanism of slow axonal transport and its relation to fast axonal transport.

Cytoplasmic protein transport in axons ('slow axonal transport') is essential for neuronal homeostasis, and involves Kinesin-1, the same motor for membranous organelle transport ('fast axonal transport'). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin-1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ-like domain of the kinesin light chain in the Kinesin-1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin-1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant-negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction.

Functional near-infrared spectroscopy reveals altered hemispheric laterality in relation to schizotypy during verbal fluency task.

Previous functional neuroimaging studies have demonstrated that patients with schizophrenia and those with schizotypal personality disorder (SPD) show reduced laterality, or relative right hemispheric dominance, during the performance of cognitive activation tasks; however, neuroimaging studies looking at non-clinical schizotypy have been few. We have recently reported that schizotypal traits at a non-clinical level are associated with right prefrontal dominance during a letter version of the verbal fluency task (VFT), but it is unknown whether such relationship between schizotypy and functional laterality would be observed across various cognitive tasks. Here we examined the relationships of schizotypal traits as measured by the Schizotypal Personality Questionnaire (SPQ) in healthy adults with hemispheric lateralization of prefrontal activation during letter and category VFTs, using near-infrared spectroscopy. Thirty-two participants were divided into high- (n=16) and low- (n=16) SPQ groups by the median split of the total SPQ score. The high-SPQ group, but not low-SPQ group, showed significantly right-greater-than-left asymmetry of prefrontal activation during letter VFT, whereas such pronounced hemispheric asymmetry in relation to schizotypy was not found during category VFT. These results indicate that non-clinical schizotypy is related to right prefrontal preference during the letter version of VFT in particular, suggesting that the association between schizotypal traits and functional laterality may vary depending on cognitive activation tasks.

[Constituents relating to anti-oxidative and alpha-glucosidase inhibitory activities in Yacon aerial part extract].

Hot water extract of the aerial part of Yacon (Smallanthus sonchifolia, Compositae) showed potent free radical-scavenging activity and inhibitory effects on lipid peroxidation in rat brain homogenate. The most potent antioxidative activity focused on the 50% MeOH-eluted fraction on DIAION HP-20 column chromatography. The structure of the major component in the fraction was identified as 2,3,5-tricaffeoylaltraric acid (TCAA) based on spectroscopic evidence. The antioxidative activity of TCAA is superior to that of natural antioxidants such as (+/-)-catechin, alpha-tocopherol, and ellagic acid, and TCAA also showed selective maltase-inhibitory activity (IC(50) 49 microg/ml). As the hypoglycemic activity of Yacon extract was described in a previous report, the present results showing that the aerial part of Yacon has strong antioxidative activity may encourage its potential use as a food supplement to prevent type II diabetes.

Antimicrobial activity of 2-arylbenzofurans from Morus species against methicillin-resistant Staphylococcus aureus.

Nine 2-arylbenzofurans isolated from Morus species were tested for their antimicrobial activities against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), Micrococcus luteus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Among these compounds, chalcomoracin (a leaf phytoalexine of mulberry tree) exhibited considerable antibacterial activity against MRSAs (MICs 0.78 mug/ml).

Antimicrobial activities of hydrophobic 2-arylbenzofurans and an isoflavone against vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus.

Eight 2-arylbenzofurans and an isoflavone isolated from medicinal plants were tested for their antimicrobial activities against vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). Among these compounds, six hydrophobic 2-arylbenzofurans (log P = 4.4-8.7) exhibited considerable antibacterial activity against five VRE strains(VanA-, VanB-, and VanC-phenotypes) (MICs = 3.13-6.25 microg/mL). Five compounds also showed antibacterial activity against ten MRSA strains (MIC80 = 3.13 microg/mL).

Antimicrobial activity of hydrophobic xanthones from Cudrania cochinchinensis against Bacillus subtilis and methicillin-resistant Staphylococcus aureus.

Ten xanthones with one or two isoprenoid groups and a prenylated benzophenone isolated from roots of Cudrania cochinchinensis (Moraceae) were tested for their antimicrobial activities against Bacillus subtilis and methicillin-resistant Staphylococcus aureus (MRSA). Among these compounds, gerontoxanthone H exhibited considerable antibacterial activity against B. subtilis (MIC = 1.56 microg/ml). Four xanthones, gerontoxanthone I, toxyloxanthone C, cudraxanthone S, and 1,3,7-trihydroxy-2-prenylxanthone, showed weak antibacterial activity against the bacterium (MICs = 3.13-6.25 microg/ml). These compounds also exhibited similar MIC values against methicillin-sensitive S. aureus, MRSAs, and Micrococcus luteus.

Where does slow axonal transport go?

Axonal transport is the specialized and well-developed intracellular transport system for regulated and/or long-distance transport based on generalized cellular machineries. Among them, slow axonal transport conveys cytoplasmic proteins. The motor molecule, the nature of transporting complex and the transport regulation mechanism for slow transport are still unclarified. There has been a dispute regarding the nature of transporting complex of cytoskeletal proteins, polymer-sliding hypothesis versus subunit-transport theory. Recent data supporting the hypothesis of polymer sliding in cultured neurons only reconfirm the previously reported structure and this inference suffers from the lack of ultrastructural evidence and the direct relevance to the physiological slow transport phenomenon in vivo. Observation of the moving cytoskeletal proteins in vivo using transgenic mice or squid giant axons revealed that subunits do move in a microtubule-dependent manner, strongly indicating the involvement of microtubule-based motor kinesin. If the slow transport rate reflects the intermittent fast transport dependent on kinesin motor, we have to investigate the molecular constituents of the transporting complex in more detail and evaluate why the motor and cargo interaction is so unstable. This kind of weak and fluctuating interaction between various molecular pairs could not be detected by conventional techniques, thus necessitating the establishment of a new experimental system before approaching the molecular regulation problem.

Kinesin superfamily protein 2A (KIF2A) functions in suppression of collateral branch extension.

Through interactions with microtubules, the kinesin superfamily of proteins (KIFs) could have multiple roles in neuronal function and development. During neuronal development, postmitotic neurons develop primary axons extending toward targets, while other collateral branches remain short. Although the process of collateral branching is important for correct wiring of the brain, the mechanisms involved are not well understood. In this study, we analyzed kif2a(-/-) mice, whose brains showed multiple phenotypes, including aberrant axonal branching due to overextension of collateral branches. In kif2a(-/-) growth cones, microtubule-depolymerizing activity decreased. Moreover, many individual microtubules showed abnormal behavior at the kif2a(-/-) cell edge. Based on these results, we propose that KIF2A regulates microtubule dynamics at the growth cone edge by depolymerizing microtubules and that it plays an important role in the suppression of collateral branch extension.

Antimicrobial activity of licorice flavonoids against methicillin-resistant Staphylococcus aureus.

Nineteen flavonoids isolated from licorice (Glycyrrhiza glabra, G. inflata and G. uralensis) were tested for their antimicrobial activities against methicillin sensitive Staphylococcus aureus, methicillin resistant S. aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa.

Anti-Helicobacter pylori flavonoids from licorice extract.

Licorice is the most used crude drug in Kampo medicines (traditional Chinese medicines modified in Japan). The extract of the medicinal plant is also used as the basis of anti-ulcer medicines for treatment of peptic ulcer. Among the chemical constituents of the plant, glabridin and glabrene (components of Glycyrrhiza glabra), licochalcone A (G. inflata), licoricidin and licoisoflavone B (G. uralensis) exhibited inhibitory activity against the growth of Helicobacter pylori in vitro. These flavonoids also showed anti-H. pylori activity against a clarithromycin (CLAR) and amoxicillin (AMOX)-resistant strain. We also investigated the methanol extract of G. uralensis. From the extract, three new isoflavonoids (3-arylcoumarin, pterocarpan, and isoflavan) with a pyran ring, gancaonols A[bond]C, were isolated together with 15 known flavonoids. Among these compounds, vestitol, licoricone, 1-methoxyphaseollidin and gancaonol C exhibited anti-H. pylori activity against the CLAR and AMOX-resistant strain as well as four CLAR (AMOX)-sensitive strains. Glycyrin, formononetin, isolicoflavonol, glyasperin D, 6,8-diprenylorobol, gancaonin I, dihydrolicoisoflavone A, and gancaonol B possessed weaker anti-H. pylori activity. These compounds may be useful chemopreventive agents for peptic ulcer or gastric cancer in H. pylori-infected individuals.

Neuritogenesis of herbal (+)- and (-)-syringaresinols separated by chiral HPLC in PC12h and Neuro2a cells.

Syringaresinol isolated from Epimedium koreanum NAKA1 and Magnolia officinalis REHD. et WILS. was subjected to optical resolution by chiral HPLC to give (+)- and (-)-enantiomers. The two syringaresinol enantiomers, as well as a mixture of their glucosides, showed dose-dependent neuritogenesis in a concentration range from 0.24 to 24 microM in PC12h cells.