Thin-section- and matrix-free mass spectrometry imaging: Reproducible sample transfer using novel platinum-coated porous plate formed of glass beads.

RATIONALE: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) typically requires sample preparation such as sectioning and spraying of the matrix. Sample transfer using a Pt-coated porous plate formed of glass beads simplifies preparation and enables reproducible MSI measurements. METHODS: The surface of a sintered-glass-bead porous plate was coated with Pt on one side of the plate. Polymer additives and the cross-section of a strawberry were chosen as the sample and transferred to the Pt-coated surface of the plate. This process was completely thin-section- and matrix-free. The prepared plates were analyzed using a commercial MALDI time-of-flight instrument. RESULTS: Several constituents of the polymer additives (Irgafos 168 and Irganox 1010) and strawberry metabolites (hexose, citric acid, and sucrose) were detected without organic matrices. These ion images were obtained with a special distribution of the retained constituents. Duplicate prepared plates using the same strawberry cross-section reproduced the ion images. CONCLUSIONS: A sample transfer process using a Pt-coated porous plate formed of glass beads was demonstrated as an alternative preparation method for MSI. This process has the potential to simplify MSI preparation and achieve easily reproducible MSI measurements.

Real-time observation of the photoionization-induced water rearrangement dynamics in the 5-hydroxyindole-water cluster by time-resolved IR spectroscopy.

Solvation plays an essential role in controlling the mechanism and dynamics of chemical reactions in solution. The present study reveals that changes in the local solute-solvent interaction have a great impact on the timescale of solvent rearrangement dynamics. Time-resolved IR spectroscopy has been applied to a hydration rearrangement reaction in the monohydrated 5-hydroxyindole-water cluster induced by photoionization of the solute molecule. The water molecule changes the stable hydration site from the indolic NH site to the substituent OH site, both of which provide a strongly attractive potential for hydration. The rearrangement time constant amounts to 8 ± 2 ns, and is further slowed down by a factor of more than five at lower excess energy. These rearrangement times are slower by about three orders of magnitude than those reported for related systems where the water molecule is repelled from a repulsive part of the interaction potential toward an attractive well. The excess energy dependence of the time constant is well reproduced by RRKM theory. Differences in the reaction mechanism are discussed on the basis of energy relaxation dynamics.

Effect of an Enhanced Nose-to-Brain Delivery of Insulin on Mild and Progressive Memory Loss in the Senescence-Accelerated Mouse.

Insulin is now considered to be a new drug candidate for treating dementias, such as Alzheimer's disease, whose pathologies are linked to insulin resistance in the brain. Our recent work has clarified that a noncovalent strategy involving cell-penetrating peptides (CPPs) can increase the direct transport of insulin from the nasal cavity into the brain parenchyma. The present study aimed to determine whether the brain insulin level increased by intranasal coadministration of insulin with the CPP penetratin has potential for treating dementia. The pharmacological actions of insulin were investigated at different stages of memory impairment using a senescence-accelerated mouse-prone 8 (SAMP8) model. The results of spatial learning tests suggested that chronic intranasal administration of insulin with l-penetratin to SAMP8 slowed the progression of memory loss in the early stage of memory impairment. However, contrary to expectations, this strategy using penetratin was ineffective in recovering the severe cognitive dysfunction in the progressive stage, which involves brain accumulation of amyloid ß (Aß). Immunohistological examination of hippocampal regions of samples from SAMP8 in the progressive stage suggested that accelerated nose-to-brain insulin delivery had a partial neuroprotective function but unexpectedly increased Aß plaque deposition in the hippocampus. These findings suggest that the efficient nose-to-brain delivery of insulin combined with noncovalent CPP strategy has different effects on dementia during the mild and progressive stages of cognitive dysfunction.

Elevation of the Energy Threshold for Isomerization of 5-Hydroxyindole-(tert-butyl alcohol)1 Cluster Cations.

Isomerization between two hydrogen-bonded (H-bonded) isomers of 5-hydroxyindole-(tert-butyl alcohol)1 cluster cations ([5HI-(t-BuOH)1]+) was investigated in the gas phase. In the S0 state, jet-cooled 5HI-(t-BuOH)1 has two structural isomers, 5HI(OH)-(t-BuOH)1 and 5HI(NH)-(t-BuOH)1, in which the t-BuOH molecule is bound to the OH or the NH group of 5HI. The IR photodissociation spectrum of [5HI-(t-BuOH)1]+ generated by two-color resonant two-photon ionization (2C-R2PI) via the S1-S0 origin of 5HI(NH)-(t-BuOH)1 provided evidence of both [5HI(OH)-(t-BuOH)1]+ and [5HI(NH)-(t-BuOH)1]+ coexisting in the D0 state, indicating that [5HI(NH)-(t-BuOH)1]+ isomerizes to [5HI(OH)-(t-BuOH)1]+ after 2C-R2PI of 5HI(NH)-(t-BuOH)1. The lower limit of the energy threshold for the isomerization of [5HI(NH)-(t-BuOH)1]+ to [5HI(OH)-(t-BuOH)1]+ was experimentally determined to be 3362 ± 30 cm-1, and the corresponding energy threshold for the isomerization of [5HI(NH)-(H2O)1]+ to [5HI(OH)-(H2O)1]+ has been reported to be 2127 ± 30 cm-1. Thus, the energy threshold for the isomerization is elevated by at least 1200 cm-1 when the solvent molecule changes from H2O to t-BuOH. The elevation of the energy threshold is explained by the difference in the stabilization energies of [5HI-(t-BuOH)1]+ and [5HI-(H2O)1]+ in the initial and transition states owing to the larger proton affinity of t-BuOH than H2O.

Rearrangements of a Water Molecule in Both Directions between Two Hydrogen-Bonding Sites of 5-Hydroxyindole Cation: Experimental Determination of the Energy Threshold for the Rearrangement.

Rearrangements of a water molecule in both directions between two hydrogen-bonding (H-bonding) sites of the 5-hydroxyindole (5HI) cation was investigated in the gas phase. IR-dip spectra of jet-cooled 5HI-(H2O)1 revealed that two structural isomers, 5HI(OH)-(H2O)1 and 5HI(NH)-(H2O)1, in which a water molecule is bound to either the OH group or the NH group of 5HI, were formed in the S0 state. The IR photodissociation spectrum of [5HI-(H2O)1](+) generated by two-color resonant two-photon ionization (2C-R2PI) via the S1-S0 origin of 5HI(NH)-(H2O)1 clearly showed that [5HI(OH)-(H2O)1](+) and [5HI(NH)-(H2O)1](+) coexist in the D0 state. The appearance of [5HI(OH)-(H2O)1](+) after R2PI via the S1-S0 origin of 5HI(NH)-(H2O)1 is explained by isomerization of [5HI(NH)-(H2O)1](+) to [5HI(OH)-(H2O)1](+), which corresponds to the rearrangement of the water. In addition, isomerization in the opposite direction was also observed when [5HI-(H2O)1](+) was generated via the S1-S0 origin of 5HI(OH)-(H2O)1. The upper limit of the energy threshold for the rearrangement of the water in [5HI(NH)-(H2O)1](+) was experimentally determined to be 2127 ± 30 cm(-1) from the adiabatic ionization energy of 5HI(NH)-(H2O)1. Above the energy threshold, the water molecule in [5HI-(H2O)1](+) may fluctuate between the two preferential H-bonding sites of 5HI(+).

Simultaneous Interaction of Hydrophilic and Hydrophobic Solvents with Ethylamino Neurotransmitter Radical Cations: Infrared Spectra of Tryptamine(+)-(H2O)m-(N2)n Clusters (m,n ≤ 3).

Solvation of biomolecules by a hydrophilic and hydrophobic environment strongly affects their structure and function. Here, the structural, vibrational, and energetic properties of size-selected clusters of the microhydrated tryptamine cation with N2 ligands, TRA(+)-(H2O)m-(N2)n (m,n ≤ 3), are characterized by infrared photodissociation spectroscopy in the 2800-3800 cm(-1) range and dispersion-corrected density functional theory calculations at the ωB97X-D/cc-pVTZ level to investigate the simultaneous solvation of this prototypical neurotransmitter by dipolar water and quadrupolar N2 ligands. In the global minimum structure of TRA(+)-H2O generated by electron ionization, H2O is strongly hydrogen-bonded (H-bonded) as proton acceptor to the acidic indolic NH group. In the TRA(+)-H2O-(N2)n clusters, the weakly bonded N2 ligands do not affect the H-bonding motif of TRA(+)-H2O and are preferentially H-bonded to the OH groups of the H2O ligand, whereas stacking to the aromatic π electron system of the pyrrole ring of TRA(+) is less favorable. The natural bond orbital analysis reveals that the H-bond between the N2 ligand and the OH group of H2O cooperatively strengthens the adjacent H-bond between the indolic NH group of TRA(+) and H2O, while π stacking is slightly noncooperative. In the larger TRA(+)-(H2O)m clusters, the H2O ligands form a H-bonded solvent network attached to the indolic NH proton, again stabilized by strong cooperative effects arising from the nearby positive charge. Comparison with the corresponding neutral TRA-(H2O)m clusters illustrates the strong impact of the excess positive charge on the structure of the microhydration network.

Weak hydrogen bonding motifs of ethylamino neurotransmitter radical cations in a hydrophobic environment: infrared spectra of tryptamine(+)-(N2)n clusters (n ≤ 6).

Size-selected clusters of the tryptamine cation with N2 ligands, TRA(+)-(N2)n with n = 1-6, are investigated by infrared photodissociation (IRPD) spectroscopy in the hydride stretch range and quantum chemical calculations at the ωB97X-D/cc-pVTZ level to characterize the microsolvation of this prototypical aromatic ethylamino neurotransmitter radical cation in a nonpolar solvent. Two types of structural isomers exhibiting different interaction motifs are identified for the TRA(+)-N2 dimer, namely the TRA(+)-N2(H) global minimum, in which N2 forms a linear hydrogen bond (H-bond) to the indolic NH group, and the less stable TRA(+)-N2(π) local minima, in which N2 binds to the aromatic π electron system of the indolic pyrrole ring. The IRPD spectrum of TRA(+)-(N2)2 is consistent with contributions from two structural H-bound isomers with similar calculated stabilization energies. The first isomer, denoted as TRA(+)-(N2)2(2H), exhibits an asymmetric bifurcated planar H-bonding motif, in which both N2 ligands are attached to the indolic NH group in the aromatic plane via H-bonding and charge-quadrupole interactions. The second isomer, denoted as TRA(+)-(N2)2(H/π), has a single and nearly linear H-bond of the first N2 ligand to the indolic NH group, whereas the second ligand is π-bonded to the pyrrole ring. The natural bond orbital analysis of TRA(+)-(N2)2 reveals that the total stability of these types of clusters is not only controlled by the local H-bond strengths between the indolic NH group and the N2 ligands but also by a subtle balance between various contributing intermolecular interactions, including local H-bonds, charge-quadrupole and induction interactions, dispersion, and exchange repulsion. The systematic spectral shifts as a function of cluster size suggest that the larger TRA(+)-(N2)n clusters with n = 3-6 are composed of the strongly bound TRA(+)-(N2)2(2H) core ion to which further N2 ligands are weakly attached to either the π electron system or the indolic NH proton by stacking and charge-quadrupole forces.

Diversity and composition of flower-visiting insects and related factors in three fruit tree species.

Animal-mediated pollination is an essential ecosystem service for the production of many fruit trees. To reveal the community composition of flower-visiting wild insects which potentially contribute to fruit production and to examine the effects of geographic location, local meteorological conditions and locally introduced domesticated pollinators on them, we investigated the community composition of insects visiting the flowers (hereafter, "visitors") of apple, Japanese pear and Oriental persimmon for 1‒3 years at 20 sites around Japan. While most of the variation (82%) of the community composition was explained by tree species with a slight contribution by geographic distance (2%), maximum temperature and tree species contributed 62% and 41% of the variation in total abundance of the visitors, respectively. Though the dominant families of the visitors varied spatiotemporally, the community composition of the visitors of apple and Japanese pear clearly differed from that of Oriental persimmon. While Andrenidae and Syrphidae together accounted for 46%‒64% of the visitors of apple and Japanese pear, Apidae represented 57% of the visitors of Oriental persimmon. The taxonomic richness, diversity and evenness of the visitors were best predicted by locally introduced domesticated pollinators and local meteorological conditions of wind speed and maximum temperature. Amongst these selected factors, locally introduced domesticated pollinators could have the largest impact. It seemed to be strongly related to the reduction of taxonomic richness, diversity and evenness of the visitors, accounting for 41‒89% of the variation. Results suggested that the community composition and total abundance of potential pollinators were predominantly determined by tree species and temperature, but locally introduced domesticated pollinators could have a determinantal pressure on the taxonomic diversity of the community.

Photoionization-induced water migration in the hydrated trans-formanilide cluster cation revealed by gas-phase spectroscopy and ab initio molecular dynamics simulation.

Photoionization-induced water migration in the trans-formanilide-water 1:1 cluster, FA-(H(2)O)(1), has been investigated by using IR-dip spectroscopy, quantum chemical calculations, and ab initio molecular dynamics simulations. In the S(0) state, FA-(H(2)O)(1) has two structural isomers, FA(NH)-(H(2)O)(1) and FA(CO)-(H(2)O)(1), where a water molecule is hydrogen-bonded (H-bonded) to the NH group and the CO group, respectively. In addition, the S(1)-S(0) origin transition of FA(CO)-(H(2)O)(2), where a water dimer is H-bonded to the CO group, was observed only in the [FA-(H(2)O)(1)](+) mass channel, indicating that one of the water molecules evaporates completely in the D(0) state. These results are consistent with a previous report [Robertson, E. G. Chem. Phys. Lett., 2000, 325, 299]. In the D(0) state, however, [FA-(H(2)O)(1)](+) produced by photoionization via the S(1)-S(0) origin transitions of FA(NH)-(H(2)O)(1) and FA(CO)-(H(2)O)(1) shows essentially the same IR spectra. Compared with the theoretical calculations, [FA-(H(2)O)(1)](+) can be assigned to [FA(NH)-(H(2)O)(1)](+). This means that the water molecule in [FA-(H(2)O)(1)](+) migrates from the CO group to the NH group when [FA-(H(2)O)(1)](+) is produced by photoionization of FA(CO)-(H(2)O)(1). [FA-(H(2)O)(1)](+) produced by photoionization of FA(CO)-(H(2)O)(2) also shows the IR spectrum corresponding to [FA(NH)-(H(2)O)(1)](+). In this case, the water migration from the CO group to the NH group occurs with the evaporation of a water molecule. Ab initio molecular dynamics simulations revealed the water migration pathway in [FA-(H(2)O)(1)](+). The calculations of classical electrostatic interactions show that charge-dipole interaction between FA(+) and H(2)O induces an initial structural change in [FA-(H(2)O)(1)](+). An exchange repulsion between the lone pairs of the CO group and H(2)O in [FA-(H(2)O)(1)](+) also affects the initial direction of the water migration. These two factors play important roles in determining the initial water migration pathway.

Effective nose-to-brain delivery of exendin-4 via coadministration with cell-penetrating peptides for improving progressive cognitive dysfunction.

In a recent study, we demonstrated the potential of a cell-penetrating peptide (CPP) penetratin to deliver the peptide drug insulin to the brain via nasal administration, and its pharmacological effect on the mild cognitive dysfunction in senescence-accelerated mouse (SAMP8). However, the therapeutic potential of intranasal insulin administration was attenuated when applied to the aged SAMP8 with severe cognitive dysfunction. The present study, therefore, aimed to overcome the difficulty in treating severe cognitive dysfunction using insulin by investigating potential alternatives, glucagon-like peptide-1 (GLP-1) receptor agonists such as exendin-4. Examination using normal ddY mice demonstrated that the distribution of exendin-4 throughout the brain was dramatically increased by intranasal coadministration with the L-form of penetratin. The activation of hippocampal insulin signaling after the simultaneous nose-to-brain delivery of exendin-4 and an adequate level of insulin were confirmed by analyzing the phosphorylation of Akt. Furthermore, spatial learning ability, evaluated in the Morris water maze test after daily administration of exendin-4 with L-penetratin and supplemental insulin for 4 weeks, suggested therapeutic efficacy against severe cognitive dysfunction. The present study suggests that nose-to-brain delivery of exendin-4 with supplemental insulin, mediated by CPP coadministration, shows promise for the treatment of progressive cognitive dysfunction in SAMP8.