Tools and Methods for Investigating Synthetic Metal-Catalyzed Reactions in Living Cells.

Although abiotic catalysts are capable of promoting numerous new-to-nature reactions, only a small subset has so far been successfully integrated into living systems. Research in intracellular catalysis requires an interdisciplinary approach that takes advantage of both chemical and biological tools as well as state-of-the-art instrumentations. In this perspective, we will focus on the techniques that have made studying metal-catalyzed reactions in cells possible using representative examples from the literature. Although the lack of quantitative data in vitro and in vivo has somewhat limited progress in the catalyst development process, recent advances in characterization methods should help overcome some of these deficiencies. Given its tremendous potential, we believe that intracellular catalysis will play a more prominent role in the development of future biotechnologies and therapeutics.

p-Coumaric Acid Enhances Hypothalamic Leptin Signaling and Glucose Homeostasis in Mice via Differential Effects on AMPK Activation.

AMP-activated protein kinase (AMPK) plays a crucial role in the regulation of energy homeostasis in both peripheral metabolic organs and the central nervous system. Recent studies indicated that p-Coumaric acid (CA), a hydroxycinnamic phenolic acid, potentially activated the peripheral AMPK pathway to exert beneficial effects on glucose metabolism in vitro. However, CA's actions on central AMPK activity and whole-body glucose homeostasis have not yet been investigated. Here, we reported that CA exhibited different effects on peripheral and central AMPK activation both in vitro and in vivo. Specifically, while CA treatment promoted hepatic AMPK activation, it showed an inhibitory effect on hypothalamic AMPK activity possibly by activating the S6 kinase. Furthermore, CA treatment enhanced hypothalamic leptin sensitivity, resulting in increased proopiomelanocortin (POMC) expression, decreased agouti-related peptide (AgRP) expression, and reduced daily food intake. Overall, CA treatment improved blood glucose control, glucose tolerance, and insulin sensitivity. Together, these results suggested that CA treatment enhanced hypothalamic leptin signaling and whole-body glucose homeostasis, possibly via its differential effects on AMPK activation.

Organoiridium-quinone conjugates for facile hydrogen peroxide generation.

An organoiridium complex bearing a quinone moiety was shown to significantly accelerate the rate of H2O2 formation in the presence of air and sodium formate at low catalyst concentrations. This reaction is proposed to operate through a synergistic mechanism involving transfer hydrogenation catalysis and radical chemistry. Our bifunctional iridium complex could potentially be used in anti-cancer chemotherapy or other applications requiring rapid generation of reactive oxygen species.

Photodissociative Cross-Linking of Non-covalent Peptide-Peptide Ion Complexes in the Gas Phase.

We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355 nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide. The cross-link positions were established from collision-induced dissociation tandem mass spectra (CID-MS3) providing sequence information on the covalent adducts. Effects of the amino acid residue (Pro or Phe) and its position in the target peptide sequence were evaluated. For proline-containing peptides, interactions resulting in covalent cross-links in these complexes became more prominent as proline was moved towards the C-terminus of the target peptide sequence. The photocross-linking yields of phenylalanine-containing peptides depended on the position of both phenylalanine and photoleucine. Density functional theory calculations were used to assign structures of low-energy conformers of the (GLPMG + GLL*LK + H)+ complex. Born-Oppenheimer molecular dynamics trajectory calculations were used to capture the thermal motion in the complexes within 100 ps and determine close contacts between the incipient carbene and the H-X bonds in the target peptide. This provided atomic-level resolution of potential cross-links that aided spectra interpretation and was in agreement with experimental data. Graphical Abstract ᅟ.

Spontaneous Isomerization of Peptide Cation Radicals Following Electron Transfer Dissociation Revealed by UV-Vis Photodissociation Action Spectroscopy.

Peptide cation radicals of the z-type were produced by electron transfer dissociation (ETD) of peptide dications and studied by UV-Vis photodissociation (UVPD) action spectroscopy. Cation radicals containing the Asp (D), Asn (N), Glu (E), and Gln (Q) residues were found to spontaneously isomerize by hydrogen atom migrations upon ETD. Canonical N-terminal [z4 + H]+● fragment ion-radicals of the R-C●H-CONH- type, initially formed by N-Cα bond cleavage, were found to be minor components of the stable ion fraction. Vibronically broadened UV-Vis absorption spectra were calculated by time-dependent density functional theory for several [●DAAR + H]+ isomers and used to assign structures to the action spectra. The potential energy surface of [●DAAR + H]+ isomers was mapped by ab initio and density functional theory calculations that revealed multiple isomerization pathways by hydrogen atom migrations. The transition-state energies for the isomerizations were found to be lower than the dissociation thresholds, accounting for the isomerization in non-dissociating ions. The facile isomerization in [●XAAR + H]+ ions (X = D, N, E, and Q) was attributed to low-energy intermediates having the radical defect in the side chain that can promote hydrogen migration along backbone Cα positions. A similar side-chain mediated mechanism is suggested for the facile intermolecular hydrogen migration between the c- and [z + H]●-ETD fragments containing Asp, Asn, Glu, and Gln residues. Graphical Abstract ᅟ.

Experimental Evidence for Noncanonical Thymine Cation Radicals in the Gas Phase.

Thymine cation radicals were generated in the gas phase by collision-induced intramolecular electron transfer in [Cu(2,2':6,2″-terpyridine)(thymine)]2+• complexes and characterized by ion-molecule reactions, UV-vis photodissociation action spectroscopy, and ab initio and density functional theory calculations. The experimental results indicated the formation of a tautomer mixture consisting chiefly (77%) of noncanonical tautomers with a C-7-H2 group. The canonical 2,4-dioxo-N-1,N-3-H isomer was formed as a minor component at ca. 23%. Ab initio CCSD(T) calculations indicated that the canonical [thymine]+• ion was not the lowest-energy isomer. This contrasts with neutral thymine, for which the canonical isomer is the lowest-energy structure. Exothermic unimolecular isomerization by a methyl hydrogen migration in the canonical [thymine]+• ion required a low energy barrier, forming a C-7-H2,O-4-H isomer. Noncanonical thymine tautomers with a C-7-H2 group were also identified by calculations as low-energy isomers of 2'-deoxythymidine phosphate cation radicals. The relative energies of thymidine ion isomers were sensitive to the computational method used and were affected by solvation. The noncanonical [thymine]+• ions have extremely low adiabatic recombination energies (REadiab < 5.9 eV), making them potential ionization hole traps in ionized nucleic acids.

Stereospecific control of peptide gas-phase ion chemistry with cis and trans cyclo ornithine residues.

We report non-chiral amino acid residues cis- and trans-1,4-diaminocyclohexane-1-carboxylic acid (cyclo-ornithine, cO) that exhibit unprecedented stereospecific control of backbone dissociations of singly charged peptide cations and hydrogen-rich cation radicals produced by electron-transfer dissociation. Upon collision-induced dissociation (CID) in the slow heating regime, peptide cations containing trans-cO residues undergo facile backbone cleavages of amide bonds C-terminal to trans-cO. By contrast, peptides with cis-cO residues undergo dissociations at several amide bonds along the peptide ion backbone. Diastereoisomeric cO-containing peptides thus provide remarkably distinct tandem mass spectra. The stereospecific effect in CID of the trans-cO residue is explained by syn-facially directed proton transfer from the 4-ammonium group at cO to the C-terminal amide followed by neighboring group participation in the cleavage of the CO-NH bond, analogous to the aspartic acid and ornithine effects. Backbone dissociations of diastereoisomeric cO-containing peptide ions generate distinct [bn ]+ -type fragment ions that were characterized by CID-MS3 spectra. Stereospecific control is also reported for electron-transfer dissociation of cis- and trans-cO containing doubly charged peptide ions. The stereospecific effect upon electron transfer is related to the different conformations of doubly charged peptide ions that affect the electron attachment sites and ensuing N-Cα bond dissociations.

UV-Vis Action Spectroscopy Reveals a Conformational Collapse in Hydrogen-Rich Dinucleotide Cation Radicals.

We report the generation of deoxyriboadenosine dinucleotide cation radicals by gas-phase electron transfer to dinucleotide dications and their noncovalent complexes with crown ether ligands. Stable dinucleotide cation radicals of a novel hydrogen-rich type were generated and characterized by tandem mass spectrometry and UV-vis photodissociation (UVPD) action spectroscopy. Electron structure theory analysis indicated that upon electron attachment the dinucleotide dications underwent a conformational collapse followed by intramolecular proton migrations between the nucleobases to give species whose calculated UV-vis absorption spectra matched the UVPD action spectra. Hydrogen-rich cation radicals generated from chimeric riboadenosine 5'-diesters gave UVPD action spectra that pointed to novel zwitterionic structures consisting of aromatic π-electron anion radicals intercalated between stacked positively charged adenine rings. Analogies with DNA ionization are discussed.

Radical Reactions Affecting Polar Groups in Threonine Peptide Ions.

Peptide cation-radicals containing the threonine residue undergo radical-induced dissociations upon collisional activation and photon absorption in the 210-400 nm range. Peptide cation-radicals containing a radical defect at the N-terminal residue, [•Ala-Thr-Ala-Arg+H]+, were generated by electron transfer dissociation (ETD) of peptide dications and characterized by UV-vis photodissociation action spectroscopy combined with time-dependent density functional theory (TD-DFT) calculations of absorption spectra, including thermal vibronic band broadening. The action spectrum of [•Ala-Thr-Ala-Arg+H]+ ions was indicative of the canonical structure of an N-terminally deaminated radical whereas isomeric structures differing in the position of the radical defect and amide bond geometry were excluded. This indicated that exothermic electron transfer to threonine peptide ions did not induce radical isomerizations in the fragment cation-radicals. Several isomeric structures, ion-molecule complexes, and transition states for isomerizations and dissociations were generated and analyzed by DFT and Møller-Plesset perturbational ab initio calculations to aid interpretation of the major dissociations by loss of water, hydroxyl radical, C3H6NO•, C3H7NO, and backbone cleavages. Born-Oppenheimer molecular dynamics (BOMD) in combination with DFT gradient geometry optimizations and intrinsic reaction coordinate analysis were used to search for low-energy cation-radical conformers and transition states. BOMD was also employed to analyze the reaction trajectory for loss of water from ion-molecule complexes.

Near-UV Photodissociation of Tryptic Peptide Cation Radicals. Scope and Effects of Amino Acid Residues and Radical Sites.

Peptide cation-radical fragment ions of the z-type, [●AXAR+], [●AXAK+], and [●XAR+], where X = A, C, D, E, F, G, H, K, L, M, N, P, Y, and W, were generated by electron transfer dissociation of peptide dications and investigated by MS3-near-ultraviolet photodissociation (UVPD) at 355 nm. Laser-pulse dependence measurements indicated that the ion populations were homogeneous for most X residues except phenylalanine. UVPD resulted in dissociations of backbone CO─NH bonds that were accompanied by hydrogen atom transfer, producing fragment ions of the [yn]+ type. Compared with collision-induced dissociation, UVPD yielded less side-chain dissociations even for residues that are sensitive to radical-induced side-chain bond cleavages. The backbone dissociations are triggered by transitions to second (B) excited electronic states in the peptide ion R-CH●-CONH- chromophores that are resonant with the 355-nm photon energy. Electron promotion increases the polarity of the B excited states, R-CH+-C●(O-)NH-, and steers the reaction to proceed by transfer of protons from proximate acidic Cα and amide nitrogen positions. Graphical Abstract ᅟ.

UV Action Spectroscopy of Gas-Phase Peptide Radicals.

UV photodissociation (UVPD) action spectroscopy is reported to provide a sensitive tool for the detection of radical sites in gas-phase peptide ions. UVPD action spectra of peptide cation radicals of the z-type generated by electron-transfer dissociation point to the presence of multiple structures formed as a result of spontaneous isomerizations by hydrogen atom migration. N-terminal Cα radicals are identified as the dominant components, but the content of isomers differing in the radical defect position in the backbone or side chain depends on the nature of the aromatic residue with phenylalanine being more prone to isomerization than tryptophan. These results illustrate that spontaneous hydrogen atom migrations can occur in peptide cation-radicals upon electron-transfer dissociation.

Serine effects on collision-induced dissociation and photodissociation of peptide cation radicals of the z+• -type.

The serine residue displays specific effects on the dissociations of peptide fragment cation-radicals of the z+• type which are produced by electron transfer dissociation. Energy-resolved collision-induced dissociation (ER-CID), time-resolved infrared multiphoton dissociation (TR-IRMPD), and single-photon UV photodissociation at 355 nm revealed several competitive dissociation pathways consisting of loss of OH radical, water, and backbone cleavages occurring at N-terminal and C-terminal positions relative to the serine residue. The activation modes using slow-heating and UV photon absorption resulted in different relative intensities of fragment ions. This indicated that the dissociations proceeded through several channels with different energy-dependent kinetics. The experimental data were interpreted with the help of electron structure calculations that provided fully optimized structures and relative energies for cis and trans amide isomers of the z4+• ions as well as isomerization, dissociation, and transition state energies. UV photon absorption by the z4+• ions was due to Cα-radical amide groups created by ETD that provided a new chromophore absorbing at 355 nm.

Combining Near-UV Photodissociation with Electron Transfer. Reduction of the Diazirine Ring in a Photomethionine-Labeled Peptide Ion.

Electron transfer dissociation of peptide ions with the diazirine-containing residue photomethionine (M*) results in side-chain dissociations by loss of C3H7N2 radicals in addition to standard backbone cleavages. The side-chain dissociations are particularly prominent upon activation of long-lived, charge-reduced, cation radicals (GM*GGR + 2H)(+•). Investigation of these cation radicals by near-UV photodissociation and collisional activation revealed different fragmentation products and mechanisms resulting from these ion activation modes. The dissociations observed for photomethionine were dramatically different from those previously reported for the lower homologue photoleucine; here, a difference by a single methylene group in the side chain had a large effect on the chemistries of the cation radicals upon ETD and further activation. ETD intermediates and products were probed by tandem 355-nm UV photodissociation-collision induced dissociation and found to contain chromophores that resulted from electron attachment to the diazirine ring. The nature of the newly formed chromophores and ion energetics and kinetics were investigated by electron structure calculations combining ab initio and density functional theory methods and Rice-Ramsperger-Kassel-Marcus (RRKM) theory. The dramatic difference between the dissociations of L* and M* containing peptide cation radicals is explained by electronic effects that play a role in stabilizing critical reaction intermediates and steer the dissociations into kinetically favored reaction channels. In addition, a new alternating UVPD-ETD-UVPD MS(4) experiment is introduced and utilized for ion structure elucidation.

Probing peptide cation-radicals by near-UV photodissociation in the gas phase. Structure elucidation of histidine radical chromophores formed by electron transfer reduction.

Electron transfer reduction of gas-phase ions generated from histidine-containing peptides forms stable cation-radicals that absorb light at 355 nm, as studied for AAHAR, AAHAK, DSHAK, FHEK, HHGYK, and HHSHR. Laser photodissociation of mass-selected cation-radicals chiefly resulted in loss of H atoms, contrasting dissociations induced by slow collisional heating. The 355 nm absorption was due to new chromophores created by electron transfer and radical rearrangements in the cation-radicals. The chromophores were identified by time-dependent density functional theory calculations as 2H,3H-imidazoline and 2H-dihydrophenol radicals, formed by hydrogen atom transfer to the histidine and tyrosine side chain groups, respectively. These radicals undergo facile C-H bond dissociations upon photon absorption. In contrast, dissociations of histidine peptide cation-radicals containing the 1H,3H-imidazoline ring prefer loss of 4-methylimidazole via a multistep reaction pathway. The isomeric cation-radicals can be distinguished by a combination of collision-induced dissociation and near-UV photodissociation. The TD-DFT excitation energies in model imidazoline radicals were benchmarked on EOM-CCSD energies, and a satisfactory agreement was found for the M06-2X and ωB97XD functionals. The combination of electron transfer, photodissociation, collisional activation, and theory is presented as a powerful tool for studying structures and electronic properties of peptide cation-radicals in the gas phase.

Dimensions of gender relations and reproductive health inequity perceived by female undergraduate students in the Mekong Delta of Vietnam: a qualitative exploration.

INTRODUCTION: Increasing evidence indicates that gender equity has a significant influence on women's health; yet few culturally specific indicators of gender relations exist which are applicable to health. This study explores dimensions of gender relations perceived by female undergraduate students in southern Vietnamese culture, and qualitatively examines how this perceived gender inequity may influence females' sexual or reproductive health. METHODS: Sixty-two female undergraduate students from two universities participated in eight focus group discussions to talk about their perspectives regarding national and local gender equity issues. RESULTS: Although overall gender gaps in the Mekong Delta were perceived to have decreased in comparison to previous times, several specific dimensions of gender relations were emergent in students' discussions. Perceived dimensions of gender relations were comparable to theoretical structures of the Theory of Gender and Power, and to findings from several reports describing the actual inferiority of women. Allocation of housework and social paid work represented salient dimensions of labor. The most salient dimension of power related to women in positions of authority. Salient dimensions of cathexis related to son preference, women's vulnerability to blame or criticism, and double standards or expectations. Findings also suggested that gender inequity potentially influenced women's sexual and reproductive health as regards to health information seeking, gynecological care access, contraceptive use responsibility, and child bearing. CONCLUSION: Further investigations of the associations between gender relations and different women's sexual and reproductive health outcomes in this region are needed. It may be important to address gender relations as a distal determinant in health interventions in order to promote gender-based equity in sexual and reproductive health.

Implementation of a genetically tuned neural platform in optimizing fluorescence from receptor-ligand binding interactions on microchips.

This paper describes the use of a genetically tuned neural network platform to optimize the fluorescence realized upon binding 5-carboxyfluorescein-D-Ala-D-Ala-D-Ala (5-FAM-(D-Ala)(3) ) (1) to the antibiotic teicoplanin from Actinoplanes teichomyceticus electrostatically attached to a microfluidic channel originally modified with 3-aminopropyltriethoxysilane. Here, three parameters: (i) the length of time teicoplanin was in the microchannel; (ii) the length of time 1 was in the microchannel, thereby, in equilibrium with teicoplanin, and; (iii) the amount of time buffer was flushed through the microchannel to wash out any unbound 1 remaining in the channel, are examined at a constant concentration of 1, with neural network methodology applied to optimize fluorescence. Optimal neural structure provided a best fit model, both for the training set (r(2) = 0.985) and testing set (r(2) = 0.967) data. Simulated results were experimentally validated demonstrating efficiency of the neural network approach and proved superior to the use of multiple linear regression and neural networks using standard back propagation.

Perceived gender inequality, sexual communication self-efficacy, and sexual behaviour among female undergraduate students in the Mekong Delta of Vietnam.

BACKGROUND: Worldwide, the literature on sexual behaviour has documented associations between gender-based relationship inequality and sexual communication ability and the actual use of condoms or other contraceptives among young women. This study aimed to examine these associations among undergraduate female students in the Mekong Delta of Vietnam. METHODS: A cross-sectional survey of 1181 female third-year students from two universities in the Mekong Delta was conducted. Latent variable modelling and logistic regression were employed to examine the hypothesised associations. RESULTS: Among the 72.4% of students who had ever had boyfriends, 44.8% indicated that their boyfriends had asked for sex, 13% had had penile-vaginal sex and 10.3% had had oral sex. For those who had had penile-vaginal sex, 33% did not use any contraceptive method, including condoms, during their first sexual intercourse. The greater a student's perception that women were subordinate to men, the lower her self-efficacy for sexual communication and the lower her actual frequency of discussing safer sex matters and asking her partner to use a condom. Sexual communication self-efficacy was associated with actual contraceptive use (P=0.039) but only marginally with condom use (P=0.092) at first sexual intercourse. CONCLUSION: Sexual health promotion strategies should address the influence of gender relations on young women's sexual communication self-efficacy and the subsequent impact on actual contraceptive and condom use.