Multiply Charged Cation Attachment to Facilitate Mass Measurement in Negative-Mode Native Mass Spectrometry.

Native mass spectrometry (MS) is usually conducted in the positive-ion mode; however, in some cases, it is advantageous to use the negative-ion polarity. Challenges associated with native MS using ensemble measurements (i.e., the measurement of many ions at a time as opposed to the measurement of the charge and the mass-to-charge ratio of individual ions) include narrow charge state distributions with the potential for an overlap in neighboring charge states. These issues can either compromise or preclude confident charge state (and hence mass) determination. Charge state determination in challenging instances can be enabled via the attachment of multiply charged ions of opposite polarity. Multiply charged ion attachment facilitates the resolution of charge states and generates mass-to-charge (m/z) information across a broad m/z range. In this work, we demonstrated the attachment of multiply charged cations to anionic complexes generated under native MS conditions. To illustrate the flexibility available in selecting the mass and charge of the reagents, the 15+ and 20+ charge states of horse skeletal muscle apomyoglobin and the 20+ and 30+ charge states of bovine carbonic anhydrase were demonstrated to attach to model complex anions derived from either ß-galactosidase or GroEL. The exclusive attachment of reagent ions is observed with no evidence for proton transfer, which is the key for the unambiguous interpretation of the post-ion/ion reaction product ion spectrum. To illustrate the application to mixtures of complex ions, the 10+ charge state of bovine ubiquitin was attached to mixtures of anions generated from the 30S and 50S particles of the Escherichia coli ribosome. Six and five major components were revealed, respectively. In the case of the 50S anion population, it was shown that the attachment of two 30+ cations of carbonic anhydrase revealed the same information as the attachment of six 10+ cations of ubiquitin. In neither case was the intact 50S particle observed. Rather, particles with different combinations of missing components were observed. This work demonstrated the utility of multiply charged cation attachment to facilitate charge state assignments in native MS ensemble measurements of heterogeneous mixtures.

Variations on the balloon analogue risk task: A censored regression analysis.

In the present project, we reexamined the balloon analogue risk task (BART) by evaluating three variations on the task: one that does not require pumping, one that controls for trial duration, and another that withholds feedback on popping until the end of each trial. To accurately assess the censored data produced by the BART, performance was compared across these variations using Bayesian analysis with censored regression. The first experiment compared a task that required pumping to one that did not, and revealed that the tendency to respond earlier than is optimal does not reflect an avoidance of effort. The second experiment included a condition in which the duration of each trial was held constant by continuing to automatically inflate a balloon to its maximum size after a cash-in response; feedback on the pop time was withheld until the end of each trial. This condition revealed that the tendency to respond earlier is not driven by a desire to finish the task quickly by cashing in early, but the results also strongly suggested that the immediate experience of popping created a greater aversion to risk (although this condition difference was inconsequential by the end of the experiment). The article concludes by considering the implications of these results for cognitive neuroscience approaches to understanding performance on the BART.

Optimal go/no-go ratios to maximize false alarms.

Despite the ubiquity of go/no-go tasks in the study of behavioral inhibition, there is a lack of evidence regarding the impact of key design characteristics, including the go/no-go ratio, intertrial interval, and number of types of go stimuli, on the production of different response classes of central interest. In the present study we sought to empirically determine the optimal conditions to maximize the production of a rare outcome of considerable interest to researchers: false alarms. As predicted, the shortest intertrial intervals (450 ms), intermediate go/no-go ratios (2:1 to 4:1), and the use of multiple types of go stimuli produced the greatest numbers of false alarms. These results are placed within the context of behavioral changes during learning.

Sodium butyrate stimulates NHE8 expression via its role on activating NHE8 basal promoter activity.

Butyrate is a major metabolite in colonic lumen. It is produced from bacterial fermentation of dietary fiber. Butyrate has been shown to stimulate electroneutral sodium absorption through its regulation on sodium/hydrogen exchanger 3 (NHE3). Although NHE8, the newest addition of intestinal NHE family, is involved in sodium absorption in the intestinal tract, whether butyrate modulates NHE8 expression in the intestinal epithelial cells is not known. In the current study, we showed that butyrate treatment strongly induced NHE8 protein and NHE8 mRNA expression in human intestinal epithelial cells. Transfection with the human NHE8 promoter reporter constructs showed that butyrate treatment stimulated reporter gene expression at an amount comparable with its stimulation of NHE8 mRNA expression. Interestingly, a similar result was also observed in human NHE8 promoter transfected cells after trichostatin (TSA) treatment. Gel mobility shift assay identified an enhanced Sp3 protein binding on the human NHE8 basal promoter region upon butyrate stimulation. Furthermore, Sp3 acetylation modification is involved in butyrate-mediated NHE8 activation in Caco-2 cells. Our findings suggest that the mechanism of butyrate action on NHE8 expression involves enhanced Sp3 interaction at the basal promoter region of the human NHE8 gene promoter to activate NHE8 gene transcription. Thus butyrate is involved in intestinal regulation of NHE8 resulting enhanced sodium absorption.

X-ray Crystallographic, Multifrequency Electron Paramagnetic Resonance, and Density Functional Theory Characterization of the Ni(P(Cy)2N(tBu)2)2(n+) Hydrogen Oxidation Catalyst in the Ni(I) Oxidation State.

The Ni(I) hydrogen oxidation catalyst [Ni(P(Cy)2N(tBu)2)2](+) (1(+); P(Cy)2N(tBu)2 = 1,5-di(tert-butyl)-3,7-dicyclohexyl-1,5-diaza-3,7-diphosphacyclooctane) has been studied using a combination of electron paramagnetic resonance (EPR) techniques (X-, Q-, and D-band, electron-nuclear double resonance, hyperfine sublevel correlation spectroscopy), X-ray crystallography, and density functional theory (DFT) calculations. Crystallographic and DFT studies indicate that the molecular structure of 1(+) is highly symmetrical. EPR spectroscopy has allowed determination of the electronic g tensor and the spin density distribution on the ligands, and revealed that the Ni(I) center does not interact strongly with the potentially coordinating solvents acetonitrile and butyronitrile. The EPR spectra and magnetic parameters of 1(+) are found to be distinctly different from those for the related compound [Ni(P(Ph)2N(Ph)2)2](+) (4(+)). One significant contributor to these differences is that the molecular structure of 4(+) is unsymmetrical, unlike that of 1(+). DFT calculations on derivatives in which the R and R' groups are systematically varied have allowed elucidation of structure/substituent relationships and their corresponding influence on the magnetic resonance parameters.

Altered residues in key proteins influence the expression and activity of the nitrogenase complex in an adaptive CO2 fixation-deficient mutant strain of Rhodobacter sphaeroides.

Previously, the RubisCO-compromised spontaneous adaptive Rhodobacter sphaeroides mutant, strain 16PHC, was shown to derepress the expression of genes that encode the nitrogenase complex under normal repressive conditions. As a result of this adaptation, the active nitrogenase complex restored redox balance, thus allowing strain 16PHC to grow under photoheterotrophic conditions in the absence of an exogenous electron acceptor. A combination of whole genome pyrosequencing and whole genome microarray analyses was employed to identify possible loci responsible for the observed phenotype. Mutations were found in two genes, glnA and nifA, whose products are involved in the regulatory cascade that controls nitrogenase complex gene expression. In addition, a nucleotide reversion within the nifK gene, which encodes a subunit of the nitrogenase complex, was also identified. Subsequent genetic, physiological and biochemical studies revealed alterations that led to derepression of the synthesis of an active nitrogenase complex in strain 16PHC.

Gas-Phase Covalent Bond Formation via Nucleophilic Substitution: A Dissociation Kinetics Study of Leaving Groups, Isomeric R Groups, and Nucleophilic Sites.

Nucleophilic substitution covalent modification ion/ion reactions were carried out in a linear quadrupole ion trap between the doubly protonated peptides KGAILKGAILR, RARARAA, and RKRARAA and isomers of either singly deprotonated 3- or 4-sulfobenzoic acid (n-SBA) esterified with either N-hydroxysuccinimide (NHS) or 1-hydroxy-7-aza-benzotriazole (HOBt). The cation/anion attachment product, through which the covalent reaction occurs, was isolated and subjected to dipolar DC (DDC) activation to generate covalently modified product over the ranges of DDC activation energies and times. The resulting survival yields were used to determine reaction rates, and Tolmachev's effective ion temperature was used to extract Arrhenius and Eyring activation parameters. It was found that the kinetics determined under these conditions are highly sensitive to the identities and locations of the nucleophilic sites on the peptides, the leaving groups on the reagent, and the location of the attachment sites on the reagent and analyte. Depending upon the identity of the analyte/reagent combination, significant variations in activation energy or entropy (or both) were both found to underlie the measured rate differences. The determination of dissociation kinetics under DDC conditions and application of Tolmachev's effective ion temperature treatment enables unique insights into the dynamics of gas-phase covalent bond formation via ion/ion reactions.

Gas-Phase Ion/Ion Chemistry as a Probe for the Presence of Carboxylate Groups in Polypeptide Cations.

The reactivity of 1-hydroxybenzoyl triazole (HOBt) esters with the carboxylate functionality present in peptides is demonstrated in the gas phase with a doubly deprotonated dianion. The reaction forms an anhydride linkage at the carboxylate site. Upon ion trap collisional-induced dissociation (CID) of the modified peptide, the resulting spectrum shows a nominal loss of the mass of the reagent and a water molecule. Analogous phenomenology was also noted for model peptide cations that likely contain zwitterionic/salt-bridged motifs in reactions with a negatively charged HOBt ester. Control experiments indicate that a carboxylate group is the likely reactive site, rather than other possible nucleophilic sites present in the peptide. These observations suggest that HOBt ester chemistry may be used as a chemical probe for the presence and location of carboxylate groups in net positively charged polypeptide ions. As an illustration, deprotonated sulfobenzoyl HOBt was reacted with the [M+7H]7+ ion of ubiquitin. The ion was shown to react with the reagent and CID of the covalent reaction product yielded an abundant [M+6H-H2O]6+ ion. Comparison of the CID product ion spectrum of this ion with that of the water loss product generated from CID of the unmodified [M+6H]6+ ion revealed the glutamic acid at residue 64 as a reactive site, suggesting that it is present in the deprotonated form. Graphical Abstract ᅟ.

The temporal dynamics of waiting when reward is increasing.

The temporal dynamics of waiting are complex. The present study used a video game involving contingencies that produced differential reinforcement of wait times by arranging for the magnitude of the reward to be related to the duration of each inter-response time. In previous research, when outcomes were gradually increasing in value from a minimum to a maximum, two modes of behavior are observed: waiting as little as possible before cashing in (i.e., responding rapidly) or waiting until the maximum is available (i.e., responding slowly). When outcomes were either a smaller sooner or larger later reward, two modes of behavior were again observed which corresponded to choosing either reward immediately after it was available. In the present study, outcome values increased linearly for a period of time, leveled off, increased abruptly, and then increased linearly. This configuration produced three modes of wait times in people - responding immediately, responding immediately after the abrupt increase, and responding when the maximum possible reward was achieved on each trial. Three factors were evaluated as possible causes of each behavioral mode, a desire for immediacy or action, the trade-off between molecular and molar maximization, and ease of responding.

Hot under the collar: The impact of heat on game play.

High temperatures have been documented to affect behavior in a variety of ways depending on the nature of the task. We extended this prior research by examining the effects of dynamically changing temperature on various aspects of performance in a video game task. In the span of approximately an hour, temperature was gradually increased, stayed constant for a period of time, and gradually decreased to baseline. The gaming task was a variation on one used to assess impulsivity in participants thus allowing the possibility of assessing the effects of temperature on impulsive choice. Rather than heat increasing impulsivity and thus decreasing wait times, participants showed increases in wait times as temperature increased which either suggests that participants were becoming more self-controlled under heat or that the documented negative impact of heat on motor functioning was dominating their performance. Importantly, the participant's sensitivity to the changing task requirements was not affected by changes in temperature.

In Situ XPS Chemical Analysis of MnSiO3 Copper Diffusion Barrier Layer Formation and Simultaneous Fabrication of Metal Oxide Semiconductor Electrical Test MOS Structures.

Copper/SiO2/Si metal-oxide-semiconductor (MOS) devices both with and without a MnSiO3 barrier layer at the Cu/SiO2 interface have been fabricated in an ultrahigh vacuum X-ray photoelectron spectroscopy (XPS) system, which allows interface chemical characterization of the barrier formation process to be directly correlated with electrical testing of barrier layer effectiveness. Capacitance voltage (CV) analysis, before and after tube furnace anneals of the fabricated MOS structures showed that the presence of the MnSiO3 barrier layer significantly improved electric stability of the device structures. Evidence of improved adhesion of the deposited copper layer to the MnSiO3 surface compared to the clean SiO2 surface was apparent both from tape tests and while probing the samples during electrical testing. Secondary ion mass spectroscopy (SIMS) depth profiling measurements of the MOS test structures reveal distinct differences of copper diffusion into the SiO2 dielectric layers following the thermal anneal depending on the presence of the MnSiO3 barrier layer.

Pulsed-Plasma Physical Vapor Deposition Approach Toward the Facile Synthesis of Multilayer and Monolayer Graphene for Anticoagulation Applications.

We demonstrate the growth of multilayer and single-layer graphene on copper foil using bipolar pulsed direct current (DC) magnetron sputtering of a graphite target in pure argon atmosphere. Single-layer graphene (SG) and few-layer graphene (FLG) films are deposited at temperatures ranging from 700 °C to 920 °C within <30 min. We find that the deposition and post-deposition annealing temperatures influence the layer thickness and quality of the graphene films formed. The films were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and optical transmission spectroscopy techniques. Based on the above studies, a diffusion-controlled mechanism was proposed for the graphene growth. A single-step whole blood assay was used to investigate the anticoagulant activity of graphene surfaces. Platelet adhesion, activation, and morphological changes on the graphene/glass surfaces, compared to bare glass, were analyzed using fluorescence microscopy and SEM techniques. We have found significant suppression of the platelet adhesion, activation, and aggregation on the graphene-covered surfaces, compared to the bare glass, indicating the anticoagulant activity of the deposited graphene films. Our production technique represents an industrially relevant method for the growth of SG and FLG for various applications including the biomedical field.

A delay discounting task produces a greater likelihood of waiting than a deferred gratification task.

A first-person-shooter video game was adapted for the study of choice between smaller sooner and larger later outcomes to compare the behavioral patterns produced by deferred gratification (DG) and delay discounting (DD) tasks. Participants played a game in which they could either fire their weapon sooner and do a small amount of damage or wait a few seconds to fire their weapon and do a larger amount of damage. For the DD task, a failure to fire within one second committed the player to waiting for the larger later outcome thus removing the opportunity to defect during the delay that is present in the DG task. The incentive structure changed multiple times during game play so that at times the optimal decision was to choose the smaller sooner outcome whereas at other times the optimal decision was to wait for the larger later outcome. Players assigned to the DD task showed a greater tendency to wait and lower sensitivity to the changing incentives.

Outcome probability versus magnitude: when waiting benefits one at the cost of the other.

Using a continuous impulsivity and risk platform (CIRP) that was constructed using a video game engine, choice was assessed under conditions in which waiting produced a continuously increasing probability of an outcome with a continuously decreasing magnitude (Experiment 1) or a continuously increasing magnitude of an outcome with a continuously decreasing probability (Experiment 2). Performance in both experiments reflected a greater desire for a higher probability even though the corresponding wait times produced substantive decreases in overall performance. These tendencies are considered to principally reflect hyperbolic discounting of probability, power discounting of magnitude, and the mathematical consequences of different response rates. Behavior in the CIRP is compared and contrasted with that in the Balloon Analogue Risk Task (BART).