Glucagonotropic and Glucagonostatic Effects of KATP Channel Closure and Potassium Depolarization.

The role of depolarization in the inverse glucose-dependence of glucagon secretion was investigated by comparing the effects of KATP channel block and of high potassium. The secretion of glucagon and insulin by perifused mouse islets was simultaneously measured. Lowering glucose raised glucagon secretion before it decreased insulin secretion, suggesting an alpha cell-intrinsic signal recognition. Raising glucose affected glucagon and insulin secretion at the same time. However, depolarization by tolbutamide, gliclazide, or 15 mM KCl increased insulin secretion before the glucagon secretion receded. In contrast to the robust depolarizing effect of arginine and KCl (15 and 40 mM) on single alpha cells, tolbutamide was of variable efficacy. Only when applied before other depolarizing agents had tolbutamide a consistent depolarizing effect and regularly increased the cytosolic Ca2+ concentration. When tested on inside-out patches tolbutamide was as effective on alpha cells as on beta cells. In the presence of 1 µM clonidine, to separate insulinotropic from glucagonotropic effects, both 500 µM tolbutamide and 30 µM gliclazide increased glucagon secretion significantly, but transiently. The additional presence of 15 or 40 mM KCl in contrast led to a marked and lasting increase of the glucagon secretion. The glucagon secretion by SUR1 knockout islets was not increased by tolbutamide, whereas 40 mM KCl was of unchanged efficiency. In conclusion a strong and sustained depolarization is compatible with a marked and lasting glucagon secretion. KATP channel closure in alpha cells is less readily achieved than in beta cells, which may explain the moderate and transient glucagonotropic effect.

EPR Distance Measurements on Long Non-coding RNAs Empowered by Genetic Alphabet Expansion Transcription.

We present herein a novel nitroxide spin label-containing RNA triphosphate TPT3NO and its application for site-specific spin-labeling of RNA through in vitro transcription using an expanded genetic alphabet. Our strategy allows the facile preparation of spin-labeled RNAs with sizes ranging from short RNA oligonucleotides to large, complex RNA molecules with over 370 nucleotides by standard in vitro transcription. As a proof of concept, inter-spin distance distributions are measured by pulsed electron paramagnetic resonance (EPR) spectroscopy in short self-complementary RNA sequences and in a well-studied 185 nucleotide non-coding RNA, the B. subtilis glmS ribozyme. The approach is then applied to probe for the first time the folding of the 377 nucleotide A-region of the long non-coding RNA Xist, by PELDOR.

Towards Reverse Transcription with an Expanded Genetic Alphabet.

Unnatural base pairs (UBPs) strikingly augment the natural genetic alphabet. The development of particular hydrophobic UBPs even allows insertion and stable propagation in bacteria. Those UBPs expand the chemical scope of DNA and RNA, and thus, could enable the evolution of novel aptamers or ribozymes by in vitro selection (systematic evolution of ligands by exponential enrichment, SELEX). However, the application of such UBPs in reverse transcription (rtc), which is a key step for RNA-based SELEX, has not been reported so far. The implication of Romesberg's NaM:TPT3 base pair in rtc reactions is presented by testing five commercially available reverse transcriptases (RTs). The employed RTs predominantly pause at the site of the unnatural nucleotide rTPT3 not being able to accept the dNaM building block as a substrate. This allows verification of the unnatural base position in RNA and an estimation of their abundance. In contrast, primer extension from an rNaM-containing template results in considerably more full-length cDNA. Furthermore, RTs that could potentially be able to handle an expanded genetic alphabet based on NaM:TPT3 are presented.

Posttranscriptional spin labeling of RNA by tetrazine-based cycloaddition.

The site-specific introduction of spin labels into RNA for distance measurements by EPR gives insight into its solution structure. We here present a method for spin labeling of in vitro transcribed RNA. Distance distributions between two nitroxide spin labels are determined by PELDOR in a self-complementary RNA duplex.

Iluminated by foreign letters - Strategies for site-specific cyclopropene modification of large functional RNAs via in vitro transcription.

The synthesis of sequence-specifically modified long RNA molecules, which cannot entirely be prepared via solid phase synthesis methods is experimentally challenging. We are using a new approach based on an expanded genetic alphabet preparing site-specifically modified RNA molecules via standard in vitro transcription. In this report, the site-specific labeling of functional RNAs, in particular ribozymes and a long non-coding RNA with cyclopropene moieties, is presented. We provide detailed instructions for RNA labeling via in vitro transcription and include required analytical methods to verify production and identity of the transcript. We further present post-transcriptional inverse electron demand Diels-Alder cycloaddition reactions on the cyclopropene-modified sequences and discuss applications of the genetic alphabet expansion transcription for in vitro preparation of labeled functional RNAs with complex foldings. In detail, the glmS and CPEB3 ribozymes were site-specifically decorated with methyl cyclopropene moieties using the unnatural TPT3CP triphosphate and were proven to be still functional. In addition, the structurally complex A region of the Xist lncRNA (401nt) was site-specifically modified with methyl cyclopropene and detected by fluorescence after cycloaddition reaction with a tetrazine-BODIPY conjugate.

Subscales of the Barratt Impulsiveness Scale differentially relate to the Big Five factors of personality.

The place of impulsiveness in multidimensional personality frameworks is still unclear. In particular, no consensus has yet been reached with regard to the relation of impulsiveness to Neuroticism and Extraversion. We aim to contribute to a clearer understanding of these relationships by accounting for the multidimensional structure of impulsiveness. In three independent studies, we related the subscales of the Barratt Impulsiveness Scale (BIS) to the Big Five factors of personality. Study 1 investigated the associations between the BIS subscales and the Big Five factors as measured by the NEO Five-Factor Inventory (NEO-FFI) in a student sample (N = 113). Selective positive correlations emerged between motor impulsiveness and Extraversion and between attentional impulsiveness and Neuroticism. This pattern of results was replicated in Study 2 (N = 132) using a 10-item short version of the Big Five Inventory. In Study 3, we analyzed BIS and NEO-FFI data obtained from a sample of patients with pathological buying (N = 68). In these patients, the relationship between motor impulsiveness and Extraversion was significantly weakened when compared to the non-clinical samples. At the same time, the relationship between attentional impulsiveness and Neuroticism was substantially stronger in the clinical sample. Our studies highlight the utility of the BIS subscales for clarifying the relationship between impulsiveness and the Big Five personality factors. We conclude that impulsiveness might occupy multiple places in multidimensional personality frameworks, which need to be specified to improve the interpretability of impulsiveness scales.

Road crossing behavior under traffic light conflict: Modulating effects of green light duration and signal congruency.

A large number of pedestrians and cyclists regularly ignore the traffic lights to cross the road illegally. In a recent analysis, illegal road crossing behavior has been shown to be enhanced in the presence of incongruent stimulus configurations. Pedestrians and cyclists are more likely to cross against a red light when exposed to an irrelevant conflicting green light. Here, we present experimental and observational data on the factors moderating the risk associated with incongruent traffic lights. In an observational study, we demonstrated that the conflict-related increase in illegal crossing rates is reduced when pedestrian and cyclist green light periods are long. In a laboratory experiment, we manipulated the color of the irrelevant signals to expose participants to different degrees of incongruency. Results revealed that individuals' performance gradually varied as a function of incongruency, suggesting that the negative impact of a conflicting green light can be reduced by slightly adjusting its color. Our findings highlight that the observation of real-world behavior at intersections and the experimental analysis of psychological processes under controlled laboratory conditions can complement each other in identifying risk factors of risky road crossing behavior. Based on this combination, our study elaborates on promising measures to improve safety at signalized intersections.

Selective Cooperation in the Supermarket : Field Experimental Evidence for Indirect Reciprocity.

Numerous laboratory experiments suggest that mechanisms of indirect reciprocity might account for human cooperation. However, conclusive field data supporting the predictions of indirect reciprocity in everyday life situations is still scarce. Here, we attempt to compensate for this lack by examining the determinants of cooperative behavior in a German supermarket. Our methods were as follows: Confederates of the experimenter lined up at the checkout, apparently to buy a single item. As an act of cooperation, the waiting person in front (the potential helper) could allow the confederate to go ahead. By this means, the potential helper could take a cost (additional waiting time) by providing the confederate with a benefit (saved waiting time). We recorded the potential helpers' behavior and the number of items they purchased as a quantitative measure proportional to the confederate's benefit. Moreover, in a field experimental design, we varied the confederates' image by manipulating the item they purchased (beer vs. water). As predicted, the more waiting time they could save, the more likely the confederates were to receive cooperation. This relationship was moderated by the confederates' image. Cost-to-benefit ratios were required to be more favorable for beer-purchasing individuals to receive cooperation. Our results demonstrate that everyday human cooperation can be studied unobtrusively in the field and that cooperation among strangers is selective in a way that is consistent with current models of indirect reciprocity.

Site-specific enzymatic introduction of a norbornene modified unnatural base into RNA and application in post-transcriptional labeling.

Inverse electron demand Diels-Alder cycloadditions have proven to be extremely useful for mild and additive-free orthogonal labeling of biomolecules, amongst others, for RNA labeling in vitro and in a cellular context. Here we present a method for site-specific introduction of an alkene modification into RNA via T7 in vitro transcription. For this, an unnatural, hydrophobic base pairing system developed by Romesberg and coworkers was modified introducing one or two norbornene moieties at predefined positions into RNA oligonucleotides in an in vitro transcription reaction. This allows post-transcriptional functionalization of these RNA molecules with tetrazine derivatives containing for instance fluorophores or biotin.

Mapping self-reported to behavioral impulsiveness: the role of task parameters.

Despite being regarded as indicators of a common psychological capacity, behavioral and self-reported measures of impulsiveness have been found to barely correlate with each other. Acknowledging the construct's multidimensional nature, the present study set out to map dissociable components of behavioral self-control (delay discounting, response inhibition) onto lower-order facets of self-reported impulsiveness. In addition, we examined whether the relationship between response inhibition and self-reported impulsiveness depends on the balance between facilitative and interfering priming processes involved in a laboratory task. In two consecutive studies, 185 participants completed laboratory self-control tasks as well as self-report questionnaires designed to measure facets of impulsiveness. Correlational analyses revealed an association between subscales of the Barratt Impulsiveness Scale (BIS) and response inhibition in a go/no-go paradigm involving simultaneously presented task-irrelevant distractors. This association vanished when an onset asynchrony between distractor and target stimuli was introduced. Previous findings regarding correlations between BIS subscales and delay discounting or intra-individual response variability could not be replicated. Results indicate that the relationship between response inhibition and self-reported impulsiveness critically varies as a function of subtle task parameters. Focusing on these procedural details and the multidimensionality of self-reported impulsiveness might allow for a more differentiated analysis of the convergent validity of self-control measures.

Sweet delusion. Glucose drinks fail to counteract ego depletion.

Initial acts of self-control have repeatedly been shown to reduce individuals' performance on a consecutive self-control task. In addition, sugar containing drinks have been demonstrated to counteract this so-called ego-depletion effect, both when being ingested and when merely being sensed in the oral cavity. However, since the underlying evidence is less compelling than suggested, replications are crucially required. In Experiment 1, 70 participants consumed a drink containing either sugar or a non-caloric sweetener between two administrations of delay-discounting tasks. Experiment 2 (N=115) was designed to unravel the psychological function of oral glucose sensing by manipulating the temporal delay between a glucose mouth rinse and the administration of the consecutive self-control task. Despite applying powerful research designs, no effect of sugar sensing or ingestion on ego depletion could be detected. These findings add to previous challenges of the glucose model of self-control and highlight the need for independent replications.

The dark side of stimulus control--associations between contradictory stimulus configurations and pedestrians' and cyclists' illegal street crossing behavior.

Since illegal pedestrian behavior represents a major source of accidents, research investigating possible reasons and risk factors for crossing against the lights is pivotal for enhancing safety in traffic. The present approach regards behavior at signalized intersections as a result of multiple stimulus discrimination. Hence, it is expected that at crossings divided by a median refuge the excitatory potential of a "consecutive green light" or "oncoming pedestrians" (S+*) attenuates the inhibition of crossing behavior induced by the relevant red light (S-). Standardized observations at critical intersections in Braunschweig, Germany, were conducted to investigate these hypotheses. Comparing outside traffic participants' behavior in the presence of different stimulus configurations identified the assumed S+* as substantial risk factors for illegal crossings. Moreover, the presented model of stimulus control integrates past risk factor research and may help develop future prevention measures.