Voltage- and substrate-dependent interactions between sites in putative re-entrant domains of a Na+-coupled phosphate cotransporter

A common structural feature characterises sodium-coupled inorganic phosphate cotransporters of the SLC34 family (NaPi-IIa/b/c): a pair of inverted regions in the N- and C-terminal halves of the protein. These regions are hypothesised to contain re-entrant domains that associate to allow alternating access of the substrates from either side of the membrane. To investigate if these domains interact during the NaPi-II transport cycle, we introduced novel cysteines at three functionally important sites associated with the predicted re-entrant domains of the flounder NaPi-IIb for the purpose of fluorescent labelling and cross-linking. Single and double mutants were expressed in Xenopus oocytes and their function analysed using electrophysiological and real-time fluorometric assays. The substitution at the cytosolic end of the first re-entrant domain induced a large hyperpolarizing shift in the voltage dependence of steady-state and presteady-state kinetics, whereas the two substitutions at the external face were less critical. By using Cu-phenanthroline to induce disulfide bridge formation, we observed a loss of transport activity that depended on the presence of sodium in the incubation medium. This suggested that external sodium increased the probability of NaPi-IIb occupying a conformation that favours interaction between sites in the re-entrant domains. Furthermore, voltage-dependent fluorescence data supported the hypothesis that a localised interaction between the two domains occurs that depends on the membrane potential and substrate present: we found that the fluorescence intensity reported by a labelled cysteine in one domain was dependent on the side chain substituted at a functionally critical site in the opposed domai

Conferring electrogenicity to the electroneutral phosphate cotransporter NaPi-IIc (SLC34A3) reveals an internal cation release step

The SLC34 family of Na+-dependent inorganic phosphate cotransporters comprises two electrogenic isoforms (NaPi-IIa, NaPi-IIb) and an electroneutral isoform (NaPi-IIc). Both fulfill essential physiological roles in mammalian phosphate homeostasis. By substitution of three conserved amino acids, found in all electrogenic isoforms, at corresponding sites in NaPi-IIc, electrogenicity was re-established and the Na+/P i stoichiometry increased from 2:1 to 3:1. However, this engineered electrogenic construct (AAD-IIc) had a reduced apparent P i affinity and different presteady-state kinetics from the wild-type NaPi-IIa/b. We investigated AAD-IIc using electrophysiology and voltage clamp fluorometry to elucidate the compromised behavior. The activation energy for cotransport was threefold higher than for NaPi-IIc and 1.5-fold higher than for NaPi-IIa and the temperature dependence of presteady-state charge displacements suggested that the large activation energy was associated with the empty carrier reorientation. AAD-IIc shows a weak interaction of external Na+ ions with the electric field, and thus retains the electroneutral cooperative interaction of two Na+ ions preceding external P i binding of NaPi-IIc. Most of the presteady-state charge movement was accounted for by the empty carrier (in the absence of external P i ), and the cytosolic release of one Na+ ion (in the presence of P i ). Simulations using a kinetic model recapitulated the presteady-state and steady-state behavior and allowed identification of two critical partial reactions: the final release of Na+ to the cytosol and external P i binding. Fluorometric recordings from AAD-IIc mutants with Cys substituted at functionally important sites established that AAD-IIc undergoes substrate- and voltage-dependent conformational changes that correlated qualitatively with its presteady-state kinetic

An ERP Investigation on Visuotactile Interactions in Peripersonal and Extrapersonal Space: Evidence for the Spatial Rule

The spatial rule of multisensory integration holds that cross-modal stimuli presented from the same spatial location result in enhanced multisensory integration. The present study investigated whether processing within the somatosensory cortex reflects the strength of cross-modal visuotactile interactions depending on the spatial relationship between visual and tactile stimuli. Visual stimuli were task-irrelevant and were presented simultaneously with touch in peripersonal and extrapersonal space, in the same or opposite hemispace with respect to the tactile stimuli. Participants directed their attention to one of their hands to detect infrequent tactile target stimuli at that hand while ignoring tactile targets at the unattended hand, all tactile nontarget stimuli, and any visual stimuli. Enhancement of ERPs recorded over and close to the somatosensory cortex was present as early as 100 msec after onset of stimuli (i.e., overlapping with the P100 component) when visual stimuli were presented next to the site of tactile stimulation (i.e., perihand space) compared to when these were presented at different locations in peripersonal or extrapersonal space. Therefore, this study provides electrophysiological support for the spatial rule of visual–tactile interaction in human participants. Importantly, these early cross-modal spatial effects occurred regardless of the locus of attention. In addition, and in line with previous research, we found attentional modulations of somatosensory processing only to be present in the time range of the N140 component and for longer latencies with an enhanced negativity for tactile stimuli at attended compared to unattended locations. Taken together, the pattern of the results from this study suggests that visuotactile spatial effects on somatosensory processing occur prior and independent of tactile–spatial attention

The shape of the change: Cumulative and incremental changes in daily mood during mobile-app-supported mindfulness training

Understanding of the exact trajectories of mood improvements during mindfulness practice helps to optimize mindfulness-based interventions. The Mindfulness-to-Meaning model expects mood improvements to be linear, incremental, and cumulative. Our findings align with this expectation. We used multilevel growth curve models to analyze daily changes in positive mood reported by 190 Polish participants during 42 days of a mobile-app-supported, mindfulness-based intervention. The daily positive mood increased among 83.68% of participants. Participants who started the training reported worse mood improved more and faster than participants with better mood at the baseline. Dispositional mindfulness and narcissism – individual difference variables associated with high vs. low emotion regulation ability, respectively – were not associated with mood improvement trajectories. A small group of participants (16.32%) showed a steady decline in positive mood during the intervention. The results underscore the importance of a more comprehensive understanding of individual variability in benefiting from mindfulness-based interventions

When far is near: ERP correlates of crossmodal spatial interactions between tactile and mirror-reflected visual stimuli

Visuo-tactile integration occurs in a privileged way in peripersonal space, namely when visual and tactile stimuli are in spatial proximity. Here, we investigated whether crossmodal spatial effects (i.e. stronger crossmodal interactions for spatially congruent compared to incongruent visual and tactile stimuli) are also present when visual stimuli presented near the body are indirectly viewed in a mirror, thus appearing in far space. Participants had to attend to one of their hands throughout a block of stimuli in order to detect infrequent tactile target stimuli at that hand while ignoring tactile targets at the unattended hand, all tactile non-target stimuli, and any visual stimuli. Visual stimuli were presented simultaneously with tactile stimuli, in the same (congruent) or opposite (incongruent) hemispace with respect to the tactile stimuli. In one group of participants the visual stimuli were delivered near the participants’ hands and were observed as indirect mirror reflections (‘mirror’ condition), while in the other group these were presented at a distance from the hands (‘far’ condition). The main finding was that crossmodal spatial modulations of ERPs recorded over and close to somatosensory cortex were present in the ‘mirror’ condition but not the ‘far’ condition. That is, ERPs were enhanced in response to tactile stimuli coupled with spatially congruent versus incongruent visual stimuli when the latter were viewed through a mirror. These effects emerged around 190 ms after stimuli onset, and were modulated by the focus of spatial attention. These results provide evidence that visual stimuli observed in far space via a mirror are coded as near-thebody stimuli according to their known rather than to their perceived location. This suggests that crossmodal interactions between vision and touch may be modulated by previous knowledge of reflecting surfaces (i.e. top-down processing)

ERP correlates of tactile spatial attention differ under intra- and intermodal conditions

To investigate whether the mechanisms underlying endogenous tactile spatial attention differ under pure tactile compared to mixed modality conditions event-related brain potentials (ERPs) were recorded to bilateral tactile and visual cues and tactile imperative stimuli. In the cue-stimulus interval the anterior directing attention negativity (ADAN) was present contralateral to the side of the attentional shift. Importantly, under pure tactile conditions this component persisted until imperative stimulus onset, while it diminished under intermodal conditions. Furthermore, post-tactile stimulus onset attentional modulations were present for the P100 component and later latencies under intermodal conditions. In contrast, under pure tactile conditions attentional modulations only emerged for the N140 component and later latencies. It is suggested that mechanisms underlying attentional orienting and selection are not entirely supramodal but depend in part on the modalities involved

Endogenous cholinergic inputs and local circuit mechanisms govern the phasic mesolimbic dopamine response to nicotine

Nicotine exerts its reinforcing action by stimulating nicotinic acetylcholine receptors (nAChRs) and boosting dopamine (DA) output from the ventral tegmental area (VTA). Recent data have led to a debate about the principal pathway of nicotine action: direct stimulation of the DAergic cells through nAChR activation, or disinhibition mediated through desensitization of nAChRs on GABAergic interneurons. We use a computational model of the VTA circuitry and nAChR function to shed light on this issue. Our model illustrates that the α4β2-containing nAChRs either on DA or GABA cells can mediate the acute effects of nicotine. We account for in vitro as well as in vivo data, and predict the conditions necessary for either direct stimulation or disinhibition to be at the origin of DA activity increases. We propose key experiments to disentangle the contribution of both mechanisms. We show that the rate of endogenous acetylcholine input crucially determines the evoked DA response for both mechanisms. Together our results delineate the mechanisms by which the VTA mediates the acute rewarding properties of nicotine and suggest an acetylcholine dependence hypothesis for nicotine reinforcement.Peer reviewe

Uses of innovative modeling tools within the implementation of the marine strategy framework directive

© 2016 Lynam, Uusitalo, Patrício, Piroddi, Queirós, Teixeira, Rossberg, Sagarminaga, Hyder, Niquil, Möllmann, Wilson, Chust, Galparsoro, Forster, Veríssimo, Tedesco, Revilla and Neville. In Europe and around the world, the approach to management of the marine environment has developed from the management of single issues (e.g., species and/or pressures) toward holistic Ecosystem Based Management (EBM) that includes aims to maintain biological diversity and protect ecosystem functioning. Within the European Union, this approach is implemented through the Marine Strategy Framework Directive (MSFD, 2008/56/EC). Integrated Ecosystem Assessment is required by the Directive in order to assess Good Environmental Status (GES). Ecological modeling has a key role to play within the implementation of the MSFD, as demonstrated here by case studies covering a range of spatial scales and a selection of anthropogenic threats. Modeling studies have a strong role to play in embedding data collected at limited points within a larger spatial and temporal scale, thus enabling assessments of pelagic and seabed habitat. Furthermore, integrative studies using food web and ecosystem models are able to investigate changes in food web functioning and biological diversity in response to changes in the environment and human pressures. Modeling should be used to: support the development and selection of specific indicators; set reference points to assess state and the achievement of GES; inform adaptive monitoring programs and trial management scenarios. The modus operandi proposed shows how ecological modeling could support the decision making process leading to appropriate management measures and inform new policy

An integrated experimental and quantum-chemical investigation on the vibrational spectra of chlorofluoromethane

The vibrational analysis of the gas-phase infrared spectra of chlorofluoromethane (CH2ClF, HCFC-31) was carried out in the range 200-6200 cm(-1). The assignment of the absorption features in terms of fundamental, overtone, combination, and hot bands was performed on the medium-resolution (up to 0.2 cm(-1)) Fourier transform infrared spectra. From the absorption cross section spectra accurate values of the integrated band intensities were derived and the global warming potential of this compound was estimated, thus obtaining values of 323, 83, and 42 on a 20-, 100-, and 500-year horizon, respectively. The set of spectroscopic parameters here presented provides the basic data to model the atmospheric behavior of this greenhouse gas. In addition, the obtained vibrational properties were used to benchmark the predictions of state-of-the-art quantum-chemical computational strategies. Extrapolated complete basis set limit values for the equilibrium geometry and harmonic force field were obtained at the coupled-cluster singles and doubles level of theory augmented by a perturbative treatment of triple excitations, CCSD(T), in conjunction with a hierarchical series of correlation-consistent basis sets (cc-pVnZ, with n = T, Q, and 5), taking also into account the core-valence correlation effects and the corrections due to diffuse (aug) functions. To obtain the cubic and quartic semi-diagonal force constants, calculations employing second-order Moller-Plesset perturbation (MP2) theory, the double-hybrid density functional B2PLYP as well as CCSD(T) were performed. For all anharmonic force fields the performances of two different perturbative approaches in computing the vibrational energy levels (i.e., the generalized second order vibrational treatment, GVPT2, and the recently proposed hybrid degeneracy corrected model, HDCPT2) were evaluated and the obtained results allowed us to validate the spectroscopic predictions yielded by the HDCPT2 approach. The predictions of the deperturbed second-order perturbation approach, DVPT2, applied to the computation of infrared intensities beyond the double-harmonic approximation were compared to the accurate experimental values here determined. Anharmonic DFT and MP2 corrections to CCSD(T) intensities led to a very good agreement with the absorption cross section measurements over the whole spectral range here analysed. (C) 2013 AIP Publishing LLC