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As illustrated by the results above, through careful tailoring of G4-forming sequences we achieve a dramatic reduction of the intrinsic conformational heterogeneity of the wild-type telomeric G4s and a selective favoring of specific G4 conformations that are otherwise only marginally stable. Our single-molecule FRET experiments utilizing these sequences allowed obtaining characteristic signatures of different G4 conformations and thus permitted linking the experimentally observed E states to distinct folded G4 conformations.
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To further aid in the assignment of the observed E states, we performed MD simulations of the DNA construct used in the single-molecule experiments. DNA constructs containing hybrid 1, hybrid 2, 2-tetrad basket and chair (wild-type and mutant) G4s were simulated (Figure 2E and ‘Materials and Methods’ section for details). We find that the G4 structures are predominantly positioned on top of the duplex, i.e. with the G-tetrad plane perpendicular to the long axis of the duplex, for the DNA constructs containing the hybrid 1, hybrid 2 and chair (wild-type and mutant) conformations. However, the 2-tetrad basket conformation is predominantly tilted relative to the duplex, resulting in positioning of the G-tetrad plane almost parallel to the long axis of the duplex (Supplementary Figure S7). This feature is likely to originate due to the structural specificity of the 2-tetrad basket conformation that contains a long diagonal loop that may create some steric restrictions for the G4-duplex relative positioning. To assess the transfer efficiency for each simulated G4 construct we used the AV approach (39) (Figure 2E, Supplementary Figure S5 and ‘Materials and Methods’ section for details). The transfer efficiencies derived from the simulations can be directly compared to those obtained from single-molecule FRET experiments (Figure 2F). The experimental and simulated transfer efficiencies for the chair, hybrid 1 and hybrid 2 conformations match within uncertainty. We even observe a small difference in the simulated transfer efficiencies for the mutant (E = 0.84) and wild-type (E = 0.89) chair conformations (Supplementary Figure S5), that is also detected in the single-molecule experiments (Figure 2). This reflects the small structural differences of the two constructs and demonstrates the robustness of both approaches of assessing the transfer efficiencies. Due to the specific positioning of the 2-tetrad basket conformation relative to the duplex, the separation between the two fluorophores is larger than for other G4 conformations. It appears in a range very close to the Förster radius (R0 = 5.6 Å) (Supplementary Figure S5), where the energy transfer efficiency is mostly sensitive to distance changes. This observation can potentially explain the small discrepancy between the experimental and simulated transfer efficiencies for this particular conformation. Overall, we find a remarkable agreement between the two sets of transfer efficiencies obtained from single-molecule FRET experiments and simulations, which further supports our structural assignment.
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To obtain further mechanistic insight into the folding of G4s we extracted the probability and rate constants for each individual transition observed in the course of G4 folding (Figure 1C, Supplementary Figure S3, Supplementary Tables S2 and 3). We find that the majority of transitions occur between an unfolded and the different folded conformations i.e. the interconversion between folded conformations proceeds via an unfolding step (Figure 1C). Direct transitions between folded conformations were also observed, but are significantly less probable (< 5%) (Supplementary Table S2). It should be noted that direct interconversion between folded G4s is structurally implausible and will require at least partial unfolding (14). Therefore, we believe that the small number of direct transitions observed in our experiments may involve a rapid complete or partial unfolding step that, however, remains hidden due to limited time resolution. We find that the chair, 2-tetrad basket and hybrid 2 conformations are rapidly formed short-lived folded states (kF = 0.19-0.21 s−1 and kU = 0.15-0.35 s−1) (Supplementary Table S3) that are extensively observed at the early stages of G4 folding (Figure 1F).
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Interestingly, our cation-induced folding experiments reveal that the chair (E≈0.88) is the preferred first populated short-lived folded conformation (80% of all trajectories showing real-time folding) (Figure 1B and Supplementary Figure S1A–D), an observation that is in excellent agreement with results from recent ensemble stop-flow experiments (21). In contrast to more complex structural arrangements necessary for the formation of other conformations, the folding into the chair conformation per se can occur through a simple bending of a G-hairpin (transiently populating the unfolded ensemble) (21,22), which may preferentially favor this conformational transition. In the remaining 20% of cases the 2-tetrad basket conformation forms first, which can be explained by the fact that for the formation of this conformation only one K+ cation is required. It should be noted, however, that we estimate similar energy barriers for all folding transitions (Supplementary Table S4). In addition, we find that the unfolding of the chair G4 is slower by approximately a factor of two (kU = 0.15 s−1) compared to the unfolding of the 2-tetrad basket and hybrid conformations. This observation implies kinetic trapping of the anti-parallel chair conformation (49) and can explain the prevalence of this conformation at the beginning of the folding process (Figure 1F).
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Based on the folding/unfolding rates, we can also assess the energetics of the individual conformational transitions observed here (Figure 3 and Supplementary Table S4). The folding transitions to the 2-tetrad basket and hybrid 2 conformations are unfavorable under the current experimental conditions, while the chair is slightly favored. Our results reveal that the hybrid 1 conformation is the most stable and dominant conformation at the folding equilibrium, which stability \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$(\Delta{G_U}\sim 3\,{k_B}T)$\end{document} is in reasonable agreement with the previously reported values from ensemble (50,51) and single-molecule (25) experiments under similar buffer conditions.
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One-dimensional representation of the free energy landscape describing the multi-pathway folding of human telomeric G4s. Horizontal solid lines show energy levels of an unfolded and different folded conformations and the dashed line indicates the estimated height of the energy barrier (see ‘Materials and Methods’ section). Transitions between unfolded and folded conformations are shown as arrows. The thickness of the arrows scales with the value of rate constants of each transition. The slow unfolding transition of the hybrid 1 is shown as a dashed line. Possible transitions between folded conformations are omitted here to avoid overcrowding the figure. The color code of different G4 conformations is the same as in Figure 1B.
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By following the entire folding process (hours) of telomeric G4 at the single-molecule level and with a time resolution below 0.5 s, we could resolve several G4 states along the G4 folding pathway and provide a detailed description of their folding energetics and timescales. The combination of single-molecule experiments with different G4 forming DNA sequences and computational modeling of the experimental DNA constructs made possible the identification of several distinct folded G4 conformations of the human telomeric sequence that were not previously simultaneously observed. Single-molecule FRET is a powerful method for resolving several conformational states of complex biological systems that has been used for disentangling the structural polymorphism of telomeric G4s (14,49,52–54). However, previously, only two folded G4 states have been identified through single-molecule FRET experiments, which have been conditionally assigned to parallel and anti-parallel G4 conformations (14,49,52–54). In contrast, we identify FRET signatures of four distinct G4 conformations. The ability to directly resolve such large number of G4 conformations by FRET is not straightforward, due to similar size (Rg∼12 Å) (55) and comparable end-to-end distances for all G4 conformations. The resolution of different G4 conformations is achieved here through a careful design of the experimental system and choice of the specific positions for fluorophore labeling of the DNA constructs. Thus, our integrated approach, utilizing the power of single-molecule FRET microscopy and MD simulations, revealed the formation of two hybrid and two anti-parallel conformations in potassium containing solutions. One of the hybrid (hybrid 2) conformations and both anti-parallel conformations are kinetically favored during G4 folding, whereas the other hybrid conformation (hybrid 1) is the most thermodynamically stable structure for wild-type human telomeric G4s.
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In contrast to the moderate thermodynamic stability of G4 structures \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$(\Delta{G_U} < 3\,{k_B}T)$\end{document} the folding energy barriers are relatively large \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$(\Delta G_F^\ddagger \sim 15\,{k_B}T)$\end{document}, resulting into a slow folding process (several hours were necessary to reach thermodynamic equilibrium in our experimental conditions). Such high folding energy cost can be explained by an extreme flexibility and large degree of freedom of G4-forming sequences. Indeed, the formation of a planar G-quartet, necessary building block for G4s, may require large structural arrangements of the unfolded single-stranded DNA chain to bring together four guanine bases separated by as many as 20 nucleotides. Interestingly, we find similar folding barriers for all G4 conformations populating the energy landscape. This observation may indicate that the transition state separating the unfolded and folded states is likely to be very similar for all G4 conformations observed here. Although our experiments do not provide any direct structural features of the G4 transition state, we, however, may envision it as an electrostatically stabilized compact structure with similar global fold as for the folded G4s, but lacking most of the native contacts.
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We also observe high kinetic barriers for G4 unfolding \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$(\Delta G_U^\ddagger \sim 15 - 18\,{k_B}T)$\end{document} that allow kinetic trapping of folded G4 structures. These thermodynamic features may explain the pronounced structural polymorphism of the G4s, as a result of kinetic partitioning, involving several marginally stable folded G4 conformations.
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The observation of direct folded-to-unfolded transitions implies an extremely high degree of cooperativity of G4 folding/unfolding. Such level of cooperativity may be surprising, as previously several partially folded structures, such as G-hairpins and G-triplexes have been proposed to bridge the completely unfolded and folded G4 states, resulting in sequential folding pathways (21–24,26). These structures were also proposed to form during helicase-induced sequential unwinding of G4s (56,57). In our experiments with a G-triplex forming sequence such stable partially folded structures were not detected and are thus unlikely to contribute to any of the high E states here (Supplementary Figure S8). It should, however, be noted that we expect the unfolded state of G4s to be represented by a dynamic ensemble of a number of different unfolded and transiently formed partially folded conformations (29), possibly including G-hairpins and G-triplexes that may appear as a relatively broad peak at low transfer efficiencies in our single-molecule FRET histograms.
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Our experimental data obtained at the single-molecule level provides a detailed description of G4 folding as a complex multi-pathway process that involves numerous parallel folding/unfolding transitions and an extensive redistribution between several conformational states. Different site-specific DNA modifications can affect the energetics of the folded states and modify the conformational landscape (Figure 2). Our data show that these modified sequences exhibit similar complex folding behavior, as the one described for the wild-type sequence. The environment and specificity of folding conditions can also influence the G4 folding process. At higher potassium concentrations (100 mM KCl) similar folding behavior and conformational heterogeneity of G4s is observed (Supplementary Figure S9). Under these conditions, the folding of G4s is accelerated, compared to low salt conditions, as well as the marginally stable chair, 2-tetrad and hybrid 2 conformations get more destabilized. Overall, high potassium concentrations favor the formation of thermodynamically stable hybrid 1 conformation and result in reduction of the observed dynamics (Supplementary Figure S9). Studies in sodium-containing solutions show that G4 folding can be very dynamic and follows a multi-pathway process also in these conditions (30,58). These independent observations obtained for different DNA sequences or solutions conditions may point towards common mechanistic features of G4 folding, as described in this work.
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An extreme folding dynamics of G4s, as observed here, is certain to impact and possibly modulate their interaction with proteins (59,60) and ligands (61). Although the majority of observed folded states are only marginally stable in current conditions, we expect that they can be individually recognized and trapped through selective structure-specific interactions with different ligands (62) and proteins (63). The structural knowledge available through our experiments can potentially be used to control the folding pathway of G4 structures, i.e. through stabilization of particular G4 conformations via specifically tailored G4-ligand interactions. Therefore, this extreme folding dynamics of G4s should be considered for the development of novel G4-targeting anti-cancer therapies and for elucidating G4-proteins interactions. Our work thus provides a comprehensive thermodynamic and kinetic description of the folding of telomeric G4s, the implications of which may contribute to the structural and energetics framework necessary for future studies of G4 folding in vivo.
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The survival of the offspring is crucial for one’s reproductive success. Newborns may therefore carry specific features that act as key stimulus, which automatically captures attention and motivates actions such as caretaking. The baby schema (BS) is such a key stimulus. In the infant face it is defined as a combination of child characteristic features like big eyes, small nose, chubby cheeks, and higher forehead1. Humans bear high costs in raising their offspring, because pregnancy and production of maternal milk are energetically expensive2, which should make newborns highly relevant, at least for mothers. But since humans practice alloparental care3 and the BS is further known to be a universally relevant stimulus4 the BS should be highly significant for women in general. The increased cuteness of infant faces in comparison to adults may therefore prioritize selective attention5,6 and may enhance evaluation of these stimuli as socially relevant for actions like caretaking7.
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There is already evidence for an adaptive neural mechanism through which the perception of cuteness as a feature of the BS is most likely promoted in humans. Accordingly, cute baby faces have repeatedly yielded activations of regions of the mesolimbic reward system8,9. For example, Glocker et al.10 observed an increase of activation in the nucleus accumbens in women while evaluating pictures of babies with increasing BS (i.e., low BS < unmanipulated BS < high BS). Interestingly, the mesolimbic reward system may also be highly responsive to the influence of two hormones that have opposing roles in human nurturing behaviors. Firstly, a high oxytocin (OT) receptor density can be found in the mesolimbic reward system of social mammals11 and increased activation of the ventral tegmental area to images of crying infants after OT treatment suggested that OT influences the reward value of stimuli carrying the BS12. Further, OT administration increased the preference for pictures of young children. This preference was determined through rs53576G homozygote participants (a polymorphism in the OT receptor gene)13. Finally, OT has been consistently associated with higher maternal attachment and increased caretaking in humans14,15. The androgen testosterone (T) may also have reinforcing effects in the mesolimbic reward system16. Yet, in contrast to OT it may compromise behaviors associated with maternal care (e.g. nurturing)17. Previous studies have noted a reduced T level during early parenthood and this decline appeared to be associated with the quality of childcare18–22 (but see23).
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However, the interactive influence of T and OT on neural processing of the BS currently remains elusive. Only a few behavioral studies have so far assessed their combined influence on aspects of nurturing behaviors. In the context of paternal caretaking behavior Weisman and colleagues24 found that OT administration led to an unexpected short-term alteration of endogenous T, whereby nurturing behaviors like social gaze also increased. Given that women are the main caretakers during the first months after childbirth when infants depend on maternal milk, and yet sometimes show impairments like postpartum depression or child neglect25, it is of crucial importance to take a closer look on these interactive influences in women as well. In nulliparous women a higher endogenous T reduced selective attention to infant targets in the context of adult distractors6. Importantly, attention to infant stimuli increased after OT administration, but only in women with high endogenous T. This suggests that OT may oppose the negative effects of T on nurturing behavior in women6. There are already some indications on the behavioral level that T and OT may have gender-specific impacts on parental behavior. Gordon and colleagues26 found that high T levels in fathers negatively influenced the association between OT and paternal behavior, whereas in mothers high T levels evoked positive associations. Nevertheless, the neural mechanisms underlying the effect of the OT by T interaction on selective attention to infants in general and the highly relevant key stimulus BS in particular remains elusive.
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In order to further our understanding of the nature of hormonal interactions in nurturing behavior, the present study used functional magnetic resonance imaging (fMRI) and a double-blind placebo-controlled OT administration between-subjects protocol to examine the influence of exogenous OT and endogenous T on selective attention to the BS in nulliparous women. To assess selective attention to the BS we used an implicit association task. Implicit association tasks have the advantage that the degree of attentional capture by an infant as opposed to an adult face can be determined implicitly by the mean reaction time (RT) of the participant, with a shorter RT indicating faster attentional processing of the baby schema and increased action. Previous neuroimaging studies combined passive viewing and explicit evaluation tasks (e.g., cuteness perception on a Likert-Scale), which are more vulnerable to experimenter demand effect. Further, these studies do not allow the assessment of the mechanism that promotes faster reactions to infants. Building on previous findings6 we hypothesized that OT administration would compensate the negative effects of high endogenous T on attentional processing of the BS, probably through the modulation of activation in key nodes of the mesolimbic reward system.
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The influence of the endogenous T concentration (measured out of one saliva sample that was collected directly before administration) on attention to babies was analyzed with a 3 (morph type: low BS, natural BS and high BS) × 2 (treatment: OT vs. placebo) × 2 (T: median split of the sample that was collected directly before administration) ANOVA. Of particular interest was the significant three-way interaction between the factors “morph type”, “treatment” and “T” (F2,104 = 4.359; p = 0.015; see Table S1 for details). Building on previous research6 we assumed that women with high endogenous T derive greater benefit from OT treatment than women with low endogenous T and hence will become more sensitive for the BS. We found that OT-treated women with high endogenous T exhibited an attentional preference for the natural BS (positive Delta-RT: MhighT = 6.4 ± SEM 24.2 ms) compared to OT-treated women with low endogenous T (positive Delta-RT: MlowT = −50.6 ± SEM 16 ms) but these results remained on statistical trend level [Bonferroni-corrected statistical threshold was p ≤ 0.0166 (t27 = 1.86, p = 0.03; one-tailed, a priori hypothesis based on6)]. In the placebo group women with low endogenous T directed less attention to the plus morphed babies (MlowT = −39.1 ± SEM 21.3 ms) than women with high T concentrations (MhighT = 23.4 ± SEM 14.6 ms; t25 = 2.29, p = 0.015, one-tailed, a priori hypothesis based on6). The placebo-treated participants with high or low T did not show any differences in the preference for the natural BS.
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Drug treatment did not influence the participants RTs to geometric figures. Yet, the type of target (F2,110 = 39.485; p < 0.001; np2 = 0.418) and the interaction between target and distractor (F2,110 = 25.871; p < 0.001; np2 = 0.32) influenced the participants’ RTs in the control task (see also Table S2). These results are consistent with the assumption that simple geometric forms could have emotional meanings and may even trigger an attentional bias27.
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When contrasting “baby vs. adult target” for the different morph types, we found an effect of treatment in the direct comparison of the OT and placebo group in the left frontolateral cortex (MNI-coordinates [t-value; pFWEcorr] −33, 5, 49 [4.62; 0.023]), but only for the high BS condition (Fig. 1). The identified region was located at the intersection of the precentral sulcus and inferior frontal sulcus, which corresponds to the inferior frontal junction (IFJ)28. Additional t-tests on the parameter estimates extracted from the left IFJ, confirmed the difference in activation between the OT and the placebo group in the high BS condition (t55 = 4.43; p < 0.001) (Fig. 1). We could not find any difference in the direct comparison of OT > placebo in the unmanipulated or the low BS conditions.Figure 1Increased activation of the inferior frontal junction (IFJ) for baby targets that were morphed towards higher BS > adult targets in the comparison of the OT and placebo group (MNI coordinates X, Y, Z [t-value]: −33, 5, 49 [4.62]). Activation is displayed in MNI space on an axial slice. Parameter estimates were extracted from spheres at the local maximum with a radius of 3 mm. They were significantly different between the OT and the placebo group for the high BS condition.
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Increased activation of the inferior frontal junction (IFJ) for baby targets that were morphed towards higher BS > adult targets in the comparison of the OT and placebo group (MNI coordinates X, Y, Z [t-value]: −33, 5, 49 [4.62]). Activation is displayed in MNI space on an axial slice. Parameter estimates were extracted from spheres at the local maximum with a radius of 3 mm. They were significantly different between the OT and the placebo group for the high BS condition.
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The SPM multiple regression analysis was used to analyze the influence of the degree of selective attention to babies on brain activation. For this, we used the mean Delta RT of all baby conditions from each subject and correlated them with the individual activation in the contrast of “baby vs. adult target”, separately for OT- and for placebo-treated participants. We found increased activation in the right putamen of OT-treated women (MNI-coordinates [t-value; pFWEcorr] 30, −1, 13 [5.65; 0.052]; Fig. 2a) that was positively related to selective attention to babies, but barely missed the FWE-corrected threshold in the whole-brain analysis. Yet, when extracting the parameter estimates from the right putamen, a significant positive correlation with selective attention to babies emerged (Fig. 2a). We also found a positive correlation with activation in the left putamen at the statistical threshold of p < 0.001, uncorrected (MNI-coordinates [t-value] −30, 14, 10 [4.85]; Fig. 2a). In the placebo group, two homologous clusters emerged in the posterior temporal cortex of which one survived the FWE correction on cluster level and one barely missed the statistical threshold (MNI-coordinates [t-value; pFWEcorr] 48, −22, −2 [5.69; 0.067]; MNI-coordinates [t-value; pFWEcorr] −66, −49, −2 [4.99; < 0.001]).Figure 2Degree of selective attention to babies for OT treated participants (in blue) or placebo treated participants (in red) with high or low T concentrations. (a) Activation in the left and the right putamen for baby relative to adult targets positively correlated with selective attention to babies after OT treatment (N = 29). Parameter estimates from local activation maxima (L:–21 14–11; R: 30–1 13; spheres with 6 mm radius) for the right and the left putamen are also displayed for illustration purposes. (b) Positive correlation of activation in the left and right putamen with selective attention to babies for OT treated women with high endogenous T concentrations (N = 16). (c) OT treated women with low endogenous T concentrations (N = 13) did not show activation of the putamen in relation to the relative RTs (i.e., selective attention to babies).
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Degree of selective attention to babies for OT treated participants (in blue) or placebo treated participants (in red) with high or low T concentrations. (a) Activation in the left and the right putamen for baby relative to adult targets positively correlated with selective attention to babies after OT treatment (N = 29). Parameter estimates from local activation maxima (L:–21 14–11; R: 30–1 13; spheres with 6 mm radius) for the right and the left putamen are also displayed for illustration purposes. (b) Positive correlation of activation in the left and right putamen with selective attention to babies for OT treated women with high endogenous T concentrations (N = 16). (c) OT treated women with low endogenous T concentrations (N = 13) did not show activation of the putamen in relation to the relative RTs (i.e., selective attention to babies).
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Following the behavioral results, we subdivided the sample in women with either low or high endogenous T concentrations. OT-treated women with high endogenous T showed significantly enhanced activation in the left and right putamen in the contrast of “baby vs. adult target” in association with the individual Delta-RTs as an indicator of enhanced selective attention to babies (i.e., a positive correlation). The positive correlation with the left putamen also surpassed the statistical criterion (MNI-coordinates [t-value; pFWEcorr] −21, 14, −11 [5.97; 0.005]; Fig. 2b), while the one with the right putamen was significant at p < 0.001, uncorrected (MNI-coordinates [t-value] 30, −1, 13 [5.33]; Fig. 2b). OT-treated participants with low endogenous T and participants of the placebo group did not show this positive correlation between activation in the putamen and selective attention to babies. The brain-behavior correlations based on the parameter estimates extracted from the left and the right putamen supported these results (Fig. 2c; Table S3).
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The current study investigated the influence of exogenous OT and its interaction with endogenous T on brain activity during processing of infant faces, which varied in the intensity of BS. We found a positive effect of drug treatment in the direct comparison of OT versus placebo using the contrast of “baby versus adult target” in the IFJ, but only in the high BS condition. This finding supports the idea that OT may enhance attention to increased BS and may further promote one’s motivation to act. Additionally, we found a positive correlation between selective attention to babies, as indicated by increasing Delta-RTs, and increased activation in the left putamen following OT treatment. Interestingly, this correlation was specific for women with high endogenous T. Collectively, these findings may provide a hint to the neural mechanism by which OT may support the sensitivity for the BS and may counteract the negative effects of T in the modulation of nurturing behavior.
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Based on our previous study6 we expected that OT administration would modulate attention to infant faces especially in women with high endogenous T. In line with this hypothesis, we found an interaction between the degree of BS, the treatment group and endogenous T level. OT-treated women with high endogenous T showed an attentional bias towards babies (positive Delta-RTs) compared to OT-treated women with low endogenous T (negative Delta-RTs), but only for the natural BS and this result remained on statistical trend level. We could not find further differences in the attention to babies between high and low endogenous T concentrations in the OT group, but the Delta RTs of the conditions with lower and higher BS were positive in both T groups. We suppose that adult pictures could also represent salient stimuli for nulliparous women in reproductive age, which may have rendered any behavioral effects rather small (see8 for review). Yet, the presence of a statistical trend in the OT-treated women with high endogenous T may conform to the idea that a system influenced by high levels of T may be more receptive to OT treatment, because in the female brain T could be directly converted into estradiol and thus modulate OT receptor density, which has been demonstrated in rats29.
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We found a positive effect of treatment (OT > placebo) on processing of targets with higher BS in the left IFJ. The IFJ is located in the frontolateral cortex between the premotor and the prefrontal cortex and has been implicated in cognitive control30,31. Recent research further demonstrated its specific involvement in the control of selective visual attention32, action perception33 and the detection of behaviorally salient cues34. In addition, the IFJ has been associated with the understanding of action35, which may be considered as one basis of social cognition and empathy. Previous studies also detected increased activation during processing of infant faces in areas near our IFJ cluster. Caria and colleagues36 located activation in the precentral cortex (BA 6; MNI coordinates: 36–6 45) in the contrast of “unknown infant > adult faces”. A study on synchrony and specificity between the maternal and paternal brain found activation in the inferior frontal gyrus (IFG) for both sexes in response to own-infant-parent-interaction videos37. Finally, the IFG has also been related to OT- induced emotional empathy38. In our study, the activation of the IFJ was limited to babies with a high BS. Previous observations indicating that babies with stronger BS were treated preferentially39 and perceived as cuter40,41 provide some space for speculation, that the stronger BS could provoke an increased motivation to act. The findings of Glocker et al.10 who also found activation in the precentral gyrus (MNI coordinates: −42 0 29; −46 6 33; −51 8 36) across the three BS conditions, which was located near our cluster, could also provide indications for this speculation. Yet, it is difficult to distinguish between participants who searched for the target or avoided the distractors, which makes it difficult to interpret the difference between the OT group (positive parameter estimates) and the placebo group (negative parameter estimates). This would be of great interest and should be taken into account in a future study.
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Additionally, we observed a positive correlation between activity in the left putamen and increased selective attention to babies in OT-treated women. The putamen belongs to the mesolimbic reward system and is part of the striatum. Equivalent to many other studies the detected activation appeared only in participants that were treated with OT15. We suppose that this finding supports the idea that OT increases the reward value of babies, probably as an adaptation to motivate caretaking behavior through mesolimbic-reward pathways37. This assessment is also supported by previous reports that have demonstrated OT-dependent activations in areas of the reward system as consequence of listening to crying babies (amygdala and insula42), to laughing babies (amygdala43) and processing of child pictures (globus pallidus44). Yet, the OT-related response in the putamen did not parametrically change with the amount of BS inherent to the presented pictures. This indicates a rather general mechanism that may ensure reflexive attention to babies regardless of their cuteness.
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Even more importantly, the positive correlation between increased selective attention to babies and activation in the left putamen only remained significant in the high endogenous T group after OT administration. Only a few behavioral studies have so far examined the interactions between OT and T in women, and, to the best of our knowledge, our study is the first that used functional neuroimaging to assess the underlying neural mechanism. Here we used the T sample that was collected directly before administration to capture the current T state of the participant and not the habitual concentration, because we could not control for daytime fluctuations. This method also allows the comparability with previous research26,45,46. The single T sample collection before administration provided the opportunity to analyze the real-time T concentration at the measuring time. But, for the same reasons, we cannot exclude further T fluctuations that might have occurred during scanning, which may have weakened the stability of this single measurement and might therefore constitute a potential limitation of this study. However, the T concentration before administration and post-test concentration after measurement were highly correlated (r = 0.447; p < 0.001). This may suggest that T concentration may have been relatively stable during the actual scanning period.
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| 100.0 |
The present study shows that the reward system may be involved in this attentional bias towards babies. Little is known about the precise interaction of OT and T in the brain. In the female brain the majority of T may be converted to estradiol via aromatase47, and estradiol can increase the number of OT receptors in the brain29. Yet, the observation of higher systemic T concentrations in women may indicate an impaired ability to convert it to estradiol, thus either leaving a higher amount of T in the circuitry that may counteract maternal behavior through an unknown mechanism or by leaving the number of OT receptors unaffected by reduced influence of estradiol. In a similar vein one may speculate that high OT concentrations may serve as a compensatory mechanism for high T concentrations, we presume that women with low T concentrations were less sensitive to OT administration, because low T concentrations should support nurturing behavior per se6. This would indeed explain why we could not find any activation in the brain reward system in women with low endogenous T after OT administration. But since babies nevertheless remain a strong social cue, this does not explain why neither in the low T nor in the placebo group the baby pictures were rewarding by themselves. We suppose that the young adult stimuli may also be socially salient for young participants, for instance as potential mates, and therefore could be equally rewarding and evoke similar activation in the brain reward system48. Our experimental design does not allow the isolation of the response to adult or infant faces, since pictures were always presented in combination, but only enables the identification of activational differences during processing of these stimuli. So we cannot answer this question. However, for reasons discussed before we presume that the brain of women with a high endogenous T concentration may be more sensitive to OT administration than women with a low endogenous T. One could therefore speculate that same age adult stimuli as well as infant pictures both constitute a strong social stimulus, but OT could function as an enhancer of reproductive behavior and direct attention towards infant stimuli in women with high T level, who may be more sensitive to OT. In this study, we focused on nulliparous women that reacted to unfamiliar infant or adult stimuli to investigate the neuroendocrine basics of the key stimulus in general. As oxytocin is especially known to influence attachment behavior15 it could be of great interest to use the same paradigm with familiar infants in further research. The results of this study are therefore limited to the general mechanism of the key stimulus BS and are not necessarily transferable to attachment behavior in mothers or other caretakers. Gordon et al.26 found a positive interaction between increased T levels and OT on maternal touch in mothers, while the effect in fathers was in the opposite direction. Their results may indicate that the interaction between T and OT may be sexually dimorphic. Therefore, our results on nulliparous women may resemble those in mothers but it is possible that they deviate to the results in fathers and men who have never sired a child. Although the findings of Gordon and colleagues26 were focused on parents, we assume that the results in mothers support our previous findings that OT seemed to promote adult-infant discrimination and increased attention to baby pictures, but only in women with high endogenous T6. OT is a peptide hormone that is critically involved in mother-infant attachment49. Especially lactating mothers show increased OT levels50. The artificial increase through intranasal administration could be closely related to the concentration found in new mothers (see Weisman et al.51 for the active drug effects reflected in saliva). Although there already exists excellent research about the active drug effects and reflection in saliva49–51, a direct comparison of mothers and non-mothers salivary OT concentration with and without OT administration would be of great interest. But yet, aside from oxytocin, postpartum hormonal changes have large transitions which makes the results of nulliparous women not necessarily comparable with mothers52,53.
|
study
| 99.94 |
The detected activations in the putamen correspond well with the literature and accentuate the previous research on oxytocin and maternal behavior8,9,54. Since we know very little about the interactions of T and OT in the female brain, we abstained from using an a priori region-of-interest approach and analyzed our data on whole brain level. However, findings in the IFJ are not common in the context of OT administration studies and in an a priori region-of-interest approach the IFJ would have probably not been included. This might limit the interpretation of this finding, although the activation of the IFJ survived the correction for multiple testing on a whole-brain basis. Future studies will therefore be necessary to show if this finding can be replicated in this context.
|
study
| 100.0 |
All of the participating women used hormonal contraceptives and were tested in the pill-phase. This was done to control for a potential pregnancy and to prevent cyclic changes in steroid hormone levels. But it’s important to note that previous research showed that the intake of oral contraceptives increased performance in affective responsiveness and that affective responsiveness was positively influenced by oral contraceptives (pill-intake phase versus pill-free week)55. Further, testosterone in females on oral contraceptives may be downregulated56. Therefore, using participants on hormonal contraceptives for an OT-intervention study may not represent the ideal model to detect OT sensitivity, that may be modulated by T. Yet, ethical concerns (increased pregnancy risk in women with a natural cycle), the fact that T level fluctuates across the natural cycle (e.g., rises during the first half), as well as our previous results in women on oral contraceptives6, led to our decision to test only women who received hormonal contraception in this between-subjects fMRI design. As a future direction, it would be of great interest to further examine normal cycling women and the influence of the intake of oral contraceptives on the recent results.
|
study
| 100.0 |
Collectively, the present findings support the idea of an adaptive hormonal mechanism that promotes selective attention to babies. Here, we demonstrate that an increased BS was associated with enhanced activation of the IFJ after OT treatment, a brain region implicated in cognitive control and the motivation to act. We also found that OT may modulate selective attention to babies through the recruitment of the reward system. We found a positive correlation between activity in the putamen and attention to baby faces. However, after accounting for endogenous T level, this correlation only remained significant in women with a high T concentration. These results are consistent with our previous findings6 and may indicate that OT possibly compensates high T concentrations and the reduced attention to infant stimuli through an enhancement of the reward value of babies.
|
study
| 100.0 |
Sixty nulliparous female university students (mean age ± SD = 24.63 ± 3.08 years) participated in the study. All participants were right handed, healthy, and were not taking any medication except from hormonal contraceptives to prevent cyclic changes in T level. They also performed a pregnancy test on the test day and met the criteria to participate in an fMRI study.
|
study
| 100.0 |
Administration of OT was placebo-controlled and double-blind (see Fig. 3 for experimental schedule). Participants brought three saliva samples from home sampling and provided two further saliva samples at the test place (one before administration and one after measurement). In the scanner participants performed two versions of a target detection paradigm (see also Supplementary Material and Methods).Figure 3Time schedule of the experimental procedure. 60 participants were invited for testing. 57 participants could be included into the analysis (29 of the OT group; 28 of the placebo group). 20 of the participants performed the RMET test before the fMRI measurement and 37 participants performed the RMET test after the fMRI measurement.
|
study
| 100.0 |
Time schedule of the experimental procedure. 60 participants were invited for testing. 57 participants could be included into the analysis (29 of the OT group; 28 of the placebo group). 20 of the participants performed the RMET test before the fMRI measurement and 37 participants performed the RMET test after the fMRI measurement.
|
study
| 99.94 |
The target detection paradigm was based on the odd-one-out principle (Fig. 4 for schematic illustration). In the first task subjects were asked to identify the odd-one-out on a display of four human faces, which was either an adult face (target) out of three infant faces (distractors) or an infant target out of three adult distractors, as fast and accurate as possible. The BS of the infant faces was parametrically manipulated (see also6 for the procedure). The same odd-one-out procedure was repeated in the baseline task, in which task geometric shapes (triangles, squares, or circles) were used (Supplementary Material and Methods for detailed information). The first paradigm was 16.4 minutes long and the baseline task was 6.4 minutes long.Figure 4Illustration of the target detection paradigm with example images. The three picture of babies stand for the distractors here (photographs of either 3 adult faces or 3 infant faces were shown; the 3 infant faces had the same amount of BS [higher, unmanipulated or lower BS]). The adult picture on the left stand for target here, which could be either an adult or infant face of higher, unmanipulated or lower BS. The task was to identify the target that did not fit to the other pictures via button press.
|
study
| 100.0 |
Illustration of the target detection paradigm with example images. The three picture of babies stand for the distractors here (photographs of either 3 adult faces or 3 infant faces were shown; the 3 infant faces had the same amount of BS [higher, unmanipulated or lower BS]). The adult picture on the left stand for target here, which could be either an adult or infant face of higher, unmanipulated or lower BS. The task was to identify the target that did not fit to the other pictures via button press.
|
other
| 99.7 |
The salivary T samples were collected on the test day and analyzed in our in-house laboratory with a T luminescence immunoassay from IBL International (TECAN group global; Hamburg, Germany) (see Supplementary Material and Methods for detailed information).
|
study
| 99.9 |
OT was administered double-blind and placebo controlled in a between-subjects design. The participants self-administered 3 puffs in each nostril alternately of the unlabeled nasal spray. The amount of OT corresponded to 24 IU. The placebo spray consisted of chlorobutanol-hemihydrat (0.5%) with no active treatment. To ensure treatment efficiency57, exposure time was 45 min until the fMRI measurement began.
|
other
| 50.94 |
IBM SPSS statistics 19 was used to analyze the behavioral data. To represent selective attention to babies of different BS conditions, the relative reaction times (Deltas-RTs) were calculated by subtracting the low distracting condition (i.e., infant target and three adult distractors) from the high distracting condition (i.e., adult target and three baby faces as distractors) (see also6). For a subject with an increased sensitivity for the BS, we predicted higher Delta-RTs, resulting from the combined effect of increased distraction by infant faces in the adult target condition and more rapid selection of the infant target in the first condition.
|
study
| 100.0 |
Following the procedures used previously26,46,58,our analysis of T content focused on the one saliva sample that was collected directly before treatment. Based on the endogenous T concentration, we calculated the median to separate the participants in two groups of either high or low endogenous T concentration (see6 for a similar procedure).
|
study
| 100.0 |
We used a repeated measures ANOVA and post hoc t-tests to assess the effects of the factors ‘morph type’ (low BS, unmanipulated BS, high BS), ’treatment group’ (OT or placebo) and ‘endogenous T concentration’ (high or low T) on the Delta-RTs. Bonferroni correction yielded a corrected statistical threshold of p ≤ 0.0166 and a corresponding statistical trend level of p ≤ 0.033.
|
study
| 100.0 |
For analysis of the baseline task with geometrical shapes we performed a repeated measures ANOVA with the factors ‘target figure’ (triangles, squares, or circles), ‘distractor figures’ (triangles, squares, or circles) and ‘treatment group’ (OT or placebo) on the RTs.
|
study
| 100.0 |
Statistical effects are considered significant at p < 0.05 (two-tailed), if not otherwise indicated. If the sphericity assumption was not met in the ANOVA, we report the Greenhouse-Geisser corrected values. Since all data followed the assumed normal distribution, we used post-hoc t-tests.
|
study
| 99.94 |
On the first level, we used a general linear model (GLM) for statistical analysis of event-related activity. A vector with the temporal onsets of the experimental conditions was convolved with a canonical hemodynamic response function (hrf) to produce the predicted hemodynamic response to each experimental condition. The conditions ‘target’ (adult or infant), ‘morph type’ of the baby (higher, unmanipulated or lower BS), ‘gender’ (female or male) and the baseline task with the geometric figures were modeled as regressors. Linear t-contrasts were defined for assessing the specific effects of the varying amount of BS.
|
study
| 100.0 |
On the second level, a random effects analysis was performed. For this we used a full factorial 3 by 2 repeated-measures analysis of variance (ANOVA) that included the within-subject factor ‘morph type’ (3 steps) and the between-subject factor ‘treatment group’ (2 steps). T-tests tested for specific differences between conditions.
|
study
| 100.0 |
We also assessed whole-brain correlations for analysis of the association between treatment-related activations and selective attention to babies (Delta RTs). For this purpose we used the multiple regression routine implemented in SPM8. For the display of these behavior-brain correlations individual parameter estimates were extracted with marsbar-0.44.
|
study
| 100.0 |
Postoperative delirium (POD) is a very common complication in operative disciplines, especially in those elderly patients after cardiac surgery . The described prevalence of POD varies between 30 and 80% in elderly patients after cardiac surgeries [2, 3] and 15%–53% in elderly surgical patients . Numerous studies have revealed that POD is significantly associated with increased complication incidence, long-term cognitive impairment, prolonged hospital length of stay, elevated costs, and overall mortality [5–7]. To predict the implications of POD and improve the quality of care, attempting to determine independent risk factors for POD is of great importance. A number of previous studies have been performed regarding predicative factors for POD; however no consensus has been made until now probably due to the complicated pathogenesis of POD . Previous studies have reported that delirium is associated with elevated proinflammatory cytokines and proteins involved in the stress response in medical or surgical patients. C-reactive protein (CRP), one of the most common markers for systemic inflammation, has been indicated as independent risk factor for delirium following vascular surgery and hip surgery . However, the relationship between CRP and POD in patients undergoing laparoscopic surgery for colon carcinoma still remains relatively unknown, which was just the objective of this present study.
|
study
| 99.7 |
This present study protocol was approved by the Medical Institutional Ethics Committee of Jiangsu province and Taizhou People's Hospital. Those elderly patients (aged ≥ 65 years) scheduled to undergo selective laparoscopic surgery for colon carcinoma in Taizhou People's Hospital from April, 2014, to January, 2017, were prospectively recruited in this present study. All the participants were required to offer the signed informed consent. Exclusion criteria were described as follows: (1) with major depression; (2) with preexisting or a history of dementia delirium; (3) with cognitive impairment which was defined with a Modified Mini-Mental State Examination (MMSE) score < 24; (4) with clinically neurologic disorder or psychosis. 182 eligible patients were included into our study; 22 of them were excluded for varied reasons (informed consent refusal, missing information, etc.). In total, 160 elderly patients undergoing laparoscopic surgery for colon carcinoma were included into the final analysis, which was shown in the patient CONSORT (Figure 1).
|
study
| 100.0 |
Demographic and medical characteristics (including age, gender, and education) were evaluated. The modified Charlson's Comorbidity Index (MCCI) was utilized for the medical comorbidities assessment by summing points . POD was evaluated using the Confusion Assessment Method-Intensive Care Unit (CAM-ICU) by calculating CAM scores . POD assessment was performed once a day (in the evening) for the first 3 days and at 7th day after surgery, respectively. A positive POD diagnosis was given when patients had a positive result at least for once within 1 week of the assessment. The intraoperative variables (operation time, anesthesia time, blood loss, etc.), postoperative complications (wound infection, urinary tract infection, pulmonary infection, etc.), and postoperative adverse cardiovascular events (such as myocardial infarction, arrhythmias, and heart failure) were also detailed, recorded, and analyzed.
|
study
| 100.0 |
To avoid the interferential impacts by anesthesia, all the enrolled patients underwent the operation under general anesthesia by the same anesthesia team. With no premedication, intravenous midazolam, propofol, sufentanil, and rocuronium were used for inducing anesthesia. Anesthesia was maintained with sevoflurane, propofol, remifentanil, and dexmedetomidine. The serial blood collection preoperatively and on postoperative days 1, 2, and 3 was conducted from all enrolled participants. Blood samples were stored on ice in heparinized tubes and immediately centrifuged (1500g at 4°C for 15 minutes). The separated plasma samples from cellular material were then stored at −80°C until assayed. CRP concentrations were measured by using human enzyme linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA). The ELISA was carried out in accordance with the manufacturers' instructions by the same laboratory assistant who was completely blinded to this study.
|
study
| 100.0 |
The data analysis was performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Categorical data were expressed as number (with percentage, n%) and compared with Chi-square test or Fisher exact test. Continuous data were presented as mean levels (with standard deviation) or median (with interquartile range) and compared via the Mann–Whitney U-test or Student's t-test appropriately. The univariate and multiple logistic regression analyses were plotted to evaluate the predicative validity of pre-, intra-, or postoperative variables for POD. All statistical tests were bilateral probability and P < 0.05 was considered significant.
|
study
| 99.94 |
The demographic and clinical characteristics of the enrolled elderly patients were exhibited in Table 1 in detail. 39 of the 160 patients had suffered POD with a POD incidence of 24.4%, which was similar to other previous studies . The mean age of all the enrolled patients was 70.1 years, and significant difference in age between the patients with or without POD was found. The preoperative MCCI score was significantly higher in patients who suffered POD when compared with those without POD. In addition, the preoperative MMSE score and percentage of heavy drinkers were significantly higher in the delirious group than the nondelirious group. No statistically significant differences were found in the gender, education, body mass index, smoking habits, and ASA physical status between the patients with POD or not.
|
study
| 100.0 |
A significant difference exists between the patients in delirious group or nondelirious group with respect to postoperative incision infection. The incidence of pulmonary infection or cardiovascular events after the surgery seemed significantly associated with the occurrence of POD. When we compared other postoperative complications, no significant differences were observed between groups with respect to the prevalence of urinary tract infection, bowel obstruction, anastomotic leakage, and postoperative bleeding.
|
study
| 100.0 |
The postoperative CRP concentrations were many times as high as preoperative levels, which indicated a strong effect of operation on CRP concentrations. The results also revealed that patients with POD showed a significantly higher CRP concentration preoperatively and on postoperative day 2 than those without POD.
|
study
| 100.0 |
As shown in Table 2, all potential predicative factors mentioned above were summarized by univariate analysis. The results from the univariate logistic regression analysis indicated that age, MCCI, preoperative MMSE score, postoperative cardiovascular events, and CRP concentrations preoperatively or on postoperative day 2 were associated with POD. With these six variables introduced into the final multivariate analysis, the results suggested preoperative CRP concentrations as the independent predicator for POD in patients undergoing laparoscopic surgery for colon carcinoma (OR: 5.87; 95% CI: 2.22–11.4; P = 0.018).
|
study
| 100.0 |
To our knowledge, this current study demonstrated that plasma CRP concentrations emerged as an independent factor for POD in the elderly patients undergoing laparoscopic surgery for colon carcinoma for the first time. Recently published studies have indicated a positive correlation between POD and early mortality after surgery, which emphasizes the importance of POD prediction.
|
study
| 100.0 |
Previous studies have revealed age as a well-established predictor for POD with no certain mechanisms [16, 17]. In this present study, the patients who underwent POD also had a higher age than those without POD. However, the final multivariate analysis did not indicate age as a predicator as expected, which might be explained by the small age range or different operation types of the enrolled participants. Preoperative cognitive function decline is considered as a risk factor for postoperative cognitive problems and later life equality . Our results from univariate analysis instead of multivariate analysis also showed a close association between preoperative MMSE score and POD. Previous studies have also indicated that longer operation and anesthesia time predict delirium after cardiac surgery due to increased cytokines release and operation complexity , which was not quite in accordance with our results.
|
study
| 99.94 |
Our results showed that patients with POD had higher CRP concentrations preoperatively and on postoperative day 2. However, the configured multivariate logistic regression model suggested plasma CRP concentrations as a predictor of POD preoperatively instead of postoperative day 2. Therefore, preoperative plasma CRP concentrations may be an important risk biomarker for POD prediction in elderly patients with colon carcinoma after laparoscopic surgery. The pathophysiologic preexisted differences in the inflammatory activity during patients before the surgery might lead to different incidences of POD. This might support our findings from a pathophysiologic standpoint and offer new important targets for investigation. However, the association between preoperative CRP concentrations and POD still remains controversial until now with different conclusions in different patient samples. Previous literature examining the relationship between POD and CRP has suggested the potential predicative role of preoperative and postoperative CRP concentrations for POD in older patients undergoing major elective surgery . Other reports conducted in small samples undergoing hip and vascular surgery showed positive associations between postoperative CRP concentrations and POD [21, 22], which is not so aligned with our results. In contrast to our findings, another two studies conducted in small cohorts observed no significant association between POD and preoperative CRP concentrations [12, 23]. No close correlations were observed in critically ill medical patients . Different small sample sizes of cohort-based studies, different age ranges, different surgery types, and some other confounding factors may be potential explanations for the disparate conclusions between other previous reports and our study. Those individuals with a heightened inflammatory response are at greater risk of POD occurrence as proposed by current POD pathophysiology models . With no well-defined etiology of multifactorial POD, the hypothesis of inflammatory processes leading to neuroinflammation has gained wide attraction in recent years .
|
study
| 98.56 |
This study has some limitations. First, this study is conducted in a single-center and has a relatively small sample size in comparison with other multicenter researches. Second, the group, age range, disease diagnosis, and operation types were all relatively specific. Furthermore, the inclusion criteria of this study were not so strict and some comorbidities (such as arthritis, infections, and inflammatory diseases) might affect the results. Last, why the involved mechanisms preoperative CRP concentrations can serve as a predicator for POD still remains unclear.
|
study
| 100.0 |
In conclusion, this present study highlighted the predictive role of preoperative CRP concentrations for POD in elderly patients undergoing laparoscopic surgery for colon carcinoma. Our evidence suggested its potential role for risk stratification before the surgery from a clinical point. More intensive assessments and preventive interventions could be recommended in those patients with high risk.
|
study
| 99.94 |
Norbormide (NRB, see Figure 1A for structure) is a unique vasoactive compound endowed with species-specific vasoconstrictor activity that targets the peripheral blood vessels of the rat (Clarke, 1965; Roszkowski, 1965; Bova et al., 1996). It also provokes a vasorelaxant effect on arteries of several non-rat species, as well as on rat large-caliber vessels (Roszkowski, 1965; Poos et al., 1966; Cavalli et al., 2004). NRB is a mixture of up to eight stereoisomers, four endo-isomers and four exo-isomers; among them, only the endo-isomers demonstrate vasocontractile and rat toxicant activities (Poos et al., 1966), whereas both endo- and exo-isomers have vasodilatatory activity in rat aorta and in non-rat arteries (Cavalli et al., 2004). The contractile effect of NRB on rat peripheral blood vessels is endothelium-independent, and is of myogenic nature as confirmed by its action on single rat caudal artery myocytes (Fusi et al., 2002; Cavalli et al., 2004). NRB binding sites and the molecular mechanisms involved in its biological effects in vascular tissue have yet to be fully characterized; it has been proposed that NRB-induced vasoconstriction may result from an interaction with a phospholipase C-coupled receptor (Bova et al., 2001), whereas its relaxant effect may be caused by an inhibitory action on plasma membrane voltage-dependent L-type Ca2+ channels (Fusi et al., 2002). NRB has also been shown to activate the mitochondrial permeability transition pore (mPTP) in isolated rat mitochondria, but not in mitochondria obtained from either guinea-pigs or mice. This suggests that the effect on the mPTP, like NRB-vasoconstriction, is also species-specific. However, since this effect could also be induced in mitochondria isolated from different organs of the rat (Ricchelli et al., 2005; Zulian et al., 2007, 2011), unlike vasoconstriction, mPTP activation is not tissue-specific. It is therefore unclear whether NRB-induced activation of the mPTP constitutes a role in the vasoconstrictor effect of NRB.
|
study
| 85.1 |
Chemical structures of (A) Norbormide and (B) its fluorescent derivative NRB-AF12. (C) Live cell imaging of LX2 hepatic stellate cells fluorescently labeled with 500 ηM NRB-AF12 for 30 min. Magnification 40x; scale bar 20 μm. (D) LX2 cells stained with NRB-AF12 were fixed and imaged by confocal microscopy. Magnification 60x; scale bar 20 μm.
|
other
| 91.44 |
Correspondingly, addressing the localization of NRB could help characterize the molecular mechanisms underlying the species- and tissue-selective actions of NRB; the original primary objective of this study was therefore to develop a fluorescent derivative of NRB (NRB-AF12, see Figure 1B for structure) in order to compare its subcellular distribution in NRB-sensitive and NRB-insensitive cells. However, since preliminary results indicated a strong overlapping fluorescent distribution between NRB-AF12 and that of ER-Tracker® (ER-Tr), a commercially available fluorescent probe widely used as a selective marker for endoplasmic reticulum (ER), we extended our goals to include a comparison of the staining profiles of the two dyes. The results indicate that NRB-AF12 and ER-Tr show a near identical pattern of fluorescence distribution in both NRB-sensitive and NRB-insensitive cells, and that this fluorescence profile differs between the two cell types. Furthermore, we found that both dyes label not only ER (as would be expected with ER-Tr), but also Golgi apparatus and mitochondria. In addition, unlike ER-Tr, NRB-AF12 was also able to label lysosomes and endosomes.
|
study
| 100.0 |
To a solution of endo-NRB (1.0 g, 2.0 mmol) in DMF (6 ml) was added NaH (160 mg, 4.0 mmol, 60% w/w in oil), and the resulting mixture stirred at room temperature for 15 min, then at 80°C for a further 15 min A solution of 4-[(tert-butoxycarbonyl)amino]butyl methanesulfonate (Cao et al., 2016) (0.59 g, 2.2 mmol) in DMF (2 ml) was added, and the resulting mixture stirred at 100°C for 4 h. The reaction mixture was then allowed to cool, diluted with water and extracted with EtOAc. The separated organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and the solvent removed in vacuo. Purification by column chromatography (hexane/EtOAc, 1:1) afforded N-4′-[(tert-butoxycarbonyl)amino] butyl-5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide as an endo-exclusive mixture of four stereoisomers (white solid; 1.09 g, 1.60 mmol, 80%). 1H NMR (400 MHz, CDCl3) δ 1.30-1.68 (13H, m, C(CH3)3 and NCH2CH2CH2CH2NH), 3.05-3.20 (2H, m, NCH2CH2CH2CH2NH), 3.24-3.70 (4.2H, m, H-2, H-3, W/H-4 and NCH2CH2CH2CH2NH), 3.85–3.90 (0.4H, m, U/H-1 and V/H-1), 3.91-3.95 (0.4H, m, Y/H-4), 4.10–4.13 (0.1H, m, U/H-4), 4.26–4.30 (0.3H, m, V/H-4), 4.42–4.48 (0.6H, m, W/H-1 and Y/H-1), 5.55–5.67 (1.7H, m, OH and V/H-6 and Y/H-6), 6.02–6.04 (0.1H, m, U/H-6), 6.05–6.07 (0.2H, m, W/H-6), 6.70–7.62 (16H, m, ArH), 8.66–8.39 (2H, m, αPyr). To a solution of N-4′-[(tert-butoxycarbonyl)amino]butyl-5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide (0.34 g, 0.5 mmol) in DCM (10 ml) was added TFA (2 ml), and the mixture stirred at room temperature for 3 h. The solvent was then removed in vacuo to afford N-4′-aminobutyl-5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide trifluoroacetate as an endo-exclusive mixture of four stereoisomers [colorless gum (quant.)], which was used without further purification. To a solution of N-4′-aminobutyl-5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide trifluoroacetate (100 mg, 0.14 mmol) in DCM (4 ml) was added DIPEA (73 μl, 0.42 mmol), and the mixture stirred at room temperature for 30 min. NBD-Cl (4-chloro-7-nitrobenzofurazan) (28 mg, 0.14 mmol) was added and the resulting mixture stirred at room temperature for a further 6 h. The mixture was then diluted with DCM, washed with saturated aq. NaHCO3, then brine, and the separated organic phase dried over anhydrous Na2SO4, filtered and the solvent removed in vacuo. Purification by column chromatography (DCM) afforded 5-(α-hydroxy-α-2-pyridylbenzyl)-N-4′-[(7″-nitro-2″,1″,3″-benzoxadiazol-4”-yl)amino]butyl-7- (α-2- pyridylbenzylidene)-5-norbornene-2,3-dicarboximide (NRB-AF12) as an endo-exclusive mixture of four stereoisomers (orange solid; 50 mg, 0.067 mmol, 48%). M.p. 108-112°C; 1H NMR (400 MHz, CDCl3/MeOD 50:1 v/v) δ 1.61–1.84 (4H, m, NCH2CH2CH2CH2N), 3.34–3.73 (6.2H, m, NCH2CH2CH2CH2N, H-2, H-3 and W/H-4), 3.89–3.98 (0.7H, m, V/H-1 and Y/H-4), 3.99–4.02 (0.1H, m, U/H-1), 4.18–4.24 (0.4H, m, V/H-4 and U/H-4), 4.28–4.38 (0.6H, m, Y/H-1 and W/H-1), 5.56–5.62 (0.7H, m, V/H-6 and Y/H-6), 6.07–6.09 (0.1H, m, U/H-6), 6.12–6.14 (0.2H, m, W/H-6), 6.14–6.24 (1H, m, H-5″), 6.79–7.75 (16H, m, ArH), 8.37–8.65 (3H, m, αPyr and H-6″).
|
study
| 100.0 |
LX2 (a human immortalized hepatic stellate cell line), HSC-T6 (a rat immortalized hepatic stellate cell line), and HeLa (a human cervical carcinoma cell line) cells were cultured at 37°C in an atmosphere of 5% CO2 in Dulbecco's Modified Eagle Medium (Euroclone - Milan, Italy) supplemented with 10% Fetal Bovine Serum (Euroclone), 2 mM L-Glutamine and antibiotics. HepG2 cells (a human hepatocarcinoma cell line) were cultured at 37°C in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (Euroclone) containing 10% Fetal Bovine Serum, 2 mM L-Glutamine and antibiotics.
|
study
| 99.94 |
Smooth muscle cells were freshly isolated from the rat tail main artery under the following conditions: a 5-mm long piece of artery was incubated at 37°C for 40–45 min in 2 ml of 0.1 mM Ca2+ external solution containing 20 mM taurine, 1.35 mg/ml collagenase (type XI), 1 mg/ml soybean trypsin inhibitor, and 1 mg/ml BSA, which was lightly bubbled with a 95% O2–5% CO2 gas mixture to gently agitate the enzyme solution. Cells were stored in 0.05 mM Ca2+ external solution containing 20 mM taurine and 0.5 mg/ml BSA at 4°C under normal atmosphere, and were used for experiments within 2 days following isolation (Fusi et al., 2016).
|
study
| 100.0 |
All cell lines tested were stained immediately before imaging. Briefly, cells were trypsinized, counted and seeded in glass bottom μ-Dishes (Ibidi - Munich, Germany) at 2 × 104 cells/dish, and incubated for 24 h at 37°C in an atmosphere of 5% CO2. Cells were rinsed with Dulbecco's Modified Eagle Medium without phenol red (Euroclone) and loaded with 500 ηM NRB-AF12, 500 ηM ER-Tr red or green (#E34250 and #E34251; Thermo Fisher), and 500 ηM pHrodo red (#P10361; Thermo Fisher) at 10 μg/ml. In another experiment, LX2 cells were seeded in glass bottom μ-Dishes at 2 × 104 cells/dish and incubated for 24 h at 37°C in an atmosphere of 5% CO2. Cells were transfected with either 500 ηg of MitoDS-red or KDEL-red vector using Lipofectamine LTX Reagent (Invitrogen), according to the manufacturer specifications. At 24-h post-transfection, cells were stained with either 500 ηM NRB-AF12 or 500 ηM ER-Tr green. Cells were mounted in a Okolab microscope stage heated chamber warmed to 37°C. All images were collected with a Yokogawa CSU-X1 spinning disk confocal on a Nikon Ti-E inverted microscope equipped with a Plan Apo 60x NA 1.4 objective. Probes were excited with a 488 and 561 nm Lasers. To separate the individual emissions a dichroic filter 405/488/561/635-25 (Semrock, Rochester, NY) followed by a 440-40/521-21/607-34/700-45 filter (Semrock, Rochester, NY) were used and subsequently filtered further using band-pass filters (Semrock BP-525/30-25 and BP-607/36-25 for the green and red channels, respectively). Images were acquired with an Andor Technology iXon3 DU-897-BV EMCCD camera. For time-lapse experiments, images were collected every 10 s, using an exposure time of 500 ms.
|
study
| 99.94 |
To evaluate cell retention of the probe post-fixation/permeabilization, LX2 cells (2 × 104) were plated on a 12 mm glass coverslip placed in a well of a 24 well-plate and incubated for 24 h at 37°C in an atmosphere of 5% CO2. The cells were then treated with 500 ηM NRB-AF12 for 30 min, fixed with 4% paraformaldehyde for 15 min at room temperature and immediately mounted on microscope slides, or fixed with 4% paraformaldehyde, permeabilized with either 0.1% Triton X-100 (10 min at room temperature) or ice cold methanol (5 min at −20°C), and subsequently mounted.
|
study
| 99.94 |
In order to evaluate NRB-AF12 co-localization with endoplasmic reticulum, Golgi apparatus and with the sulfonylurea receptor (SUR) subunits of the ATP-sensitive potassium channels, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with ice cold methanol for 5 min at −20°C. ER was highlighted with antibodies directed against the ER resident protein calreticulin (1:200; Abcam #ab2907); Golgi apparatus was highlighted with antibodies directed against the Golgi apparatus structural protein GM130 (1:500; BD Biomedsciences #610822); SUR2 subunits of KATP channel were highlighted using either anti-SUR2A (1:750; Abcam #ab174629) or anti-SUR2B (1:750; Abcam #ab174631) antibodies. All primary antibodies were incubated for 90 min at room temperature. Following a PBS wash, cells were incubated with Cy™3 goat anti-rabbit IgG fluorescent secondary antibody (1:500; Jackson ImmunoResearch #111-165-003) or with Cy™3 goat anti mouse IgG fluorescent secondary antibody (1:500; KPL #072-01-18-06) for 60 min at room temperature in the presence of either 500 ηM NRB-AF12 or 500 ηM ER-Tr green. Finally, coverslips were mounted on microscope slides using Mowiol 40–88 (Sigma Aldrich). Confocal images were acquired with a Laser Scanning Confocal Microscope (LSCM) D-Eclipse C1 SHV Nikon system equipped with a Nikon Eclipse E600 microscope and 3 laser diode modules (488/543/637 nm), using a CFI Plan Apochromat 60x NA 1.4 objective and analyzed using NIS Elements software (Nikon) and the open-source platform for biological-image analysis Fiji software.
|
study
| 99.94 |
Drosophila strain W, stock number 5905 was obtained from Bloomington stock center (Indiana University, USA) and used to perform live imaging experiments. Fly larvae having reached the third instar stage whilst feeding on standard food were dissected in hemolymph-like HL3 saline (70 mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 20 mM MgCl2, 10 mM NaHCO3, 5 mM trehalose, 115 mM sucrose, 5 mM sodium HEPES, pH 7.2). For live tissue imaging ER-Tr red and NRB-AF12 were added to the medium of dissected larvae and images acquired using a Nikon eclipse C1 confocal microscope and a Nikon Fluor 60x NA 1.00 water objective.
|
other
| 97.75 |
1 × 105 LX2 cells, 1 × 105 HepG2 cells and 5 × 104 HSC-T6 cells were plated in 12 well-plates in complete culture medium and incubated for 24 h at 37°C. Cells were then treated with increasing doses (0.1, 1 and 10 μM) of NRB, NRB-AF12, glibenclamide and ER-Tr for 24 h. Cell viability was assessed by the Trypan Blue exclusion method. Briefly, cells were trypsinized, resuspended in complete medium, diluted 1:2 with a 0.4% Trypan Blue solution and finally counted using a hemacytometer under a light-microscope.
|
study
| 99.94 |
In HSNO (Hazardous Substances and New Organisms) terms, a substance is considered hazardous if it triggers any one of the threshold levels for any of the hazardous properties listed in the USA Environmental Protection Agency (EPA) guidelines, one of which is toxicity. NRB is extremely toxic to rats and as such is classified as an “extremely hazardous chemical” in the US and elsewhere. However, one of the key features of NRB is that its toxic activity is uniquely limited to rats, having little or no activity in any other species tested (including humans). In fact NRB's lethal activity is even further restricted as it seems only to be toxic to species within the Rattus genus, with other rat genera being little or not affected. As a consequence, the NRB and the NRB derivatives reported in this research paper poses little or no hazard to the researchers, and as such no special conditions or safety precautions are required to work with these substances other than those required to conform to standard laboratory practices.
|
study
| 97.94 |
All values are expressed as means ± standard error (SE) of n observations/group. Analysis of co-localization was performed using Pearson's correlation coefficient. Comparisons of more than two groups were made with a one-way ANOVA using post-hoc Tukey's test. Comparison of two groups was obtained by the Student's t-test for unpaired data when appropriate. Differences were considered statistically significant at values of P < 0.05.
|
study
| 99.94 |
Figure 1C shows phase contrast microscopy images and conventional epifluorescence microscopy images of living LX2 cells incubated with 500 ηM NRB-AF12. The images clearly show a meshwork distribution of the fluorescence with a sharp demarcation of the nuclei and an accumulation in perinuclear areas; moreover, NRB-AF12 did not stain either the nuclei or the plasma membrane. NRB-AF12 internalization into the cell was fast, commencing after only a few seconds and reaching completion within a few minutes, following its addition to the incubation medium. A similar fluorescence pattern was also observed in fixed LX2 cells (Figure 1D), however, the NRB-AF12 signal was not retained post-permeabilization (data not shown). The sub-cellular distribution of NRB-AF12 was investigated in more detail in LX2 cells using confocal microscopy in combination with fluorescent probes and/or antibodies targeted to specific subcellular structures. To evaluate the distribution of NRB-AF12 in the ER, LX2 cells were transfected with the ER-reporter red fluorescent protein (RFP)-KDEL plasmid and subsequently loaded with the dye. The results, reported in Figures 2A,D, show that the NRB-AF12 signal significantly overlapped with that of (RFP)-KDEL (Pearson's Coefficient: 0.21 ± 0.10; n ≥ 10), but a larger staining distribution of NRB-AF12 was observed which indicated other targets in addition to ER. Similar results were observed in LX2 fixed cells labeled with NRB-AF12 and immunostained for the ER resident protein Calreticulin (Pearson's Coefficient: 0.3 ± 0.06; n ≥ 10) (Figures 2B–D). We also compared the staining pattern of NRB-AF12 with that of ER-Tr, a commercial fluorescent probe widely used to label ER. Surprisingly, co-labeling of LX2 cells with NRB-AF12 and ER-Tr resulted in a strong overlap between fluorescent dyes (Figure 2C), confirmed by the high rate of co-localization index (Pearson's coefficient: 0.75 ± 0.02; n ≥ 15). This remarkable similarity between the two dyes was further supported by the observation that ER-Tr, as well as NRB-AF12, were not selective for the ER, as demonstrated by co-staining LX2 cells with ER-Tr plus either RFP-KDEL (Figure 3A) or Calreticulin (Figure 3B) (Pearson's coefficient: 0.23 ± 0.1 and 0.29 ± 0.03, respectively for RFP-KDEL and Calreticulin; n ≥ 10; Figure 3C).
|
study
| 100.0 |
(A) Live cell imaging of LX2 cells transfected with ER-reporter RFP-KDEL plasmid and subsequently stained with NRB-AF12. (B) Immunostaining of LX2 cells for the endoplasmic reticulum marker calreticulin; cells were counter-labeled with NRB-AF12. (C) Confocal live cell imaging of LX2 cells fluorescently labeled with 500 ηM NRB-AF12 and 500 ηM ER-Tr red. Insets show magnification of the pictures. Magnification 60x; scale bar 10 μm. (D) Quantification of NRB-AF12 co-localization with RFP-KDEL, Calreticulin and ER-Tr is shown as Pearson's coefficient. (n ≥ 10).
|
study
| 100.0 |
(A) Live cell imaging of LX2 cells transfected with ER-reporter RFP-KDEL plasmid and subsequently stained with ER-Tr. (B) Immunostaining of LX2 cells for the endoplasmic reticulum marker calreticulin; cells were counter-labeled with NRB-AF12. Magnification 60x; scale bar 10 μm. (C) Quantification of ER-Tr co-localization with RFP-KDEL and Calreticulin is shown as Pearson's coefficient (n ≥ 10).
|
study
| 99.94 |
The NRB-AF12 and ER-Tr subcellular distribution patterns and co-localization rates were confirmed using other NRB-insensitive cells (i.e., HepG2, HSC-T6, and HeLa cells; Figures 4A–C), and using NRB-sensitive cells such as freshly isolated rat caudal artery myocytes (Figure 4D). Interestingly, in this latter cell type, both fluorescent compounds stained not only intracellular structures, but also the plasma membrane. Co-localization rates of NRB-AF12 and ER-Tr in HepG2, HSC-T6, HeLa and primary rat vascular smooth muscle cells is reported in Figure 4E as Pearson's coefficient. These results indicates that NRB-AF12 labels intracellular structures with a pattern of distribution that is apparently very similar to that of ER-Tr, and is not confined to ER.
|
study
| 100.0 |
Representative live cells microphotographs showing co-localization between NRB-AF12 (left column) and ER-Tr red (mid column) in (A) HepG2, (B) HSC-T6, (C) HeLa, and (D) primary rat vascular smooth muscle cells (VSMC) from caudal artery. For each panel, merged fluorescence is shown in right column. Magnification 60x; scale bar, 10 μm. (E) Quantification of NRB-AF12 and ER-Tr co-localization is shown as Pearson's coefficient (n ≥ 15).
|
study
| 99.94 |
The Golgi apparatus was investigated by co-staining fixed cells with either NRB-AF12 or ER-Tr, and antibodies directed against the Golgi structural protein GM130 (α-GM130). Data obtained, shown in Figure 5A, indicates a co-localization of NRB-AF12 and α-GM130 (Pearson's coefficient: 0.53 ± 0.02; n ≥ 10; Figure 5C). Similar results were obtained when α-GM130 was used in combination with ER-Tr (Pearson's coefficient: 0.58 ± 0.02; n ≥ 10; Figures 5B,C).
|
study
| 100.0 |
Mitochondrial NRB-AF12 and ER-Tr localization was investigated in living LX2 cells transfected with the mitochondrial marker MitoDS-red plasmid, and then loaded with either NRB-AF12 or ER-Tr. As depicted in Figure 6, both dyes labeled mitochondria to similar levels (Pearson's Coefficient: 0.56 ± 0.05 and 0.54 ± 0.03, respectively for NRB-AF12 and ER-Tr; n ≥ 10).
|
study
| 100.0 |
Live cell imaging of liver myofibroblasts transfected with the mitochondrial marker Mito-Ds red and then stained using (A) NRB-AF12 or (B) ER-Tr green. Magnification 60x; scale bar 10 μm. (C) Co-localization of NRB-AF12 and ER-Tr within mitochondria is reported as Pearson's coefficient (n ≥ 10).
|
study
| 99.94 |
Next, we investigated whether NRB-AF12 and ER-Tr could label endosomes and lysosomes. For this purpose, we performed live cell imaging experiments in LX2 cells using a commercial fluorescent probe which has been previously demonstrated to highlight vesicles involved in endocytosis. As shown in Figure 7, we could not find any staining of endocytic vesicles when cells were loaded with ER-Tr (Pearson's coefficient: −0.33 ± 0.05 and −0.43 ± 0.04, respectively for endosomes and lysosomes; n ≥ 10). In contrast, NRB-AF12 showed an appreciable co-localization rate with both endosomal and lysosomal vesicles (Pearson's coefficient 0.25 ± 0.13 and 0.15 ± 0.15, respectively; n ≥ 10). Interestingly, the data revealed a high index of dispersion of the co-localization coefficients (CI 95%: −0.05 – 0.55 and −0.19 – 0.5, for endosomes and lysosomes respectively; n ≥ 10), indicating that not all endosomes/lysosomes vesicles were stained by the dye.
|
study
| 100.0 |
Confocal live cell microphotographs of LX2 cells stained with the fluorescent dye for endosomal (empty arrowhead) and lysosomal (full arrowhead) compartments pHrodo red and (A) NRB-AF12 or (B) ER-Tr green. Magnification 60x; scale bar 10 μm. (C,D) Co-localization of NRB-AF12 and ER-Tr Green with endosome and lysosome vesicles is shown as Pearson's coefficient (*p < 0.05; n ≥ 10).
|
study
| 99.94 |
Since NRB-AF12 and ER-Tr showed the same cellular labeling pattern, we hypothesized a common target for these compounds. ER-Tr probes are conjugates of glibenclamide bearing different fluorophores, BODIPY FL (ER-Tr green) and BODIPY TR (ER-Tr red). Glibenclamide is an antidiabetic drug belonging to the class of sulfonylureas that binds the sulfonylurea receptor (SUR) subunits of ATP-sensitive potassium channels (Proks et al., 2002). Accordingly, we therefore explored the likelihood of whether NRB could also target these SURs by analyzing the co-localization between NRB-AF12 and the 2 major subunits SUR2A and SUR2B. We focused our attention on these SUR isoforms because of their intracellular distribution (Bao et al., 2011) and the role of the plasmalemmal SUR2B in the regulation of vascular contraction (Morrissey et al., 2005; Teramoto, 2006; Jackson, 2008; Tinker et al., 2014). Our results indicate that in LX2 cells NRB-AF12 co-localized with both SUR2A and SUR2B, with a greater correlation for the latter (Pearson's coefficient: 0.28 ± 0.02 and 0.36 ± 0.01, respectively for SUR2A and SUR2B; n ≥ 10) (Figures 8A,C,E); comparable results were also obtained with ER-Tr (Pearson's coefficient: 0.22 ± 0.04 and 0.32 ± 0.02, respectively for SUR2A and SUR2B; n ≥ 10) (Figure 8B,D,F). It is worth noting that the cellular distribution of NRB-AF12 and ER-Tr fluorescence was larger than that observed with SUR2, suggesting non-SUR2 additional targets for both dyes.
|
study
| 100.0 |
(A,B) Confocal pictures of LX2 fixed cells immunostained for the KATP channels subunit SUR2A and counter-labeled with (A) NRB-AF12 or (B) ER-Tr green. (C,D) Immunostaining of LX2 cells for SUR2B subunit of KATP channels; cells were counter-labeled with (C) NRB-AF12 or (D) ER-Tr green. Insets show magnification of the pictures. Magnification 60x; scale bar 10 μm. (E,F) Pearson's coefficient showing co-localization rate of (E) NRB-AF12 or (F) ER-Tr green with either SURs isoforms (n ≥ 15).
|
study
| 100.0 |
The overlapping fluorescence distributions of AF-12 and ER-Tr prompted us to investigate whether NRB and glibenclamide could compete with their respective fluorescent derivatives in LX2 cells. To this end, LX2 cells were pre-treated with high concentrations (100 μM) of either NRB or glibenclamide, and then loaded with either NRB-AF12 or ER-Tr. Interestingly, pre-treatment with the non-labeled compounds failed to prevent fluorescent labeling following addition of the fluorescent derivatives (data not shown).
|
study
| 100.0 |
We also explored the fluorescence features of NRB-AF12 in intact tissues of dissected larvae of Drosophila melanogaster. Third instar larvae were labeled with NRB-AF12 and ER-Tr dissolved in the hemolymph-like HL3 saline, added to the medium following dissection and imaged with an immersion objective. As shown in Figure 9, a bright fluorescence was observed when larvae were treated with the fluorescent probes: In vivo live imaging of D. melanogaster muscle fibers (Figure 9A) and tracheal system (Figure 9B) revealed a sarcoplasmic reticulum distribution of the dyes, with NRB-AF12 and ER-Tr fluorescence significantly overlapping, as was observed in cell culture (Johnson et al., 2015). Finally, we investigated some physical properties of NRB-AF-12 such as penetration, internalization rate, retention time, washout time-course in living cells, as well as the induction of cytotoxicity, in order to establish the usefulness and reliability of the dye. Video 1 in Supplementary Material, shows that in living LX2 cells AF-12 is internalized in minutes and is retained in the cells for at least 3 h following its removal from the culture medium. Furthermore, re-exposure of the cells to dye, gave the same results, in terms of fluorescence distribution, compared to the first exposure. NRB-AF12 was apparently not toxic in both human and rat cell lines, even at concentrations much higher than those (500 nM) utilized for labeling (Figures 11B–D).
|
study
| 100.0 |
Confocal live imaging of Drosophila (A) muscles and (B) tracheal system labeled with NRB-AF12 (green) and ER-Tr red (red). Both fluorescent dyes, added to the dissected larva in HL3 physiological solution, show an overlapping pattern of distribution as highlight by the yellow signal of the merged images (*: muscle; white arrowhead: trachea; N: nucleus). Magnification 60x; scale bar 10 μm.
|
other
| 84.25 |
This study was undertaken to verify if a fluorescent derivative of NRB could furnish information about the cellular target/s involved in the selective rat-specific and tissue-selective action of unlabeled NRB. To this end, we attached the NBD fluorophore to the NRB molecule. The NBD group is known to bleach very rapidly in respect to other commercially available fluorophores, nevertheless we decided to use NBD because of its relatively small size, in the attempt to minimize the risk of inducing a distortion of the chemical-physical properties of NRB. We compared the fluorescence distribution of NBD-NRB (NRB-AF12) observed in NRB-sensitive cells (freshly isolated rat caudal artery myocytes) with that observed in NRB-insensitive cells (several cultured cell lines). Preliminary experiments conducted in our lab indicated that NRB-AF12 has the same species- and tissue-specific contractile properties of unlabeled NRB (manuscript in preparation), and therefore is a reliable tool for the purpose of this work.
|
study
| 100.0 |
Given that the cellular labeling patterns of NRB-AF12 and ER-Tr are near identical, it could be proposed that the two dyes share a common cellular target/s. ER-Tr is a BODIPY derivative of glibenclamide, a widely used antidiabetic drug belonging to the sulfonylurea family. Glibenclamide is a non-selective inhibitor of ATP-sensitive potassium channels (Proks et al., 2002), a multimeric protein complex consisting of four pore-lining inward rectifier α subunits (Kir6.1/2) and four regulatory sulfonylurea-binding β subunits (SUR1/2A/2B) that belong to the ATP-binding cassette family (Yokoshiki et al., 1998; Zerangue et al., 1999; Shi et al., 2005; Ng et al., 2010). Co-assembly of different Kir and SUR subunits generates ion channel combinations with different channel properties, and with different cell compartment or tissue distribution (Yokoshiki et al., 1998), such as the SUR1/Kir6.2 pancreatic type, the SUR2A/Kir6.2 cardiac type and the SUR2B/Kir6.1 vascular smooth muscle type. The ER-Tr and NRB-AF12 fluorescence characteristics may therefore reflect the cellular distribution of KATP channels. Inhibition of plasma membrane ATP-sensitive potassium channels leads to an increased intracellular K+ concentration, plasma membrane depolarization, opening of voltage-dependent calcium channels, and increased intracellular Ca2+ concentration (Ko et al., 2008) that, in vascular smooth muscle, triggers the contractile process. It has been reported that vascular smooth muscle plasma membrane selectively expresses SUR2B subunits of KATP channels (Morrissey et al., 2005; Teramoto, 2006; Tinker et al., 2014). These data, together with the finding (this study) that NRB-AF12 localizes to the plasma membrane only in NRB-sensitive cells, might explain the species- and tissue-selective activity of NRB, by hypothesizing an inhibitory effect of the drug on rat-specific SUR2B-containing KATP channels expressed on the plasma membrane of the rat peripheral artery myocytes. In contrast to this hypothesis is the fact that neither unlabeled NRB nor glibenclamide are able to displace NRB-AF12 or ER-Tr from their binding site, which presents the prospect of a different/additional site of action from/to KATP channels. It also raises the question as to whether the fluorescently labeled compounds have the same binding characteristics of the corresponding unlabeled compounds. However, the lack of displacement is in itself not sufficient to rule out the possibility that NRB-AF12 does bind to KATP channels, with there being at least two plausible explanations: (1) NRB-AF12 and NRB could bind to different sites within the SUR2B moiety, thus allowing the attachment of both compounds; (2) the fluorophore group of NRB-AF12 may have multiple binding sites preventing its displacement by unlabeled NRB. The existence of more than one binding site, at least for NRB-AF12, is supported by the fact that this dye, in contrast to ER-Tr, was able to label both endosomes and lysosomes. As with SUR2-containing channels, NRB-AF12 appears to be similarly targeted to the endosomal/lysosomal pathway, supporting the idea that this compound can be recycled by endocytosis as has been shown for the KATP channel subunits (Bao et al., 2011).
|
study
| 99.94 |
Live cell imaging represents an important technique in the study of biological processes; the use of fluorescent proteins or dyes provides an important tool for the in vivo visualization, in space, and time, of virtually any cellular mechanism or structure, avoiding artifacts or sample alteration that could occur with fixation methods. In this study we show that NRB-AF12 is endowed with features that make this dye an eligible prototype of new fluorescent probes, alternatives to ER-Tr, for live cell imaging (Figure 10). Furthermore, in NRB-sensitive single rat vascular myocytes, this dye, due to its constriction effect (manuscript in preparation), could also be employed to study the morphologic changes that occur in labeled intracellular organelles, as well as in the plasma membrane, during the contractile process. Whatever the binding site/s for NRB-AF12, this dye shows a cellular distribution near identical to that of ER-Tr in real-time fluorescent imaging using different cultured cell lines and freshly isolated rat caudal artery myocytes. NRB-AF12 labeling was also observed in fixed and permeabilized cells, allowing its use in immunocytochemistry co-localization experiments. In all cells tested, NRB-AF12 stained not only ER, but also other subcellular compartments such as Golgi apparatus, mitochondria, and organelles involved in the endocytic pathway (i.e., endosomes and lysosomes). NRB-AF12 internalization rate was extremely fast, since cell incubation with the fluorescent compound resulted in a well-defined meshwork staining of the cellular structures within a few minutes, and could be observed up to 3 h following its removal from the culture medium (Figure 11A). Another useful feature is that NRB-AF12 exhibited no apparent cytotoxicity, a serious disadvantage that often occurs using fluorescent probes for live cell imaging. Cell viability experiments demonstrated the dye to be biocompatible, having no harmful effects in the different cell lines (both human and rat) after 24-h incubation, even at concentrations (10 μM) much higher than those (500 ηM) needed to visualize cellular organelles (Figures 11B–D). Furthermore, the lack of cytotoxicity of NRB-AF12 allows the same sample to be repeatedly stained with the dye (for example at different time points within an experiment), without morphological alteration of cell structures or cell death; concurrently, the ability of NRB to effectively label its cellular target is preserved on re-exposure to the dye (Figure 11A).
|
study
| 99.94 |
(A) LX2 cells were incubated for 30 min with NRB-AF12 or ER-Tr green; after replacing the staining solution with probe-free medium, evaluation of NRB-AF12 and ER-Tr extent of fluorescent signal and photostability was performed. (B) LX2, (C) HSC-T6, and (D) HepG2 cells were treated for 24 h with increasing doses (0.1, 1 and 10 μM) of NRB, NRB-AF12, glibenclamide or ER-Tr. Compounds toxicity was evaluated by trypan blue exclusion test of cell viability. NT, not treated cells.
|
study
| 100.0 |
Finally, imaging experiments performed in dissected larvae of D. melanogaster demonstrated NRB-AF12 adaptability in complex biological systems. The live imaging of fly tissues showed that NRB-AF12 maintain the same subcellular distribution of ER-Tr also in vivo, supporting the theory of a common target. Moreover, we observed a significant signal in the Drosophila tracheal system where Drosophila Sur (Dsur) is highly expressed (Nasonkin et al., 1999). Dsur, encodes a glibenclamide-sensitive potassium channel and has been identified as the functional orthologue of the mammalian SUR2 (Kim and Rulifson, 2004). On the basis of these favorable properties, we propose NRB-AF12 as an alternative fluorescent dye to ER-Tr, and a new improved tool for labeling intracellular organelles.
|
study
| 100.0 |
In conclusion, this study presents evidence to demonstrate that NRB-AF12, a fluorescent derivative of the selective rat toxicant NRB, has a highly similar fluorescence distribution to ER-Tr, a fluorescent derivative of glibenclamide, a known KATP channel inhibitor. On the basis of these results we hypothesize common cellular binding sites for NRB and glibenclamide that may potentially lead to common biological properties. Furthermore, we propose NRB-AF12 as a prototype for the development of new fluorescent probes selective for intracellular structures/organelles. Studies are in progress in our lab to investigate these aspects.
|
study
| 100.0 |
CD and GO contributed equally to this work. SB, CD, and MP conceived the study. AFe, MB, DR, BH, and GR designed and performed synthesis of NRB-AF12. CD, GO, GM, FF, AFo, GC, and SD designed and performed experiments. SB, CD, and GO analyzed and interpreted data. SB, CD, GO, DR, and BH wrote the manuscript. All authors approved the final version of the manuscript.
|
other
| 99.94 |
Soil erosion by water is one of the most widespread and major ecological environmental problems worldwide, which results in reduced agricultural productivity, increased water pollution, and unsustainable development1–5. Soil erosion is a complex physical process, the fundamental explanations are the comprehensive interaction between precipitation and the surface of watersheds6,7. In a mathematical sense, it is a highly non-linear mapping relationship from the watershed surface and rainfall conditions to runoff and sediment transport8,9. Generally, the main factors affecting rainfall erosion and sediment yield include: rainfall intensity, rainfall duration, rainfall spatio-temporal characteristics, soil properties, geological conditions, vegetation, land use and antecedent soil wetting condition10,11. Rainfall is one of the most important active agents of soil erosion, due to its potential to breakdown aggregates, detach soil particles, and produce runoff12–14. At present, many scholars have conducted experimental and modelling studies in runoff and sediment yield characteristics under the conditions of different soil types, rainfall intensity, slope gradient, land use types, vegetation coverage, and water conservation measures15. However, most studies mainly explored runoff and sediment loss on runoff plots16,17. On the other hand, other studies focused on rainfall, runoff and sediment relationships at basin scales. They mainly analysed relationships between rainfall runoff and sediment yield on annual time scales18. Besides, some scholars have also constructed different rainstorm runoff-sediment models, but they did not reveal the pulsed runoff-sediment relationship of the typical loess hilly watershed, due to local characteristics19,20.
|
review
| 99.75 |
Generally, studies on runoff and sediment change on the Loess Plateau are based on model simulation or measured data analysis. The model simulation method has better prospects for causal analysis of runoff and sediment changes through flexible parameter adjustment21,22. However, the model can only be used to abstract and generalize natural phenomena, and cannot fully reveal the occurrence and evolution mechanism of natural phenomena23. Therefore, it is still an effective method to study the variation of runoff and sediment using measured data. Studies have shown that research on runoff and sediment change using measured data has the following characteristics24: (i) The spatial scales are mainly concentrated on runoff plots and slope surfaces when investigating the influence of rainfall intensity and vegetation on rainfall-runoff and sediment yield. These small-scales can not sufficiently reflect the regional variation rules of water and sediment for large spatial scales25,26. (ii) The impact of annual rainfall on runoff and sediment has been considered to be a key priority when the spatial scale expands to larger areas, but the influencing effects of rainfall intensity during one precipitation on runoff and sediment yield in large spatial scales will be weakened27,28. (iii) Statistical analysis is an important method to study rainfall-runoff interactions29. Cluster Analysis is an unsupervised learning process to find one set of similar elements in a data set, it is of great significance to policy decisions on soil and water conservation. Therefore, it is necessary to study correlations between runoff and sediment yield under individual monitoring rainstorm events in a typical small watershed of the Loess Plateau.
|
study
| 99.75 |
The Loess Plateau is located in an arid and semi-arid area. Annual rainfall ranges between 200–600 mm. Soil erosion on the Loess Plateau is very serious. Erosion is mainly caused by several heavy rainstorms during 6–9 months, and the amount of erosion by heavy rains accounts for >60% of total annual erosion30. So storm runoff erosion is the main pathway of sediment yield and the main power of sediment transport for other erosion processes31. Due to the high intensity and short duration of the few heavy rains, rainstorms on loess slopes rapidly form infiltration-excess runoff and cause very high runoff rates32. Because loess soil is highly erodible, a large amount of infiltration-excess runoff also results in excessive erosion, which makes this region one of the most eroded areas in the world33. Therefore, according to the erosion characteristics of the Loess Plateau, it has more significance for the establishment of soil erosion models and soil conservation by predicting runoff and sediment yield relationships of individual rainstorm events than studies on annual rainfall-erosion relationships34. Only clearly knowing the characteristics of erosion and sediment yield under typical pulsed runoff-erosion events will allow effective scientific management of soil and water conservation at watershed scales.
|
study
| 97.5 |
The main aims of this paper are to: (1) investigate characteristics of pulsed runoff-erosion events in a loess hilly-gully watershed, and (2) evaluate the influencing mechanisms of relationships between rainstorm and sediment yield. Results may provide the theoretical basis for the construction of rainstorm runoff and sediment yield models and for decision-making in watershed management.
|
study
| 67.0 |
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