When watching videos, our sense of reality is continuously challenged. How much can a fundamental dimension of experience such as visual flow be modified before breaking the perception of real time? Here we found a remarkable indifference to speed manipulations applied to a popular video content, a soccer match. In a condition that mimicked real-life TV watching, none of 100 naïve observers spontaneously noticed speed alterations up/down to 12%, even when asked to report motion anomalies, and showed very low sensitivity to video speed changes . When tested with a constant-stimuli speed discrimination task, JND was still high, though much reduced (9%). The presence of the original voice-over with compensation for pitch did not affect perceptual performance. Thus, our results document a rather broad tolerance to speed manipulations in video viewing, even under attentive scrutiny. This finding may have important implications. For example, it can validate video compression strategies based on sub-threshold temporal squeezing. This way, a soccer match can last only 80 min and still be perceived as natural. More generally, knowing the boundaries of natural speed perception may help to optimize the flow of artificial visual stimuli which increasingly surround us. Here we have addressed natural speed perception by measuring the detectability of speed alterations applied to a soccer match video clip.We are almost literally immersed in an artificial visual world, made in large part by motion pictures. How robust is our sense of reality with such pervasive technologies? For example, how well can we detect speed alterations in a video clip? The availability of videos, videogames and virtual reality systems, through which a variety of arbitrarily altered worlds can be experienced, calls for an appraisal of the boundaries within which video speed can be manipulated without conflicting with perception. However, although much effort has been devoted to studying motion perceptionIn the first experiment we used a rather ecological setting, with observers watching a 10-minute video clip of a soccer match without any specific task. They viewed it only once and were told that at the end they would have to answer questions concerning the match, aimed to assess attention and memory capabilities. Observers were divided into five groups (N = 25 each), and each group watched the video clip at a different speed . The original audio track with voice commentary was pitch-compensated to avoid distortions when the video was played at a different speed. In general, observers were quite compliant, as suggested by self-reported attention measures . By contrast, they were very poor at speed perception, as speed alterations went completely unnoticed when assessed through follow-up questions Q3 and Q4 (see Methods), aimed at evaluating the spontaneous noticing of speed change (0% accuracy in both cases), even though Q4 made reference to possible motion anomalies. It was necessary to explicitly mention speed in the question to register some correct responses . However, detection accuracy did not depart significantly from chance . This also held true when considering the four speed modifications separately . From these data, it would appear that observers almost completely neglected, in retrospect, up/down speed alterations up to ±12%.2(3) = 0.332, P = 0.954], which indicates that the values predicted by the logistic function fit the data well.To better quantify the perceptual performance in this first experiment, we fitted a standard logistic psychometric function to the responses to Q6 . We pooled data from all participants and used a binomial Generalized Linear Model (GLM) to fit the logistic function and parametric bootstrap to compute the 95% confidence interval of the Point of Subjective Equality (PSE) and the Just-Noticeable Difference (JND) corresponding to the psychometric function Fig. . These t6. The Hosmer-Lemeshow test was not statistically significant , which indicated a good fit. We used bootstrap to compute the 95% confidence interval of the Point of Subjective Equality (PSE) and the Just-Noticeable Difference (JND) corresponding to the psychometric function. The PSE and JND turned out to be 1.2%  and 8.8% (8.1–9.4%) of the original video speed, respectively. The results of this analysis are shown in Fig. In the second experiment we administered a psychophysical task (constant-stimuli method) involving a much larger number of observations . In this experiment, observers had to discriminate the speed of short segments of the soccer video clip with 2AFC responses (too fast or too slow). At variance with the first experiment, here observers had to explicitly and attentively evaluate the video reproduction speed. No standard was provided for speed comparison, thus observers had to rely on an internal model of the “natural” speed of a soccer match. To evaluate the performance at the population level, we used a binomial Generalized Linear Mixed Model (GLMM) with a logistic link function, which included a random intercept and slope for each participantThis pattern of results persisted even though we excluded trials with the lowest confidence . The PSE was 1.1%  and the JND 8.3% (7.7–8.9%), which suggests that subjective uncertainty did not affect speed judgments.7 or speed adaptation8, which in the constant-stimuli method depend on trial history. Indeed, PSE was significantly correlated with previous speed exposure in the first experiment . Full sequence randomization  and/or completely crossed designs  may improve parameter estimation, but at the cost of losing the advantage of testing large subject samples in “one shot”. In both cases, however, JND was rather high.Trial order (see Methods) influenced the observers’ responses: A binomial GLMM with trial order and speed as fixed effects showed that the interaction between trial order and speed was statistically significant , indicating a difference of slope of the psychometric function between the two trial orders. The JND was 7.5%  in one direction, and 10.4% (9.5–12.1%) in the other direction. The PSEs were −0.2% (−1.2–0.5%) and 3.0% (1.8–4.2%). These differences may reflect serial dependency effectsA possible reason for the poor tuning to soccer match speed could be that, despite its popularity, observers were in fact not very familiar with soccer. Indeed, self-rated soccer expertise and soccer practice were not high . However, no significant correlations emerged between JND or PSE and SES or SPS . Thus, neither generic soccer expertise nor soccer practice level seems to have affected speed judgments.2(9) = 3.783, P = 0.925], which indicates a good fit of the combined model. Despite the large uncertainty of the shape of the psychometric function in the first experiment, this analysis revealed a statically significant interaction between the experiment and the slope of the psychometric function . The speed main effect was the only other statistically significant effect . These results confirm that observers were better at noticing video speed changes in the second experiment. Indeed, these observers explicitly focused on speed alterations, which should improve perceptual performance.Likewise, the second experiment revealed a considerable perceptual tolerance to video speed. However, the JND was much smaller than in the first experiment, suggesting a higher sensitivity to speed. To ascertain this point, we compared the performances of the two experiments with a binomial GLMM by including the experiment as a fixed effect together with the speed. As before, the model included a random intercept and slope for the speed for each participant. The model assumed that the random effects were the same in the two experiments for those who participated in both experiments (see Methods). The Hosmer-Lemeshow test was not statistically significant [χA possible confounding factor is that we used a custom program to deliver the video sequence with the sound muted in the second experiment, whereas in the first experiment an open-source video player with pitch-compensated original audio-track was used (see Methods). Although the basic algorithm for speed change was identical in the two software solutions, we could not exclude subtle differences in motion rendering; also, the lack of audio-track may have changed visual sensitivity to anomalous motion in the second experiment. Therefore, we ran a control experiment to assess the impact of the different audio-visual conditions. More specifically, we repeated the second experiment (constant stimuli), but using the same setup as in the first experiment, including pitch-compensated audio-track . The results were very similar to those obtained in the second experiment, with PSE = 1.5%  and JND = 9.9% (8.8–11.2%). A direct comparison between the two experiments via a GLMM including experiment and speed as fixed effects did not reveal any statistically significant effect associated with the experiment or with the interaction between the experiment and the slope of the psychometric function . Only the speed factor was statistically significant . Thus, there was no evidence that either subtle differences in motion rendering or the audio track affected the sense of speed in any important way.9. Indeed, in the first experiment JND estimates (Q6) were quite large (∼18%). In the second experiment, when observers were explicitly asked to pay attention to speed alterations, JND was still high, although much lower than in the first experiment (∼9%). This figure did not change significantly in the control experiment, which suggests that using different software solutions or muting videos did not affect the sense of speed, at least in our experimental conditions.The results of this study documented a poor sense of speed in soccer viewing. Firstly, in retrospect participants completely missed anomalous video speeds with modifications of up/down to 12% . For comparison, consider that in experiments on inattentional blindness, where unattended elements in a visual scene, even important ones, are neglected, noticers are often a non-negligible proportion – sometimes the majority – of observers11 and in monkeys12, as well as with more or less realistic optic flow stimuli in car driving contexts16. However, we also directly compared the results obtained in a classical laboratory setting (second experiment) with those obtained in a condition that mimicked home TV watching (first experiment), showing in the latter case an even poorer sensitivity to altered video speed. Indeed, in the first experiment observers’ attention was focused on the match and not on video speed, a condition that is expected to lower sensitivity to anomalous speeds. This result underscores the importance of pairing structured psychophysical designs with tasks for retrospective noticing in an ecological condition, at the cost of “burning” participants after the critical trial, which requires a large number of participants17. Clearly, the merit of the first experiment is also its weakness: in order to mimic real-life conditions observers should be naïve, but in order to be naïve they should be tested only once. Hence, to reach definitive conclusions on real-life conditions, a very large-scale study would be required, targeting also the relevant population characteristics . Still, the fact that important speed modifications went completely neglected is remarkable. By leveraging on our JND estimates, and keeping in mind the limits of approximate estimations in the first experiment, we suggest that speed tolerance in real-life conditions can be extrapolated to be about double of that measured with the constant stimuli method.Our JND estimates are generally consistent with figures obtained under quite different laboratory conditions, stimuli and paradigms in humans20. Videos are indeed low-contrast, reduced versions of real scenes. Other explanations are possible, however, based on distortions of temporal perception with motion stimuli3, intuitive physics biases5, or motion anticipation tendencies21. Violation of speed constancy could also account for speed underestimation23.As to the small but significant speed underestimation found with the constant stimuli method, indexed by PSE, various factors may be at play. For example, speed judgments may be influenced by the brain’s perceptual tuning to low visual speeds, a bias that has been posited to emerge under uncertainty conditions with weak, low-contrast stimuli24. Such a large mismatch may have been partly intentional, as both camera operators and projectionists contributed to the cinematographic language by manipulating speed25. Indeed, altering the reproduction speed can even influence intent attribution26. At any rate, explicit speed modifications, including special effects such as instant replay, fast-motion or time lapse break our sense of reality and thus are definitely different from the sub-threshold speed changes targeted in the present study, which preserve the illusion of authenticity and a subjectively normal event flow.Interestingly, our study justifies the practice of playing 24 fps movies at 25 fps in PAL systems. Indeed, the frame rate difference (4%) would be largely sub-threshold, even if observers were to focus on speed rather than the film plot. Slightly different is the case of some old silent films that have an evident jerky appearance, which indicates that the mismatch between footage and reproducing speeds was sometimes quite large30), or apparent violations of physical laws . By contrast, it is likely that speed tolerance gets worse for more kinematically-ambiguous visual events such as water flow. This remains to be tested.Clearly, the present findings cannot be generalized, as they apply to a specific visual content, though a very common one. It is possible that speed tuning improves when more definite motion cues are available, e.g., close-up of human movements  suggests that compression should not be applied uniformly, though. For example, to remain in the domain of soccer, the normal actions shot with a very wide angle may undergo stronger compression than special camera shots. How precisely compression should be implemented in order to avoid breaking the illusion of natural speed is a matter for future investigation.8, and possibly even subtle addiction phenomena31. If liberalizing speed is the future of video technologies, we should be prepared for sub-threshold but intensive “kinetic hyper-stimulation”. Measuring speed tuning to natural speeds, in both attentive and inattentive conditions, would be a prime monitoring tool.Before applying speed-based compression indiscriminately, however, it will be important to appraise possible side-effects. For example, long-term exposure to high video speeds may lead to perceptual adaptationWe recruited 125 students enrolled in our Psychology study program . They took part in the first experiment and subsequently in the second or the control experiment on a voluntary basis, and gave informed consent prior to the beginning of the experiments. The study was conducted in accordance to the principles of the Declaration of Helsinki and the San Raffaele Ethical Committee.In the first experiment, participants were assigned to 5 groups (N = 25 each), and were blind to experimental manipulations. Participants sat in a classroom and watched a 10-min soccer video clip, in separate sessions. Each group watched the video clip played at a different speed: 0.88x, 0.96x, 1.00x, 1.04x or 1.12x. The group with speed = 1.00x consisted in fact of 2 subgroups tested in separate sessions (see below). At the end, the experimenter verbally asked the observers the following questions one by one, which they responded to in writing.Q1: Rate your concentration during the task (0–10)Q2: Which team had more ball possession?Q3: Did you notice anything strange in the video? If so, provide a brief description.Q4: Did you notice anything strange in the colour, shape or motion of the video? If so, provide a brief description.Q5: Was the video too slow/was the video too fast?Q6: Was the video too slow or too fast?Questions 1 and 2 served as generic indications of how much attention observers paid to the match, and targeted mental concentration and event following, respectively. Questions 3 and 4 quantified spontaneous noticing, the latter also providing three cues, one regarding motion. Question 5 configured a yes/no detection task, and consisted of 2 versions (see below). Question 6 configured a 2AFC discrimination task, where symmetry of the responses discourages bias. In passing from Q3 to Q6, the idea was to add specificity to each subsequent question. Thus, the Q3-Q6 sequence can be viewed as a progressively more stringent probing tool. Similar questions appear in commonly adopted protocols for subjective video quality assessment . Note that responding to Q6 is not subjected to the bias that might be present with Q3-Q5, which would manifest as a tendency to prefer the “no” response. Any bias with Q6would instead manifest as a preference for responding “too slow” or “too fast”, thus affecting PSE.In the second and the control experiments, participants  seated in a classroom watched a sequence of short segments of the same soccer video clip, whose reproduction speed was manipulated . They had to report on paper, trial by trial, whether speed appeared increased or decreased compared to the “natural”, original version, which was however never shown as standard , as well as a confidence rating for the response . Note that the 2AFC question was identical to Q6 of the first experiment. In the second experiment, half of the participants were presented a randomly generated stimulus sequence, and the other half viewed the same sequence in reverse order, in different sessions. For the control experiment, which had fewer observers, a third video sequence was used.Before the beginning of the experimental sessions, participants self-rated their soccer expertise and soccer practice levels on a 0–3 scale . These scores were normalized and expressed as percentages.The video clip was an accredited recording of an international soccer match, including the original soundtrack with voice commentary. A short 1280×720@30 fps version was obtained through video editing, lasting 9′36″. For simplicity we refer to it as to the 10-minute or short version (readers can contact the author to access the video clip). Editing also served to exclude irrelevant parts of the original video , and to place a grey patch over two frame regions containing an on-screen advertisement and the timing of the match, which could be a cue to recovering the true speed. This edited version was used in the first experiment. For the second and the control experiments, we randomly extracted 56 segments from the edited version, each lasting 15 seconds, one for each trial.https://wiki.videolan.org/) was used in the first and the control experiments, while in the second experiment a custom program was written in Matlab, using the Psychophysics Toolbox extensions. In the first experiment, we selected 5 speeds through VLC controls, nominally corresponding to speed gains of 0.88x, 0.96x, 1.00x, 1.04x and 1.12x. By comparing the true match duration (visible on the superimposed match timing) and the actual duration measured with a stopwatch in each speed condition, we empirically derived the effective video speeds achieved in the first experiment, which turned out to be 26.49, 28.81, 29.97, 31.18 and 33.87 fps, corresponding to 0.88x, 0.96x, 1x, 1.04x and 1.13x. For simplicity, we refer to this range as ±12%, and to the original speed as 30 fps. Given that calibration was performed over the entire 10-minute clip, and that stopwatch reading uncertainty can be estimated to be no more than 0.5 s, the measurement error was likely well below 0.1%. In the second experiment, we directly controlled video frame flipping, and dissociated the frame rate from the refresh rate , that is, we retained a frame for a number of refresh cycles, depending on the desired video speed. This way, we avoided two visual artefacts, namely, video tearing, which would have resulted from a crude disabling of the V-sync signal and unnatural motion, which could have resulted from frame interpolation. The frame rates achieved were 25.51, 27.00, 28.50, 30.00, 31.47, 32.97 and 34.50 fps, corresponding to speed ratios of 0.85x, 0.90x, 0.95x, 1.00x, 1.05x, 1.10x and 1.15x, thus in a range very similar to that used in the first experiment. Note that VLC does not actually implement frame interpolation and that video frame flipping is generated with the same principle that we used in the custom Matlab program, i.e., dissociating the frame rate from the refresh rate . For all experiments, a Sony fhz57 video-projector was used, driven by a Sony Vaio z equipped with an Nvidia GeForce GT330M graphic board, with V-sync enabled. The video clip was projected on a 100″ wall screen. The final projection quality was quite good, as attested by the near lack of observers’ remarks about video quality .For video clip reproduction, the open-source program VLC , while event-following was assessed through correct responses to Q2, both expressed as percentages. For spontaneous noticing (Q3 and Q4), only responses containing a reference to speed counted as positive evidence (correct responses).2 test.Detection accuracy was computed from Q5, and was defined as the ratio between the number of noticers (response “Yes!”) and the number of observers who watched the video at an altered speed. Note that question Q5 actually had 2 versions, one posed to those observers who watched a low-speed video (“Was the video too slow?”), and the other posed to those observers who a watched high-speed video (“Was the video too fast?”). This way, the task retained a yes/no detection and not a 2AFC discrimination structure. This also held for observers who watched the video clip at original speed, which were divided into two subgroups, each receiving the question in one of the two versions. We first computed a global false alarm rate, defined as the proportion of “noticers” (response “Yes!” to either question version) among those observers who watched the video clip at the original speed, regardless of the question version. The false alarm rate was also computed separately for the two question versions, which served as the correct comparison term when accuracy was measured for each individual observer’s group . False alarm rates were therefore the chance level for evaluating the detection performance with the χglm function. For the constant stimuli experiments, we used a GLMM approach to fit an individual psychometric curve to all subjects simultaneously6. To that end, we used the glmer function from the lme4 R package. In our analyses, all GLMMs included the intercept and speed in the fixed and random effects. In this framework, fixed effects represent the general tendency at the population level while random effects represent the individual deviations from the general trend. The random effects are assumed to follow a multivariate normal distribution with mean zero and unstructured variance-covariance matrix. The data did not show signs of over-dispersion, as indicated by the ratio between the Pearson residuals sum of squares and the residuals degrees-of-freedom, which was less than 1.1 in all experiments. To compare trial order in Experiment 2 or the psychometric functions across experiments, we included the corresponding factors among the fixed effects. We used the Wald test to compute the P values and parametric bootstrap with the percentile method to compute the confidence intervals (N = 500)33. Goodness of fit for GLM and GLMM models was assessed with the Hosmer-Lemeshow test, using the number of speed levels to group the data34.In the first experiment where a single response was obtained for each subject, we pooled data from all participants and fitted a logistic function with speed as predictor. To that end, we used R 0/β1 and JND = log(3)/β1 where β0 is the intercept and β1 the slope of the speed factor.The Point of Subjective Equality (PSE) was defined as the video speed at which an observer judged a speed increase or decrease as equally probable (corresponding to 50% of the psychometric function). The Just-Noticeable Difference (JND) was defined as the semi-inter-quartile difference of the psychometric function. Both PSE and JND are expressed as fractions of the original frame rate (30 fps). JND is always positive , while PSE is a signed value. PSE and JND were computed with the fixed effect parameters of the fitted models: PSE = −βThe datasets generated and/or analysed during the current study are available from the corresponding author.


The Transient Receptor Potential Melastatin Member 7 (TRPM7) is a divalent ion channel involved in cellular and systemic Mg2+ homeostasis. Abnormal expression of TRPM7 is found in numerous cancers, including colon, implicating TRPM7 in this process.Magnesium (Mg2+) status, the Mg2+ conducting channel TRPM7 in colon epithelial cells, and colon carcinogenesis, in vitro whole-cell patch clamp electrophysiology, qPCR, and pharmacological tools were used probing human colorectal adenocarcinoma HT-29 as well as normal primary mouse colon epithelial cells. This was extended to and combined with aberrant crypt foci development in an azoxymethane-induced colorectal cancer mouse model under hypomagnesemia induced by diet or pharmacologic intervention.To establish a possible link between systemic magnesium  contains supplementary material, which is available to authorized users. To test+]i Fig. . These dp < 0.01). To examine the effect of TRPM7 siRNA on TRPM7 currents, we performed patch-clamp recordings in HT-29 cells 72 h after TRPM7 siRNA transfection. As seen in Fig. p < 0.001). However, transfection with TRPM6 siRNA had no effect on TRPM7-like current . A 21-nucleotide siRNA duplex specific to human TRPM7 was used. Our q-PCR analysis showed a 67% decrease in TRPM7 mRNA in cells transfected with TRPM7 siRNA compared to scrambled siRNA control . However, transfection with TRPM6 siRNA had no effect on cell proliferation . This effect showed no significant difference between transfection of TRPM7 siRNA alone (65 ± 7.4%). Consistent with our mRNA analysis and electrophysiological recording, this result indicates that TRPM7 but not TRPM6 plays an important role in HT-29 cell proliferation.We next tested whether the activity of TRPM7 influenced the cell growth of HT-29. As seen in Fig. ion Fig. . Co-tran50 of 12.7 μM  from 2 mM to 0.05 mM significantly inhibited the cell proliferation of HT-29 (p < 0.05), which was similar to the effect of 10 μM waixA . It has been shown that the growth defect observed in TRPM7-deficient cells can be rescued by high extracellular Mg2+ supplementation via compensatory Mg2+ uptake pathways [2+. As seen in Fig. 2+ with 10 μM waixA was unable to rescue cell proliferation compared to incubation with 10 μM waixA alone . This might be explained by the low abundance or lack of alternative Mg2+ uptake mechanisms in HT-29. We therefore performed q-PCR analysis of Mg2+ transporters in HT-29 ). In the presence of physiological 390 μM [MgECs Fig.  & b. As +]I Fig.  & d. We 2+) or Mg2+-deficient (0.003% Mg2+) diet for up to 28 days. Mice were transferred into metabolic cages for 24 h before sacrifice, after which serum, urinary, fecal and bone samples were collected and assessed for Mg2+ and Ca2+ concentrations. The results revealed that mice fed the Mg2+-deficient diet excreted significant less urine and feces in 24 h than mice fed control diet while maintaining normal body weight . We conclude that a diet low in Mg2+ induces significant hypomagnesemia within 7 days, and that mice tolerate four weeks of significantly reduced Mg2+ intake.TRPM7 is emerging as a key player in cancer growth, migration and invasion. Increased expression levels of TRPM7 have been associated with poor prognosis in several cancers . Furtherht Table . Subjectiet Fig. , indicat2+ and Ca2+ analysis. The results show that, in comparison with mice of the vehicle group, mice treated with waixA had a significant decrease of Mg2+ concentration in serum for the first two days after injection, after which Mg2+ increased back to control levels , indicating that waixA increased intestinal Mg2+ absorption during that time. However, Mg2+ excretion through urine was not affected . Mice treated with waixA also had significantly reduced Ca2+ levels in feces  diet. After 1 week on their respective diet, mice were injected with either PBS alone or in conjunction with AOM (10 mg/kg) once a week for 3 weeks. The mice were sacrificed 2 weeks after the last AOM injection. To confirm hypomagnesemia, serum samples were analyzed for Mg2+ levels. As expected, all mice in the two Mg2+-deficient diet groups developed a significant decrease of Mg2+ concentrations in serum  compared to control, and, although not statistically significant (p = 0.2), tended towards increased ACF formation  and copper (Cu2+) seem to play a particular role in head and neck cancer [2+ supports HT-29 cell growth at 10 μM and inhibits it at 100 μM through a Zn2+-sensing receptor that does not seem to alter intracellular Zn2+ concentrations and involves the ERK pathway [2+) is typically associated with cell toxicity at various extracellular concentrations depending on cell type. For example, THP-1 cells, a monocyte-derived macrophage cell line, show strong cell growth suppression at 200 μM extracellular Ni2+ [2+ needs to reach 2 mM in order to perturb human osteosarcoma U2O2 or keratinocytes HaCat cell proliferation [2+ and Mg2+, and changes in cell proliferation.Perturbed metal homeostasis, in particular excess of essential metals, has been associated with carcinogenesis and metastasis . A meta-k cancer . In HT-2 pathway . Nickel lar Ni2+ . On the feration . It will2+ deficiency on early stage carcinogenesis, however, remains controversial [2+ levels in serum and altered Mg2+ absorption in colon (Figs. 2+ developed hypomagnesemia, this neither exacerbated nor reduced ACF formation (Fig. 2+ serum levels and decrease Mg2+ absorption in the colon, seemingly independent of TRPM7 (Fig. Our data confirm that waixA inhibits native TRPM7 currents in both normal primary mouse colon epithelial cells Fig.  and HT-2oversial . We hereon Figs.  & 6. Thu2+ status and propose waixA as a pharmacological tool in the study of TRPM7 in vitro and in vivo. While ACF formation remains unaffected by waixA, its impact on actual tumor growth remains to be investigated.We conclude that chemically-induced early stage colon cancer proceeds independent of systemic Mg


Recent evidence suggests that text messaging may help to reduce problem drinking as an extension to in-person services, but very little is known about the effectiveness of remote messaging on problem drinking as a stand-alone intervention, or how different types of messages may improve drinking outcomes in those seeking to moderate their alcohol consumption.We conducted an exploratory, single-blind randomized controlled pilot study comparing four different types of alcohol reduction-themed text messages sent daily to weekly drink self-tracking texts in order to determine their impact on drinking outcomes over a 12-week period in 152 participants (≈ 30 per group) seeking to reduce their drinking on the internet. Messaging interventions included: weekly drink self-tracking mobile assessment texts (MA), loss-framed texts (LF), gain-framed texts (GF), static tailored texts (ST), and adaptive tailored texts (TA). Poisson and least squares regressions were used to compare differences between each active messaging group and the MA control.When adjusting for baseline drinking, participants in all messaging groups except GF significantly reduced the number of drinks consumed per week and the number of heavy drinking days compared to MA. Only the TA and GF groups were significantly different from MA in reducing the number of drinking days. While the TA group yielded the largest effect sizes on all outcome measures, there were no significant differences between active messaging groups on any outcome measure. 79.6% of individuals enrolled in the study wanted to continue receiving messages for an additional 12 weeks at the end of the study.Results of this pilot study indicate that remote automated text messages delivered daily can help adult problem drinkers reduce drinking frequency and quantity significantly more than once-a-week self-tracking messages only, and that tailored adaptive texts yield the largest effect sizes across outcomes compared to MA. Larger samples are needed to understand differences between messaging interventions and to target their mechanisms of efficacy. The term “problem drinker” (PD) refers to individuals who experience low to moderately severe consequences from their alcohol consumption, do not exhibit serious co-occurring drug use and/or mental health disorders, and demonstrate higher psychosocial functioning than those with severe alcoholism, but still engage in heavy drinking and experience some alcohol related consequences . Most PDSMS interventions are emerging as a promising medium to deliver substance abuse-specific health interventions, as they are intrinsically proactive and can be accessed by the user at any time. Thus, goal-salient messages can be delivered at critical moments in substance abuse trajectories, such as when a craving occurs . In the There have also been some promising studies using smartphone applications for PD , 12 and Despite the recent literature on SMS for PD and other behavioral health problems, very little is known about the mechanisms that may explain the promising outcomes of SMS interventions, such as the impact of tailored message content, optimized timing of SMS delivery, and theory-driven vs. general motivational message content . In theiGain and loss-framed message content has received significant attention in the general health promotion literature and is the most studied framing paradigm for digital interventions across health domains , 14, 15.Similarly, while message tailoring has been studied in the general health promotion literature, there has been virtually no direct examination or comparison of tailored vs. untailored mobile interventions for any behavior. Tailoring can refer to personal relevance (e.g. name) or tailoring to a specific target behavior-related characteristic . Studies have generally suggested that content-tailored interventions yield better outcomes than untailored interventions, especially when content of the untailored intervention has no relevance to the recipient’s personal experiences –18. An aThe primary aim of this pilot study was to advance the literature on SMS interventions for PD by examining the effects of gain-framed (GF), loss-framed (LF), statically tailored (ST) and adaptively tailored (TA) SMS programs compared to weekly mobile assessment only (MA) on alcoholic drink reduction over a 12-week period in individuals seeking assistance on the internet to reduce their alcohol consumption to safe levels. To achieve these aims, we conducted an exploratory, single-blind, randomized controlled pilot study comparing effect sizes of weekly self-tracking mobile assessment only to the different daily messaging groups on reductions in weekly drinks, weekly heavy drinking days, number of days without drinking and drinking consequences over a 12-week period. Outcomes are designed to generate inform the development and testing of future messaging studies to reduce problem drinking.Participants were recruited between April 2014 and January 2015 through online alcohol screening and help-seeking sources, such as AlcoholScreening.org and Moderation.org. Advertisements offered individuals worried about their drinking the opportunity to screen for a research study to find out if texts could help them manage their alcohol consumption. Prospective participants were directed to the study website, which offered basic information about the study and a link to a brief screening survey in Survey Monkey. IP blocking once the web screening was completed ensured that participants could only complete the survey once from any given device. Individuals eligible to continue the screening process based on the survey were provided with the study’s email address to schedule a time to complete a 20-minute phone screening with study staff. Participants deemed eligible based on the screening call provided verbal assent and were emailed two links: one to a web-based consent form and consent form quiz, and another to the baseline survey. All participants electronically signed the consent form and any incorrect answers to the consent form quiz were reviewed to ensure comprehension. Ineligible participants were directed to online drinking resources, the SAMHSA Treatment Finder, and were offered referrals.Participants were eligible for the study if they: (1) consumed at least 13 and 15 standard drinks per week for women and men, respectively; (2) were willing to reduce their drinking to non-hazardous levels by study completion; (3) were between the ages of 21–65; (4) owned a mobile phone and were willing to receive and respond to text messages; (5) were fluent in English; and (6) could read at the 8th grade level, as determined by the consent form quiz. Participants were excluded from the study if they: (1) drank more than 45 standard drinks per week; (2) demonstrated clinically severe alcoholism ; (3) scored above a 12 on the Short Alcohol Withdrawal Scale ; (4) preStudy flow, reasons for exclusion, study assignment, and retention are presented in the Consort Flow Diagram . Within What to Expect document that corresponded to their study condition. Participants were blind to the study condition and were only aware that the type and number of messages differed between conditions. The condition-specific document trained participants on how to respond to the weekly mobile assessment (MA) texts and provided them with NIAAA guidelines for safe drinking by gender and information on what constitutes a standard drink [Once the participant completed the baseline survey, they were randomized to one of the five study conditions (see below) by the project research assistant (RA), stratified by gender and alcohol consumption. Envelopes were created based on gender  and high and low drinking, with equal chances of being selected into groups. At the halfway point of the trial, we checked group allocation and weighted the envelopes to create equal allocation across conditions. Once randomized, the RA assigned the participant into the proper messaging group, and all participants were emailed a rd drink . ParticiThe self-report internet screen included items to screen out potential participants based on age, drinking quantity and frequency via the QFV-30, a 30-day shortened time frame version of the Form-90 Quantity-Frequency measure of alcohol consumption , which hAt baseline and week 12, we assessed drinking outcomes with a more detailed version of the QFV-90, asking participants to provide an estimate of their average alcohol consumption per week and then an estimate of their average consumption by weekday . For participants randomized to the tailored adaptive group, we used responses to this item to send personalized texts on their heaviest drinking days. We assessed drinking consequences via the Short Inventory of Problems (SIP) . All meaParticipants in all conditions were given the NIAAA guidelines for safe drinking and asked to follow these guidelines to ensure that all participants were aware of the safe drinking guidelines and to offer more credibility to the mobile assessment control, as have been used in previous web-based interventions for alcohol use problems . Particiall messaging conditions received the MA as their base program.The MA group was designed to be a self-monitoring control, which has been used in both general health based SMS studies  and alcoThe loss-framed messaging program included text messages sent at 6:00 PM daily on the consequences of problem drinking. Messages were based on the existing literature and used both the social learning theory and the health belief model , which hThe gain-framed messaging program includes text messages sent at 6:00 PM daily on the benefits of reducing drinking to safe guidelines. Messages were mirrored to the LF program to provide information on the benefit that most corresponded to the opposite behavior described in each LF text message. This was also based on the social learning theory and the health belief model, with a focus on the benefits of not engaging in heavy drinking rather than the consequences of heavy drinking. An example of a GF message is, “Think of all you can achieve if you can control your drinking. Make today a day that sets the stage for change.”The ST treatment arm included tailored text messages sent at 6:00 PM daily based on individual responses to the baseline assessment. Tailoring included tailoring to the day of the week (e.g. a specific message on Friday night referencing the weekend), tailoring to the day in the program (e.g. “It’s been three weeks since you’ve signed up…”), and approximately 50% of messages were tailored based on participant gender, age, binge vs. daily drinking habits, solitary vs. social drinking habits, consumption severity, self-efficacy, motivation, craving, predicted effort required to reduce, and acceptance of the need to reduce alcohol consumption. The tailoring algorithm for continuous variables differed based on the median from our previous studies with problem drinkers.tempt for support to manage a craving to drink, drink for support when the participant had started drinking, heavy for support when the participant had started drinking heavily, win for support either when the participant succeeded at managing a craving or when the participant succeeded in drinking no more than their moderate drinking goal for the situation, and regret for support after failing to moderate.The TA intervention included all ST features along with three additional components. First, messages individuals received over the course of a week varied based on their goal achievement in the prior week. Second, two additional messages were sent that included the TA participant’s name each week at their heaviest typical drinking times. Third, participants were able to proactively text the automated system key words in order to receive just-in-time support, followed by a number of text-based check-ins. Key words included: coffee for alcohol. Messages were mirrored to the LF and GF programs as much as possible. The goal of including this program was to understand how many people would opt for NA messages and whether there would be a difference between NA and targeted content groups in terms of outcomes. This group was not included in primary analyses.An additional messaging program was included for participants who wanted to receive text messages that did not contain any reference to alcohol for reasons of stigma and privacy. Text messages were sent at 6:00 PM daily and included motivational messages on achieving one’s goals, the benefits of change, and the consequences of not changing. Mobile assessment in this group substituted the word ϕ) to allow for overdispersion and for non-normal distribution of count data [As in previous drinking moderation studies , the twount data . For theDescriptive data for the sample are presented in p < .09.), with the TA group yielding the largest effects, b = -0.40, p < .001. Multiple comparisons between treatment groups indicated that no treatment group emerged as significantly better than others, as indicated by overlapping confidence intervals. Changes from the baseline to week 12 by group are presented in Please see p =.15) when compared to MA. As with SSD, the TA group yielded the largest effects on this measure, b = -0.72, p < .0001. As before, multiple comparisons between treatment groups indicated that no treatment group emerged as significantly better than others, as indicated by overlapping confidence intervals.Assessment of the weekly sum of heavy drinking days (HDD) revealed a similar pattern, such that a significant main effect was observed for all treatment groups except GF (p = .08) when compared to MA, again with the strongest effect in the TA group, b = 0.78, p = .02. As before, multiple comparisons between treatment groups indicated that no treatment group emerged as significantly better than others, as indicated by overlapping confidence intervals.All treatment groups increased the number of days without drinking over the course of the trial, except LF  and days without drinking (.66 day increase), when compared to MA. Dichotomous analyses indicate that 31.3% reported at least moderate improvement in their drinking, which is similar to the LF group (34.6%).This is the first study to test theoretically distinct text messaging programs for any behavior change goal or health condition to weekly self-tracking assessment texts. All active messaging groups, except GF, were significantly superior to MA in reducing weekly alcohol consumption and heavy drinking days. Only the TA and GF groups were significantly superior to MA in reducing the number of drinking days per week. While TA had the largest effect sizes compared to MA, there were no significant differences between active messaging groups.Within an 8-month period, 1,149 individuals completed the screening questionnaire and 661 were eligible for the study, suggesting remote messaging interventions are an acceptable and feasible means to engage a large number of help-seeking PDs. Yet another notable finding was the remarkably high rate of participant retention from baseline to week 12 (94%) and the high level of interest expressed by participants to continue receiving messages after the treatment period. While only 64.4% of participants who completed the week 12 assessment actually signed-up, these findings are in contrast with the attrition rates associated with mobile app- and web-based digital interventions. SMS is an inherently proactive intervention, as messages automatically appear on the users’ screens, while web-pages and apps require user initiation. Thus, texting requires less user effort for the participant to receive intervention content reducing burden on the client to take the minimal necessary action to change.The primary reason for ineligibility at screening was low weekly alcohol consumption but a consistent pattern of single-night binge drinking. Given the often severe consequences of binge drinking, this group should be included in future trials. Similarly, we lost numerous potentially eligible participants between the web-screen and phone screening, suggesting a need for new methods to keep these individuals engaged in the enrollment process. Of the individuals not eligible during the phone screening, the majority were taking other substances regularly, including prescription benzodiazepines.When examined individually, all messaging groups outperformed MA on most primary outcomes. Like other messaging interventions for drinking with non-treatment seeking samples , the greThis study was not powered to detect significant differences between active groups, but the findings can inform future messaging development for health behaviors. Notably, TA consistently had the largest effect sizes among treatment groups, although effect sizes amongst treatment groups did not significantly differ. All messaging groups yielded meaningful reduction in weekly drinking, which highlights that even simple untailored daily messaging can have a positive impact on alcohol-related behavior. That these automated mobile programs significantly reduced heavy drinking has implications for improving the provision of low burden resources to heavy drinking help-seekers.The larger effect sizes, albeit not significant, of the TA group compared to others adds support for further study of just-in-time adaptive interventions to enhance behavioral outcomes , 32. HowThis was the first text messaging study to compare gain-framed vs. loss-framed mobile messaging for any behavior. We were surprised to find few differences in outcomes between these groups on any measure. While there were slight differences in the effect sizes of these groups compared to MA, they were not significant. Historically, GF and LF have been differentially used such that LF is used to motivate individuals to avoid future problems and GF to facilitate progress . Future There were no significant reductions in drinking consequences for any of the treatment groups compared to MA, though the TA group’s consequence reductions trended towards significance. This is similar to the findings of previous studies on brief in-person and web-based interventions which revealed few meaningful reductions in alcohol consequences , 35, 36.The addition of the general motivational messaging program containing no reference to alcohol represents important ground work for understanding the differential impact of content specificity vs. non-specificity on behavior change attempts. Although this group was not randomly assigned and therefore results should be viewed as exploratory, the NA group had smaller effect sizes on most outcomes than those of all other active messaging groups. This finding is in line with recent research suggesting that messaging targeted to the presenting problem was more effective in improving health outcomes than non-targeted messaging ; howeverWhile this treatment development and pilot study provide a rationale and foundation for further study of remote mobile SMS as an intervention for problem drinking, several limitations exist. First, sample sizes for each condition were small, limiting power for analyzing multiple treatment groups. Our sample was predominately white and female, limiting generalizability of findings to more diverse samples. The sample enrolled was similar in terms of gender break-down to the 1,149 total individuals screened on the web, suggesting that SMS may be particularly attractive to women seeking help or information online. This study was also very conservative with regard to who was enrolled compared to open real-world, web-based intervention studies, limiting generalizability to the heterogeneous group of PDs that might seek help on the internet. Expanding the reach of future studies to include a larger subset of treatment-seekers might result in significantly more individuals receiving messaging programs. Similarly, future research with larger samples will be needed to test effects over time before long-term efficacy can be determined.This study utilized consumption assessment methods that at present are not the gold standard metric when assessing in-person alcohol consumption . Future research with more resources should compare the reliability and validity of these brief measures when completed remotely with validated measures such as the online Timeline Follow-Back  or transS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


Xenopus embryos. Blocking HCN channels (hI currents) with pharmacological inhibitors leads to errors in organ situs. This effect is only seen when HCN4 channels are blocked early (pre-stage 10) and not by a later block (post-stage 10). Injections of HCN4-DN (dominant-negative) mRNA induce left-right defects only when injected in both blastomeres no later than the 2-cell stage. Analysis of key asymmetric genes' expression showed that the sidedness of Nodal, Lefty, and Pitx2 expression is largely unchanged by HCN4 blockade, despite the randomization of subsequent organ situs, although the area of Pitx2 expression was significantly reduced. Together these data identify a novel, developmental role for HCN4 channels and reveal a new Nodal-Lefty-Pitx2 asymmetric gene expression-independent mechanism upstream of organ positioning during embryonic left-right patterning.Laterality is a basic characteristic of all life forms, from single cell organisms to complex plants and animals. For many metazoans, consistent left-right asymmetric patterning is essential for the correct anatomy of internal organs, such as the heart, gut, and brain; disruption of left-right asymmetry patterning leads to an important class of birth defects in human patients. Laterality functions across multiple scales, where early embryonic, subcellular and chiral cytoskeletal events are coupled with asymmetric amplification mechanisms and gene regulatory networks leading to asymmetric physical forces that ultimately result in distinct left and right anatomical organ patterning. Recent studies have suggested the existence of multiple parallel pathways regulating organ asymmetry. Here, we show that an isoform of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of ion channels  is important for correct left-right patterning. HCN4 channels are present very early in  Summary: We identify a novel, developmental role for HCN4 channels and reveal a new Nodal-Lefty-Pitx2-independent, non-canonical mechanism upstream of organ positioning during embryonic left-right patterning. This article has an associated First Person interview with the first author of the paper as part of the supplementary information. Invariant left-right asymmetry is a fundamental aspect of all life, from single cell organisms to plants and animals with complex body plans like humans , 1991. CNodal-Lefty-Pitx2 as the primary node  involved in the establishment of left-right asymmetry in Xenopus embryos.Previous work has shown an important role of ion fluxes-mediated regulation of membrane voltage in determination of left-right laterality . Like otnt genes ,b. TheseXenopus embryos.Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a unique group of voltage-gated channels where the threshold voltage for opening of the channel is modulated by the metabolic state of the cell (levels of cyclic nucleotides like cAMP) . They op2014aXenopus embryos beginning at the first cleavage event (2-cell stage). Pharmacological inhibition of HCN channels (hI currents) by ZD7288 causes heterotaxia (randomization of organ situs) in Xenopus tadpoles, but only if embryos are exposed early . Similarly, injections of mRNA encoding a dominant-negative protein (which blocks HCN4 channel function) randomize organ asymmetry if injected into both blastomeres at the 2-cell stage. Remarkably, despite the early period of action (prior to asymmetric gene expression), HCN4 channel disruption does not affect the laterality of subsequent Nodal, Lefty, or Pitx2 expression, exhibiting significant latency in its effects on later organ situs. Together, these results are the first, to our knowledge, to show an early embryonic axial patterning role of this important ion channel, and implicate it in a pathway bypassing the asymmetric gene expression of Nodal, Lefty, and Pitx2 in regulating left-right asymmetry.Here we extend our recent work on HCN4 channels in cardiogenesis  using a hI currents) via a trapping mechanism (Xenopus embryos were exposed to ZD7288 (100 µM) from stage 1 through stage 10 or starting at stage 10 until stage 40, followed by anatomical laterality analysis at stage 45 (swimming tadpoles). Any instance of mirror image reversal of organ position, in the context of normal organ patterning and normal dorso-anterior index was counted as an instance of heterotaxia. Untreated embryos served as controls.Our prior work demonstrated that interference with four different ion translocators (two channels and two pumps) induces heterotaxia, the independent randomization of organ positioning along the LR axis . To testechanism . To probP<0.001, χ2) of heterotaxia in comparison to controls (∼6%) . Distribution of heterotaxia outcomes from the early ZD7288 exposure showed various combinations of asymmetric placement of gut, heart and gallbladder   A,B. In sls (∼6%) A,B. Similbladder C. Howevebladder) B,C. OverhI currents), resulted in high incidence of situs inversus. These data implicate the HCN4 channel in early (prior to stage 10) processes involved in determination of left-right patterning in the Xenopus embryo.These studies took advantage of the temporal control afforded by pharmacological experiments beginning at different timepoints. Treatment with ZD7288, which inhibits HCN (HCN4-DN (hyperpolarization-activated cyclic nucleotide-gated channel 4-dominant negative) mRNA construct that has been previously molecularly characterized and shown to inhibit the HCN4 channel current in mammalian cell culture by direct electrophysiology . We used a dominant-negative construct because many channels are present in Xenopus as maternal proteins  of heterotaxia only when injected in both blastomeres at 2-cell stage in comparison to controls (1%)  and heart (54%) were the most affected organs. In contrast to HCN4-DN, HCN2-DN mRNA-injected tadpoles showed no effect on laterality . Taken together, these data validate, in a gene-specific manner, the loss-of-function pharmacological inhibitor data implicating a role for HCN4 in LR patterning.To molecularly validate whether HCN4 channels are specifically involved in organizing left-right organ laterality, we used an ysiology . We confproteins , and thundpoints ]. HCN4-Dols (1%) A,B. Distols (1%) C, with aols (1%) B,C. OverHCN4-DN mRNA introduced even at 4-cell stage is no longer effective at randomizing the left-right axis.We then probed the time-dependency of HCN4 activity in left-right patterning by injecting at subsequent cleavage stages. We found that, even when adjusted for identical amounts and distribution of mRNA, injections performed after the second cell cleavage did not result in any significant incidence of left-right organ laterality defects in comparison to controls A. We conXenopus embryos. HCN4 channel immunohistochemistry signal was detected as early as cleavage-stage (2-cell stage) and was present through stage 9 in Xenopus embryos  pharmacological inhibitor ZD7288 (100 µM) between stages 1-10. Uninjected and untreated embryos were used as controls. The embryos were fixed at stage 21 for Nodal  of the embryos  expressing Nodal on the left side  of the embryos , showing Lefty on left side as in the set of control embryos , ZD7288 (100 µM), between stages 1-10. Uninjected and untreated embryos were used as controls [as these are known to be equivalent to water- or nonspecific mRNA-injected embryos  of the embryos  showing Pitx2 on the left side of the embryos similar to controls  of the Pitx2 transcriptional domain in the treated embryos in comparison to controls. To analyze this, we quantified the anterior to posterior spread distance and area of the Pitx2 signal in embryos  in HCN4-DN mRNA-injected and ZD7288-treated embryos  in HCN4-DN mRNA-injected and ZD7288-treated embryos  as post stage 10, HCN4 channel blocking has no effect on organ situs. Even within the first 10 stages of embryogenesis, the HCN4-DN construct induces heterotaxia only when introduced in all blastomeres during early cleavage stages, further suggesting a very early role of HCN4 in establishing left-right asymmetry. Interestingly, an early block of HCN4 with ZD7288 and HCN4-DN does not have a significant effect on the sidedness of Nodal-Lefty-Pitx2 gene expression, even though it leads to incorrect organ situs, suggesting it is acting either via a canonical pathway but bypassing Nodal and Lefty and directly affecting downstream targets or an alternative pathway of left-right asymmetry that is able to bypass the Nodal-Lefty-Pitx2 cassette completely. Blocking of HCN4 channels  induces a significant decrease in Pitx2 signal in its anterior-posterior spread and area of the signal.Here, we show that HCN4 channels are present in + and Na+ are reversed, affecting all K+ and Na+ ion channel fluxes: blocking HCN4 channel using HCN4-DN is a hyperpolarizing treatment and leads to subsequent incorrect Nodal-Lefty-Pitx2 expression and gross morphology defects of heart. In contrast, the experiments reported here made use of injections  into both blastomeres at the 2-cell stage, and the embryos were kept in low salt 0.1× MMR  throughout development. Under these conditions, an HCN4-DN-mediated block of HCN4 ion flux leads to depolarization  with normal sidedness and pattern of Nodal-Lefty-Pitx2 expression , strongly suggesting possible bioelectrically mediated Nodal-independent pathway that could correct for errors in the normal laterality-establishing transcriptional pathway.A recent series of studies and meta-analyses looking at the relation between causes of left-right abnormality, asymmetric gene expression of  defects . HoweverNodal and Lefty but incorrect organ situs after HCN4 inhibition (Pitx2 the sidedness of expression was correct (left side) but the area of expression was significantly reduced. Although Nodal and Lefty expression was unaltered, it is possible that HCN4 inhibition interferes with the function of Nodal and Lefty proteins, resulting in decreased Pitx2 expression and/or affecting other downstream pathways involved in left-right determination, thus acting through the pathway but bypassing the asymmetric gene expression of nodal and lefty genes. Alternatively, the decreased Pitx2 gene expression may be due to interference in its intronic enhancer binding function. Although Pitx2 is induced by Nodal (which is transiently expressed), its expression is maintained long after Nodal by binding of its intronic enhancer ASE with enhancers, including Nkx2 and Foxh1  expression within individual organ's tissues (particularly gut and heart) guides asymmetric morphogenesis of these organs (Xenopus)  Pitx2 signal in the lateral plate mesoderm may be affecting the Pitx2 expression in the heart and gut organs. However, unlike the heart and gut Pitx2 expression studies, we do not see morphological abnormalities in organ patterning. The gut and heart are placed in (left-right orientation) either normal or revered orientation but with fully formed morphologies.Previous studies have shown that e organs . StudiesXenopus)  have shoNodal-Lefty asymmetric gene expression , often observed in mice but rarely observed in Xenopus (situs inversus), as the left-right axis is randomly oriented with respect to the anterior-posterior and dorso-ventral axis  will result in each organ making an independent decision leading to predominantly heterotaxic individuals.Left-right asymmetry establishment can be broadly categorized into three steps: (1) symmetry breaking, (2) orientation of the axes, and (3) amplification steps . Disrupt Xenopus . Disruptral axis . Lastly,situs inversus and a small incidence of the bilateral gut (isomerism) . This is further supported by the observation that HCN4 channels are present from early cleavage stages, and their role in laterality seems to be executed during early cleavage stages. Similar to HCN4, disrupting cytoskeletal dynamics causes a major incidence of situs inversus   and 2; iinversus . It is ninversus . It is p+/K+-ATPase and V-ATPase (ATP) have been implicated. For the majority of these ion translocators, their asymmetric localization and action is post 4-cell stage, but before gastrulation. Crucially, HCN4 inhibition affects asymmetry during early embryogenesis – an observation that is incompatible with potential hypotheses about roles in regulating much later events like ciliary motion at gastrulation , as well as transgenic Xenopus in which native HCN4 is labeled with a fluorescent tag, will dissect the very early steps of HCN4 activity. New techniques for introducing material into Xenopus eggs prior to fertilization may also be useful in manipulating this process, since injections even at 1-cell stage may be attenuated in their effects by the amount of time needed to make protein from the mRNA introduced then.We found that HCN4 channels are already present in 2-cell embryos  and are uniformly expressed throughout the embryo all the way through gastrulation . Hence, al level . Moreovenodal-lefty-pitx2 to exert their effects much later. Moreover, the discovery of new ion channels that underlie the endogenous bioelectric signaling that is increasingly seen to be an important component of developmental . Xenopus embryos were housed at 14-18°C (14°C overnight after injection and subsequently at 18°C), except during drug exposure which was at 22°C, and staged according to rotocols  in 0.1× HCN4-DN was a mammalian (mouse) HCN4, modified as per 349-351 to AAA349-351) to generate HCN4-(AAA)-DN mutant from HCN4-WT using primers: 5′CACATGCTGTGCATTGAGGACGAACGTCAGGCA-3′ (forward) and 5'-TGCCTGACGTTCGTCCTCAATGCACAGCATGTG-3′ (reverse). The construct was subcloned into a pCS2 vector to transcribe into mRNAs for Xenopus microinjections.Capped synthetic mRNAs generated using mMessage mMachine kit (Ambion) were dissolved in nuclease free water and injected into embryos immersed in 3% Ficoll using standard methods . Each inXenopus embryos were analyzed as in Xenopus embryos were incubated in pharmacological blocker of HCN4 channel ZD7288 (Tocris biosciences) (100 mM stock solution in water) dissolved in 0.1× MMR  during the stages indicated in respective experiments followed by several washes with 0.1× MMR. Note that these experiments were performed at 22°C since ZD7288 effect on embryonic left-right asymmetry was found stronger at 22°C and not at 14°C and 18°C (data not shown) (t shown) . Untreat4(3) voltage reporter dyes were obtained from Invitrogen and used as per the standard protocol, including dark-field and flat-field correction (4(3) stock (1.9 mM) was dissolved 1:4000 in 0.1× MMR and the CC2-DMPE-stained embryos were then incubated in dark in this solution for at least 30 min followed by visualization under the microscope. An Olympus BX-61 microscope equipped with a Hamamatsu ORCA AG CCD camera, and controlled by Metamorph software (Molecular Devices), was used to collect signal. NIH Image J software was used to quantify the fluorescence intensities of the CC2-DMPE:DiBAC signal.CC2-DMPE and DiBACrrection . BrieflyMembrane potentials were measured using an oocyte clamp OC-725C amplifier  with a single voltage electrode. Microelectrodes were made from thin-walled borosilicate glass pulled with a flaming/brown micropipette puller  and back filled with electrode solution . Tip resistances were 80-100 MΩ. Electrode penetration of ectodermal cells was by visual guidance on a fixed-stage microscope (Zeiss) using a three-axis micromanipulator.Spatial distribution of HCN4 channel in embryos was detected by immunofluorescence for the HCN4 channel on whole embryos. Briefly, embryos were fixed overnight in MEMFA at 4°C . The embXenopus embryos were collected and fixed in MEMFA (in situ hybridization was performed as previously described (In situ anti-sense probes were generated in vitro from linearized templates using a DIG labeling mix (Roche). Chromogenic reaction times were optimized for signal to background ratio. Probes used were: Xnodal  or ANOVA (for more than two groups), as indicated with each experiment.All statistical analysis was performed using Microsoft Excel. As appropriate for each case, data were either pooled from multiple repeat experiments, with χ


This TJHP thematic series provide an overview of the history of the discovery of PACAP and its three receptors  and summ


Forty years after they were first introduced to the general public, video games have become firmly established into the mainstream of entertainment media. In parallel, early adopters and enthusiasts have become active scientists. As a result, interest in video gaming practices has shifted from an exclusive niche leisure activity to extensive social, commercial, cultural, medical, and psychiatric issues. An increasing number of studies on game-based treatments for various mental health disorders suggest that these approaches have the clear potential to promote cognitive and behavioral changes as well as symptom relief –10.Tetris, Bejeweled) had positive effects on depressive and anxious symptoms  that game and video game practice may have a positive impact through the transfer of cognitive skills acquired in the game to functional competences , 18, altAnother meta-analysis on the effect of brain-training games in older adults  showed aData on naturalistic use (in milieu use outside the scope of specific research programs) of internet-based therapies suggest that these programs are appealing for many users, particularly so for people who could benefit significantly from such treatments . Naturalvia the use of serious games tools  list of video game components that have often been related to addictive use in past research , but whiVideo games offer countless opportunities to optimize the distribution of reinforcers. The pioneering work of Schultz and colleagues on rewards  has showIn the next sections, we provide illustrations of how MMORPG-based reward distribution could be used in games designed to increase wellbeing. In the case of video games that aim to improve physical condition , a solution to compensate for the frustration of the difficult first steps of practicing (as in the violin example) would be to increase the amount of rewards at the beginning of the game. Users could initially gain in-game rewards and levels after running for short distances and, therefore, experience the subjective impression that the task is rapidly rewarding. Such a view is supported by a recent review on exergaming .A more tentative proposition would be a game designed to overcome social phobia, which could take the form of a multiplayer game where players are encouraged to have and rewarded for having face-to-face video conferences. In such a game, every social contact should be rewarded according to the reinforcement schedule depicted earlier, so that the participant fully appreciates his or her progression. Using such a game is susceptible to optimize the learning process of social exposure, in comparison to real-life situations where no direct positive feedback follows an exposure exercise (the shopkeeper will rarely congratulate the patient for sustaining their gaze and speaking clearly).These rewards mechanisms can also have downfalls. The loss of the novelty effect is one: the generalization of the so-called gamification of everyday activities tends to reduce the effect of virtual rewards, thus the incentives to receive virtual rewards could progressively decrease. It is also important to bear in mind that in-game rewards remain intangible; strategies to increase behavioral modification could link virtual rewards to real ones to increase their attractiveness . Similar strategies have been applied for a long time in contingency management for the treatment of substance use disorders with positive outcomes overall .Zombies, Run!, an audio/video game that couples running exercises to missions where the player must escape zombies chasing them. Running with headphones, the player hears where zombies are lurking and how to escape them. The more they run effectively, the more they gain levels and bonuses. A problematic use of this game could appear if the player’s purpose becomes not to enhance their running ability, but rather to progress in the game as fast as possible. As they become addicted to the in-game rewards, they will start to play the game while driving their motorcycle or car to be more effective. This supposition might sound unrealistic, but it is in fact the core mechanism of the so-called “free-to-play” games, where the player has the choice to either play a game for free, but must play for very long to get a reward, or pay to progress much faster and avoid the long wait  will not have the same visual or emotional impact as software developed by such a multidisciplinary professional team. As an illustration, a video game to treat anxiety via biofeedback, called Mindlight,savoir faire with scientific knowledge. The result is designed to propose the appealing visuals of a state-of-the-art video game with a scientific rationale.Developing a video game requires a large multidisciplinary team of programmers, artists, scenarists, 3D motion specialists, etc. Obviously, a task generated with the tools traditionally used in experimental psychology and psychiatry  become affordable and broadly available and could greatly enhance immersion. For example, people who are afraid of driving or flying could play realistic simulations to have a first experience, and eventually gain knowledge and confidence in these activities (The use of virtual avatars could also be helpful in psychiatric treatments, as, for example, evidenced by a recent study that demonstrated how using avatar-based therapy is useful to decrease persecutory auditory hallucinations (Video games give opportunities to experience sensations otherwise too dangerous, forbidden or inaccessible. tivities . This cotivities  (e.g., etivities . The useinations .The popularity of video games has been established in the past four decades and is still growing. Nowadays, successful video games are designed for entertainment, but as researchers are analyzing the mechanisms of this success, they are more and more confident on the possibilities of designing video games for wellbeing. In this article, we formulated ideas to suggest how to take advantage of the core attractive mechanisms of MMORPGs, such as conditioning reward schemes enhanced by players’ social interactions Table .We also pointed out some obstacles to the realization of video games for wellbeing, such as the lack of academic resources compared to the game industry and the risk of encouraging problematic or addictive video game use. Technological progress in the field is rapidly evolving, and virtual or augmented reality will soon provide new innovative tools to further the development of video games for wellbeing.All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.