Visuospatial neglect due to stroke is characterized by the inability to perceive stimuli emerging in the area opposite to the side of brain damage. Besides adopting conventional rehabilitation methods to treat neglect symptoms, the use of virtual reality (VR) is becoming increasingly popular. We designed a series of 9 exergames aimed to improve exploration of the neglected side of space. When new VR interventions are designed, it is important to assess the usability aspects of such management strategies within the target population. To date, most studies used questionnaires to assess user satisfaction with the intervention or product being tested. However, only a combination of both quantitative and qualitative data allows a full picture of user perspective.The purpose of this study was to quantitatively and qualitatively assess patient and therapist perspectives of a VR intervention based on the series of 9 exergames designed to explore hemineglected space. Specifically, we wanted to evaluate (1) perceived-user friendliness of the exergames, (2) attitude towards using the exergames, and (3) intention to use the exergames in the future.A total of 19 participants  evaluated the exergames they had used 5 times a week during 3 weeks. The Technology Acceptance Model (TAM) questionnaire was filled out after the intervention. Based on those responses, we conducted focus group interviews (with therapists) and individual interviews (with patients). To analyze the TAM questionnaires, we used descriptive statistics. We adopted content and comparative analysis to analyze the interviews and drew illustration maps to analyze the focus group interviews.The therapists took a more critical stance with a mean TAM questionnaire total score of 48.6 (SD 4.5) compared to the patients who had a mean total score of 56.1 (SD 12.3). The perceived user-friendliness score was 5.6 (SD 1.4) for patients and 4.9 (SD 1.4) for therapists. The attitude towards using the exergames was rated 4.8 (SD 1.9) by patients and 3.6 (SD 1.4) by therapists, respectively. The intention to use the exergames in the future was rated 3.9 (SD 2.1) by patients and 3.7 (SD 1.8) by therapists. We gained information on how to improve the exergames in the interviews.Patients and therapists perceived the exergames as user-friendly; however, using the games further with the actual test version was not perceived as conceivable. The therapists were generally more critical towards future use than the patients. Therefore, involving both users to achieve acceptable and user-friendly versions of game-based rehabilitation for the future is deemed crucial and warranted.Clinicaltrials.gov NCT02353962; https://clinicaltrials.gov/ct2/show/NCT02353962 (Archived by WebCite at http://www.webcitation.org/6soxIJlAZ) Stroke-related visuospatial neglect (VSN) due to a right-sided brain lesion (RBL) is characterized by the inability to perceive stimuli emerging in the area opposite to the side of brain damage ,2. VSN pBesides adopting conventional rehabilitation methods to treat stroke-related VSN symptoms, the use of virtual reality (VR) in their assessment and treatment is becoming increasingly popular -8. VR isA European research group, Rehabilitative Wayout In Responsive Home Environments (REWIRE), developed a game-based VR rehabilitation intervention trying to account for those challenges : (1) theWhen new VR interventions are designed, it is important to follow a phased iterative approach, wherein the usability aspects of such a management strategy, within the target population, are first assessed . UsabiliAssessing opinions from users can be done by using questionnaires or by means of interviews. The former has the advantage to assess many opinions of a representative sample but cannot tell us about the meaning behind a response. The latter is usually applied in a smaller sample but provides personal thoughts from an insider’s perspective . To dateResults of a recent systematic review state that in the posttest stage of usability evaluation, performing interviews to evaluate user perceptions of games is recommended, whereas the use of questionnaires is considered useful for evaluating user acceptance and satisfaction . As a coFor this usability study, we used a mixed methods design adopting the “sequential explanatory” design strategy . This deThere were 2 groups of users involved in this study: patients as end users and therapists as experts.The patient group included 7 adults with an ischemic (n=5 men) or hemorrhagic (n=2 women) RBL due to a first stroke with accompanying VSN symptoms as measured with the Catherine Bergego Scale (CBS) . The CBSThe expert group consisted of therapists responsible for the treatment of the stroke patients during their inpatient stay. The 12 therapists  supervised and trained the patients in the use of the REWIRE exergames during the 3-week intervention phase. Their mean age was 33.3 (SD 5.7) years (range 27-45) with a mean work experience of 6.8 (SD 5.8) years (range 0.5-20). All therapists stated being familiar with the use of computers, rating their computer knowledge as excellent (n=3), good (n=8), and poor (n=1). All 19 participants signed informed written consent before study entry. We obtained ethical approval for the study from the local Ethics Committees (Zurich No. 2014-0543 and Bern No. 389/2014) as well as from Swissmedic (2015-MD-0003). The study is registered with ClinicalTrials.gov.We installed 2 exergames training stations, one in each of the collaborating clinics . The gamWe designed a series of 9 exergames aimed to improve exploration of the neglected side of space. During the development of the exergames, we regularly tested them in healthy controls prior to implementing them in a clinical setting. Their feedback was constantly integrated in the development process until consensus was reached. In order to maintain principles of training, game progression was individually adjustable through the selection of appropriate different levels of difficulty (more demanding meant more exploration towards the hemineglected side was required) . The exeBoth therapists and patients had the opportunity to test the exergames before entering the study, followed by a training event organized by the research team to learn, for example, how to handle the game menu and Falcon Novint haptic device. During the whole intervention phase, the research staff provided telephone or personal support whenever needed, for example, to handle technical problems with the training station. The exergames intervention lasted 3 weeks and included 15 training sessions each of approximately 30 minutes duration. Patients exercised with the games under supervision of the therapists depending on their required level of support, for example, to start a new game. The neuropsychologists included exergames playing in their computer group, meaning that participating patients played the REWIRE exergames while other group members performed alternative computer tasks. The occupational therapists (OTs) supervised their patients in a one-to-one setting during individual therapy sessions. Additionally, the supervising therapist individually adjusted the intensity of playing the exergames. This was done, for example, by changing the difficulty level or game duration in the game menu or by implementing short breaks between each game if needed. Each patient selected up to four REWIRE VSN exergames from the game menu to be played in a gaming session. The choice was based on personal interest of the patient, which was assumed to enhance motivation while playing. Therefore, during the 3-week intervention time, the patient was also allowed to change games if they wanted to test another game or felt bored with the previously played one. However, we suggested the patients test all of the 9 games at least once. Rehabilitation continued during the study intervention, our exergames serving as an additional therapy option to the standard program comprising daily occupational, physical, and neuropsychological therapy.Both patients and therapists completed a questionnaire at the end of the intervention. This included 12 questions with a 7-point Likert scale , evaluating (1) perceived user-friendliness of the exergames, (2) attitude towards using the exergames, and (3) intention to use the exergames in the future. The questionnaire design was based on an abridged version of the Technology Acceptance Model (TAM). TAM is an intention-based model developed specifically for explaining user acceptance of computer technology  that we BC-T-A performed the audio-recorded individual interviews during the follow-up assessment planned for the feasibility study 4 weeks post-intervention. They focused on the patients’ everyday life experiences with right hemispheric stroke and VSN symptoms during active rehabilitation and served as an opportunity to deepen, clarify, or confirm answers that were given in the TAM questionnaire. BC-T-A, who is an occupational therapist, took an active role during the interviews, aiming to build a relationship with the participants based on confidence and co-creation. Thanks to the many opportunities to meet the patients in the past , a goodWe conducted the two focus group interviews in the collaborating rehabilitation clinics after the last patient had finished the REWIRE exergames intervention. We audio-recorded and filmed them. In comparison to individual interviews, focus groups represented group opinions influenced by social interactions and team dynamics, as therapists already knew each other well . TherefoWe expected the acceptance of and satisfaction with the neglect exergame intervention to be good, which we defined as a total mean score of more than 4 points on the Likert scale, where 1-3 points meant no agreement, 4 points neutral, and 5-7 points agreement. The maximum achievable score was 84 points, indicating perfect agreement. For the individual and focus group interviews, we hypothesized that the users (therapists and patients) would have experienced the VR-based neglect training as supportive in treating VSN.To analyze the TAM questionnaires, we used descriptive statistics in SPSS software version 23. We calculated means, standard deviations, medians, and interquartile range values as appropriate.We transcribed the individual interviews verbatim. Subsequently, we selected text passages from the entire conversation in which the interviewer (BC-T-A) and the patient discussed the use of the exergames and stored them separately. We used content and comparative analysis to analyze those passages ,36, takiWe analyzed the two focus group interviews by drawing “Focus group Illustration Maps” (FIMs) . The aimWe summarized the answers from the TAM questionnaires in This subcategory was the most positively judged among patients with a mean 5.6 (SD 1.4) points and therapists with a mean 4.9 (SD 1.4). All patients agreed on the clarity of the manual and the ease of use of the exergames, while learning to use the exergames independently would not have been easy for patients 1 and 6 , suggesting use of the mouse instead of the Novint Falcon as input device in order to solve this problem.The therapists all agreed on the game manual’s good understandability . They raThe general attitude of the patients toward the use of the exergames was 4.8 (SD 1.9) points . The theHowever, most patients preferred the conventional therapy methods over the exergames intervention, as they described it difficult to understand the purpose of the games:In the beginning, I didn’t really understand what all this meant; for what the games were good for. Then they [the therapists] explained it to me. After they had told me what aspects I had to pay attention to, then it was all good.P2I couldn’t make sense out of it [the exergames]. I always had the feeling that the things there—those tests—were meant for ones with very severe brain damage, who weren’t back on their feet yet. […] But not for me—I don’t have such severe damage!P3I don’t know what they [the exergames] would have been useful for. And no matter how much you have scored, you couldn’t see the progress you had made.P6The patients experienced conventional methods as more effective than the VR intervention:I didn’t have the impression that it [the exergames intervention] did yield much. I experienced it as being a bit silly. I had the impression that the other things simply helped me much, much more. […] The eyes were not equally challenged to move back and forth.P6The content of the games was judged as “not bad” (P4). P4 perceived the exergame “puzzle” as being difficult because the puzzle template was displayed only once at the beginning, requiring the player to piece together the puzzle out of memory. The speed of the games and the related short reaction time was another difficulty mentioned by most patients as being experienced during play. They described being initially very motivated to play the games, but over the course of the 3-week intervention, their enthusiasm decreased, as they started to perceive playing the games as “boring”  and even “childish” :You know, piling the ABC can be done by a first- or second-former! And to burst balloons that pop up out of a hole isn’t very demanding either” (P3); and “Boring! In the beginning, it was good. But most recently…it was complicated to look through this thing [chin rest], you know. The other games they had were more interesting in a way.P4Using the chin rest while playing was the main reason why most patients experienced the exergames as exhausting (mean 3.9 [SD 2.3]):This [chin rest] wasn’t useful! I couldn’t sit in an upright position and look through [the chin rest] to scan the whole computer screen. This was exhausting. You also weren’t able to turn your head.P4The suboptimal posture of the patients while using the exergames was also problematic for the therapists  and 4. IUsing the exergames regularly in the future was not viewed as conceivable yet, either among patients (mean 3.9 [SD 2.1] points) or therapists (mean 3.7 [SD 1.8] points). Most patients perceived the exergames as a good pastime and diversion that helped shorten the long days in the rehabilitation clinic  but indicated that they would prefer doing activities other than gaming once back at home: “Up there [in the rehabilitation clinic], I thought that it is way better to do this [playing the exergames] than lying in bed or sitting on a chair while doing nothing” (P6). P5 and P7 described themselves as not being “a computer freak” (P7) or “a gamer” (P5) and therefore not wanting to use the games further at home. P3 missed the relevance to real life of the games, making the following suggestion for improvements:Well, maybe tests that are more related to practice. You know, where you see: “Ah, this could be useful!” […] For example doing an exercise you will need in the future when you want to drive a car again. Reaction or such things…which will help me to go ahead.P3For some patients, the games could have been more challenging and entertaining. P7 did not experience much pleasure while playing: Not really…well, when I was successful, then I felt pleasure anyhow. Then I thought: ‘Indeed, I am not as dull as I thought!’ […] It simply worked out somehow, but not as good that I would have felt pleasure to play more.P7Remarkably, most patients were nevertheless convinced that their family and friends would support them using the exergames at home. The patients also stated that they would recommend the exergames to other patients . ReasonsThe therapists were not yet ready either to use the exergames in the future or to recommend their use to other colleagues—at least in the version used for this study—although all therapists were convinced that their workplace supports the use of VR training methods . The neuThe therapists proposed suggestions for improvements for all mentioned barriers see , which, This usability study aimed to quantitatively and qualitatively assess user perspectives (patients and therapists) of using REWIRE exergames as a novel rehabilitation intervention to treat VSN symptoms due to stroke. The findings showed that the patients as end users generally rated the use of the exergames more highly than did the therapists. Most patients experienced the games as motivating, interesting, and a welcome diversion in their daily routine during their inpatient stay in the rehabilitation clinic. The feeling of joy and motivation while playing was also described in other studies assessing user perspective in stroke patients testing a novel VR intervention ,37,38. TOur sample, however, suffered from neglect and a certain related level of anosognosia . They neMainetti et al  tested eThe lack of feedback experienced by fitter patients, combined with their perceptions of being insufficiently challenged while playing the exergames, might be reasons why they indicated preference for conventional therapy methods with a “real” therapist over this VR intervention. Their experiences of the games as “a good pastime and diversion” suggests that most patients did not see this as a rehabilitation intervention per se, supporting their preference for conventional therapy. This is in line with other findings, where stroke survivors experienced the novel games as supplementary to conventional therapy, the latter being viewed as providing beneficial rehabilitation . ConversThe REWIRE game design was rated as having limited appeal by both therapists and patients. The therapists in particular wished to have games that would be self-adaptive to patient progress in order to experience ease of responsibility. For example, they missed the opportunity to prepare a series of games that would then automatically run through during an intervention session. The lack of facility within software for patients to save and return to previously achieved difficulty levels between sessions was also noted. Those features would allow the patient to start directly at the right difficulty level and subsequently play the game independently without the therapist needing to adjust the settings before and during the training session. Although the goal of such VR interventions is to create a game menu that patients can run themselves with little input from others, it is nonetheless imperative that an expert  guides progression of the games to maintain the therapeutic basis of the intervention. In one study, where game speed and progression advanced automatically, the users were overwhelmed, which negatively influenced motivation and engagement in the game intervention .Some of our patients indicated that they missed the exergames’ relevance to real-life tasks—feedback also given by other stroke patients testing similar interventions . This isWhen supporting patients to play the REWIRE exergames, some OTs expressed uncertainty in achieving carryover effects into real-life tasks. This uncertainty was one of the reasons why they would have preferred to use time for the training of real ADL rather than game play to achieve the rehabilitation goals set for their patients. Indeed, evidence for positive carryover effects of VR interventions into real life is limited ,46. For Further reasons why the therapists would have preferred use of rehabilitation time for conventional therapy rather than for testing the novel VR intervention was the breakdown susceptibility of the software—giving them the feeling of wasting too much therapy time. Additionally, the use of the chin rest forced the patients to sit in a nonergonomic posture. This posture was also the reason why many patients got tired while playing, rather than because of cognitive challenge. When planning this study, we did not expect the chin rest to be a major problem when playing the exergames. Its use was precipitated by a need to avoid compensatory movements of the head. However, according to the feedback of all participants, the use of the chin rest for a whole therapy session of approximately 30 minutes was too exhausting. We therefore recommend the use of a chin rest for short assessments only rather than for a whole therapy session . Such teSome limitations of this study should be discussed. First, most of the stroke patients needed assistance in completion of the TAM questionnaire, either in retaining the paper-based questionnaire while it was on the table due to their hemiparesis, or in being helped to read the questions due to their VSN symptoms. Both of these issues may have influenced their responses. A touch-screen version on a tablet fixed on a table to avoid side slipping for those stroke patients who suffer from hemiparesis would allow questionnaire completion with one hand only. A button placed on the right margin of the tablet could be designed to audio-display the questions, making reading of the questions unnecessary. Second, the fact that the therapists participating in the focus group were working colleagues from the same team might also have influenced their interactions and utterances during the interview. For example, in both interview groups, the team leader was also present, which might have inhibited some participants in expressing what they really thought about the exergames. We therefore chose focus group illustration maps for data analysis. Together with the flip chart notes taken directly during the interview, those FIMs allowed a precise summary of the group statements without exposing someone through using quotes, where they might recognize the person who had said that. Third, the fact that the main researcher (BC-T-A) knew all participants quite well at the time point of the interview influenced her way of conducting the individual and focus group interviews. Holding preunderstanding about the patients’ life from former meetings during data acquisition for the feasibility study might have influenced her way of formulating questions differently than when she would have met the patient for the first time. However, the interview quality probably had improved thanks to the already established relationship. Being an occupational therapist like half of the participating therapists further influenced the flow and conduct of the focus group interviews. However, speaking the same professional language might have facilitated formulating experiences made with the patients and exergames. Fourth, the recruitment of stroke patients with VSN symptoms in a clinical setting who were fit enough to test the game-based VR intervention was quite difficult. Testing such an intervention in a later, chronic stage where most patients are in a better health condition might have been easier. However, all patients were excited to take part in a research project during their inpatient stay and they cherished being asked for their personal opinion not only in a questionnaire, but also in a face-to-face interview.Lewis and Rosie  were entThis study provided insight into user perspectives based on quantitative and qualitative statements of stroke patients suffering from VSN and therapists using novel exergames to explore the hemineglected left space in an inpatient setting. The results showed that all users perceived the REWIRE exergames as user-friendly, but that they would not necessarily entertain their use in their current format. The general attitude toward using the exergames was more positive among the patients than among the therapists. Recommendations for improvements of the exergames were mainly formulated by the therapists. Feedback suggests that once those recommendations could be realized, then the REWIRE exergames intervention could be explored using further trials. It is therefore of the utmost importance that end users (patients) and experts (therapists) are involved in order to achieve acceptable and user-friendly VR game-based rehabilitation methods.


Sounds associated with newly learned information that are replayed during non-rapid eye movement (NREM) sleep can improve recall in simple tasks. The mechanism for this improvement is presumed to be reactivation of the newly learned memory during sleep when consolidation takes place. We have developed an EEG-based closed-loop system to precisely deliver sensory stimulation at the time of down-state to up-state transitions during NREM sleep. Here, we demonstrate that applying this technology to participants performing a realistic navigation task in virtual reality results in a significant improvement in navigation efficiency after sleep that is accompanied by increases in the spectral power especially in the fast (12–15 Hz) sleep spindle band. Our results show promise for the application of sleep-based interventions to drive improvement in real-world tasks. Sleep may facilitate the transformation of recent fragile memories into stable long-term memories. Compared with an equivalent period of wake, performance in several memory domains demonstrates a greater magnitude of improvement after sleep . SeveralTemporal coupling of SOs, spindles, and hippocampal sharp wave-ripples may be a key mechanism underlying the hippocampal-neocortical dialog characteristic of systems consolidation. The drug zolpidem (Ambien) increased the temporal consistency of spindle occurrences during the down-to-up phase of slow oscillations . FurtherTargeted memory reactivation (TMR) has been successful in enhancing memory during sleep using external stimulation. The TMR approach associates sensory stimuli  with target information during encoding and then presents the same cues during sleep to facilitate memory consolidation, including visuospatial , verbal In the current study, we developed a novel method to enhance sleep spindles and spatial navigation skills using closed-loop targeted memory reactivation (CL-TMR) time-locked to the down-state to up-state transitions (DUPTs) of SOs. We tested navigation ability at multiple time points across 3 days in order to determine when the benefits of CL-TMR emerge, and how long they are observed. DUPTs were targeted to increase the likelihood of affecting spindles during the rising phase and peak of the SO. In this study, we did not investigate whether the closed-loop cue delivery confers significant benefit when compared with an open-loop approach. Multiple studies suggest that TMR may be most effective when time-locked to the DUPTs , and a rN = 37, 16 women, Mage = 25.14 years, SDage = 5.75 years) were recruited from the surrounding communities in Durham, NC; Dayton, OH, United States; and Riverside, CA, United States. They were instructed to avoid caffeine, alcohol, and naps within 24 h of any study session. Participants arrived at the laboratory around 12 pm. Data from an additional 18 participants  were excluded because of one or a combination of the following criteria: they could not complete the VR task inside the headset due to nausea, they did not receive any cues during both naps or only received cues during one of the naps, the data recordings were of poor quality , and/or they did not sleep at least 30 min with at least 15 min in stages 2 or 3. Demographic data of included participants are shown in Table 1.This study was approved by the New England Independent Review Board and all participants gave written informed consent in accordance with the Declaration of Helsinki. Healthy individuals  data were collected on a Brain Products 32-channel actiCAP electrode system and BrainAmp DC amplifier  using the standard 10–20 electrode layout. Electrocardiogram (ECG) and electrooculogram (EOG) electrodes were used for offline artifact rejection and assessment of rapid-eye movement (REM) sleep. For EOG, one electrode was placed 1 cm above the corner of the right eye and the second electrode was placed 1 cm below the corner of the left eye following recommended criteria for sleep recording . The lefFigure 1A shows an overhead map with district outlines. These six districts contained buildings with unique architectural features such that they could potentially help one identify a change in districts when moving from one to another . Pedestrians were also seen in each district and some wore clothing that could be uniquely attributed to a certain district .The virtual reality (VR) navigation task performed by participants was designed by the authors and implemented into custom-built software by Intific, Inc.  using the Unreal Engine. Participants wore an Oculus DK2 headset . The VR environment consisted of a large city with six districts; Figure 1B). A large skyscraper was placed at the northwest corner and a factory with smokestacks at the southeast corner. The west edge of the map was bordered by a beach and the east edge was bordered by mountains. Within the environment, locations of note had a sign in front with their name or logo, and were places that the participant was required to navigate to or from during the training portion of the task.Boundary landmarks were placed in the environment to encourage the use of a hippocampal-dependent boundary navigation strategy . No roads or buildings were shown on the map to prevent explicit route planning. The boundary landmarks to the east and west of the map  were designated on the map, and the cardinal direction north was designated by an “N.” The district containing the navigator’s current location had a highlighted border. The participant’s location was designated by a blue circle on the map with an arrow pointing in the direction of the participant’s heading. The location of the destination landmark was designated by a star encircled in green .A virtual cell phone interface presented a sparsely detailed map such that participants could see their own location and heading, the location of the target destination, and the borders between the six districts  and show the target destination’s location on the map. The participant could not call up the cell phone at will. Instead, the phone was automatically called up at the beginning of a route and whenever the participant crossed a boundary between districts. Other than the initial guidance provided by the phone, participants were allowed to freely explore the environment while searching for target locations during the training portions of the task. Additionally, during the training portion of the task, the cell phone was called up to alert the participant if they traveled too far along a non-optimal route. These non-optimal route alerts were turned off during testing intervals of the task. Once participants reached the target destination, they would step into a green portal and respawn at the next route’s starting location . If a participant could not complete the route within a specified time limit, the participant would respawn at the next route’s starting location at the end of the time limit.At the beginning of a route, the cell phone would present a message directing the participants to a target location . The cues were border and heading-specific. As mentioned previously, the cell phone would be called up and border of the district one was traveling into would be highlighted. This would occur at the same time the auditory cue played. A total of 18 unique cues were experienced by the participants in the environment, and the optimal routes were designed to ensure that each auditory cue would be experienced at least once during training. The cues delivered during the nap were shortened versions (700 ms long) of the cues heard during the navigation task and were naturalistic sounds  within the human audible frequency range. The relative amplitudes of each cue were calibrated so as to be of equal perceived intensity at the auditory detection threshold volume.1. This software has the benefits of interfacing with multiple EEG recording systems, including the BrainAmp DC system used for the present study, as well as an extensive library of signal processing tools for real-time analysis of signals.We developed a first-of-its-kind closed-loop system for delivery of auditory cues to drive TMR. The system times the delivery of auditory cues to DUPTs. Our CL-TMR system leverages two parallel signal processing pipelines which perform online detection of DUPT events and identification of NREM sleep state. Both pipelines were implemented using the OpenViBE open source software platformFrontal Delta corresponds to the spectral power in seven frontal channels . Occipital Alpha refers to the spectral amplitude in the frequency range 8–12 Hz at the Oz, O1, and O2 electrode positions. Global Gamma refers to the spectral amplitude in the frequency range 25–50 Hz at all 32 electrode positions.The most critical aspect to the accuracy of the system is the identification of NREM2/NREM3 sleep. We developed a novel approach to doing real-time sleep staging based on the recommended biomarkers for each stage of sleep from the American Association of Sleep Medicine . All NREN = 51 naps from 32 control participants). An experienced sleep researcher scored the nap data and the manually scored records were used as a reference to guide iterative adjustments of the thresholds of sleep stage identification in order to maximize the system’s performance across participants. These identified sleep stages were then used to gate the delivery of auditory cues with coincident detection of DUPT events.The DAG metric is computed over non-overlapping 5-s epochs and thresholded to produce the various stages of sleep between 0 (wake) and 3 (NREM3). In practice we have found the following thresholds work well in our system; wake < 0.03 (NREM1) < 0.05 (NREM2) < 0.25 (NREM3). Algorithm parameters were developed and optimized using data from Day 1 screening naps and Day 2 and Day 3 naps in control participants . However, the occurrence of DUPT events during REM is very rare resulting in very few false positives. The current false positive rate for DUPT detection is under 0.04 and occurs primarily with arousal events when participants wake from NREM2/NREM3 sleep.Figure 2 provides a schematic of the experimental procedure. All participants came for an initial screening day, Day 1, during which they gave informed consent and became acquainted with navigating in a VR environment. A subset of early participants  was also instrumented with EEG, EOG, and ECG electrodes as described previously and attempted a 90-min nap. The screening day allowed the researchers to determine whether the participants would be able to nap, fully instrumented, in an unfamiliar place and would be able to tolerate the VR headset for about 15 min or more at a time, as nausea is a common side effect. Those who passed the VR and nap screening were scheduled to continue the experiment. Those who were ineligible were compensated for their time and dismissed. Ultimately, since no differences were observed between those who took a screening day nap and those who did not, we stopped requiring the screening nap and subsequent participants only completed VR screening on Day 1.All eligible participants came in for three consecutive days of experimental sessions. On Day 2, participants were first instrumented with EEG . Vibratory feedback coincident with the sounds in the VR environment was delivered by a haptic gaming vest . The purpose of the vest was to increase the salience of the auditory cues at the district boundaries.Once instrumented, the participant began the training phase of the navigation task. Participants were instructed to pay attention to the sounds and location when crossing the district borders of the map. At the start of each of the 24 distinct routes, participants were informed of the destination on the virtual cell phone. During navigation, the “non-optimal route alert” occurred at decision points if the participant was not on the optimal (shortest) path. During training, routes were organized into blocks of contiguous routes to promote learning of the global organization of the environment; for example, if one route ended at the coffee shop, the next route would begin at the coffee shop. The 24 routes were divided into three groups of seven to nine routes each in order to give participants breaks from the headset, but participants could take as many breaks as they wished during training to prevent nausea.Following training, participants took a 20-question declarative memory pre-test which probed for knowledge about the learned VR environment before any intervention occurred. Ten questions were short-answer  and the other 10 were multiple-choice (recognition). For the purpose of the test, a “landmark” was defined as a specific location one navigated to or started from on any route during the training phase. The short-answer questions took the following forms: (1) participants were shown a picture of a landmark and asked to name the two closest landmarks to the pictured one; (2) participants were told to imagine themselves standing in a pictured location and asked to name the closest landmark to the left, right, or behind them; (3) participants were again asked to imagine standing in a pictured location, and asked to name a landmark that they could see if they were to turn in that spot; and (4) participants were shown two different landmarks and asked to name a landmark that would be passed if traveling along the optimal route from the first to the second landmark. For the multiple-choice questions, (1) participants were shown an image of the surroundings at a border crossing and asked to select the auditory cue that was heard; (2) participants were shown an image of a landmark and asked to select the direction  one must travel to get to a boundary landmark or a local landmark; and (3) participants were asked to choose the landmark that does not belong in the same district as the others.Prior to consolidation with sleep, memories are thought to be labile and prone to interference. Memory reactivation after learning, during waking periods, has been shown to reduce subsequent recall . HoweverFollowing the awake replay session, participants were required to take a nap. Electrodes were placed at the corners of the eyes for the EOG recording and the haptic vest was removed. Participants were assigned to either a control condition in which they napped without any intervention, or a CL-TMR condition in which auditory cues were delivered during the nap using the closed-loop system described above. If they were in the CL-TMR cohort, participants wore soft, flat earphones that rested over the outer ear (a confound that is elaborated on in the section “Discussion”) and the volume was calibrated prior to the nap using an adaptive staircase procedure until a volume level just above auditory threshold was reached. All participants started sleeping between 1 to 3 PM and were allowed to sleep for ∼90 min. During the nap, the volume was reduced if cues decreased the DAG measure . Conversely, if the cues did not decrease the DAG measure and no spindle response was observed, the volume was increased. Participants were awakened after about 90 min from time they fell asleep. A mandatory break of at least 30 min after the nap occurred before the final tasks in order to minimize the effects of sleep inertia. Participants used this time to complete a demographics questionnaire, the Big Five Inventory , and theFollowing the sleep inertia break, participants were given a declarative memory post-test with the same format as described for the pre-test declarative memory test but with different questions. The order of the two declarative memory tests was counterbalanced. After the test, participants underwent a second session where auditory cues were delivered in the absence of an interference task. Responses to cues were collected to try and assess any impact of the nap and intervention on awake memory replay, however, reliable and specific cue-evoked responses were not able to be identified so these analyses have been omitted in this manuscript. Finally, a navigation test in the same environment on a subset of six routes was given. Non-optimal route alerts were turned off, and speed and accuracy were recorded. Two different route sets (of 6) were tested  across participants at each post-testing interval. Thus, 12 routes were tested at Day 2 and Day 3 in each cohort, with the remaining 12 tested on Day 4. This was done to balance some of the variability in route difficulty and location within the environment across each day. At the end of the experimental session, participants were given an actigraphy device  to monitor overnight sleep. If an actigraphy device was not available, participants were asked to report the number of hours slept the previous night.This procedure was repeated for Day 3, with the only difference being that roadblocks were placed in the environment such that for 13 of the 24 routes, the participant had to identify a new optimal route. The same roadblocks were also present in the VR environment for testing on Day 3. The testing route lists on Days 2 and 3 were counterbalanced across participants using two sets of six routes to better sample the environment and maintain equivalent levels of difficulty. Thus within a cohort each testing day had 12 total routes that were tested (but only 6 in each participant for Days 3 and 4).On Day 4, participants were not instrumented. They took declarative memory tests designed to probe knowledge of the spatial layout of the landmarks in relation to the districts. On the first test, they were shown a picture of a representative building from each of the six districts and were asked to mark the location of that specific building on a map like the one shown on the virtual cell phone. On the second test, participants were given a list of all landmarks (places they could have navigated to or from during the learning and test phases) and asked to mark which district each landmark belonged in. Finally, participants were tested inside the VR for their ability to navigate the remaining 12 routes in the map without roadblocks. To avoid test/re-test confounds, in each participant the 24 routes were divided such that no route is tested twice .U tests to test our a priori hypothesis that CL-TMR would improve navigation ability. The nap data were visually scored by an experienced sleep researcher, and sleep parameters were analyzed with two-tailed Mann–Whitney U tests and the Bayesian Information Criterion as described in t-test. Because no meaningful results were found involving the declarative memory tests nor the impact of sensory interference, they will not be discussed further.In all cases where non-parametric tests were used, it is due to a lack of normality in the sample. Behavioral results were analyzed with non-parametric, two-tailed Mann–Whitney 2 and EEGlab toolboxes3  following a DUPT. Analyses requiring adjustment for false discovery rate in hypothesis testing were conducted using the approach described in With the exception of the online system developed for CL-TMR all other analyses were performed offline in MATLAB  using the BioSig toolboxolboxes3 . FollowiFigure 3A shows the operation and logic of our closed-loop TMR approach. Figure 3B shows the results of our automated sleep-staging algorithm in a single subject’s nap session. In the nap shown, NREM2/NREM3 sleep was correctly identified against visual scoring with a recall of >0.95 [true positives/] and a precision of >0.93 [true positives/]. Across participants, the recall and precision of the system were 0.89 and 0.85, respectively.Figure 3C across Day 2 naps within our CL-TMR cohort. The plot shows the mean potential across the frontal  electrodes for each DUPT which triggered a cue (n = 1190). The event related potentials (ERPs) demonstrate a high degree of consistency in the timing across participants. The mean ERP is shown in Figure 3D. The plot shows that the cues are delivered with a mean latency of 238 ± 42 ms from the time of the detection event (-80 μV crossing). This timing precedes the transition to the up-state by >100 ms on average. Note that because the ERPs are locked to the time of the actual cue delivery which is jittered with respect to the (-80 μV) detection event, the minimum negative potential of the mean ERP in the plot fails to cross -80 μV.The detection of DUPTs and latency of cue delivery is shown in Tables 3. Mann–Whitney U tests showed no group differences for any parameters either at Day 2 or at Day 3. Two parameters, the total sleep time and sleep efficiency, were trending toward significance on Day 2, but in both cases CL-TMR subjects showed better sleep compared to the controls. To support these null results, we calculated the approximate Bayes Factor (BF10) through the Bayesian Information Criterion (BIC), following the procedure described in 10 below 1, strongly supporting the absence of significant differences. Overall these results indicate that the intervention did not negatively impact sleep architecture. Additionally, we found no significant correlation between the time spent in NREM2 , NREM3 , or total sleep time  and performance during Day 2 in participants receiving the CL-TMR intervention, suggesting that performance differences were not due to differences in sleep architecture. Results comparing sleep time to performance during Day 3 and across all testing intervals were similarly uncorrelated.To investigate the impact of the intervention on the sleep architecture, all recordings were visually scored using 30-s epochs according to the AASM rules . StandarTable 4 shows the mean non-normed route times for each cohort at each training and testing interval to show the relative gains in absolute time. Figure 4 illustrates the behavioral results reported in Table 4.Because the variances across all of the route times were extremely high due to highly variable route lengths and therefore navigation times, the data were normed before statistical analysis. For every participant, each route time was divided by its across-participants mean. Analyses were performed on these normed values. U test: N1 = N2 = 12 routes, p = 0.003, Cohen’s d = 1.41). Although not statistically significant, the CL-TMR group also shows enhanced performance in terms of reduced navigation times at testing on Days 3 and 4 in comparison to controls. When considering testing across all 3 days, the CL-TMR cohort exhibits significantly improved performance, . Also notable is the reduced variability among CL-TMR participants relative to controls. This is especially visible in the Day 3 test and suggests that the intervention is delivering a consistent benefit and normalizing learning. The Day 3 training and testing intervals are subdivided into trials where the optimal path remained unchanged and those where newly placed impediments required the participant to identify a different optimal path. These changes in the environment naturally increase training and testing times on these trials for both cohorts; however, the CL-TMR group shows nominally reduced navigation time on the changed routes at training and testing compared to controls, suggesting that they may be more capable of manipulating a cognitive map to quickly identify new, more optimal routes. Higher variability in Day 3 performance on changed routes as well as a limited number of testing trials in each condition likely contributes to a lack of significance in this interval. By Day 4, control participants have made up much of the difference that is observed in Day 2. Thus, the strongest benefits of CL-TMR are observed in the immediate interval following learning.Both groups show similar training time on Day 2, however, the CL-TMR group shows significantly reduced testing time following the nap  these channels show a significant and representative increase in fast spindle energy when comparing controls to TMR participants. Power spectral density was computed from data processed with a continuous wavelet transform (Morlet wavelet) in the frequencies from 1 to 50 Hz and over the time interval from 1 s before the median time of cue delivery to 2 s after cue delivery. Figure 5 shows that the largest spectral differences exist in the fast spindle band (12–15 Hz). These differences are most pronounced in the first 500 ms following the cue onset. More modest differences can also be seen in the theta band and larger increases are observed in the low gamma band. Mean differences in the fast spindle band (12–15 Hz) during cue delivery were statistically significant  across participants but were non-significant in the 1-s window preceding the cue .We compared the responses to TMR cues with the same DUPT-locked events in control participants that received no stimulation to investigate the impact of CL-TMR. The spatial distribution of nested spindle activity may vary depending on the type of memories being consolidated . We inveFigure 6 show typical spatial bias for slow spindles to be more anterior while fast spindles are more central and posterior across all 3 days (Each participant’s mean spindle energy in the fast (12–15 Hz) and slow bands (9–12 Hz) was computed over a 1-s interval during the subsequent up state following DUPTs. The topoplots in l 3 days . OverallFigure 7 shows the Days 2 and 3 mean spindle energy topoplots for our CL-TMR participants as well as the difference between these plots and the control topoplots in Figure 6 of the same day. Overall, CL-TMR can be seen to drive increases in spindle energy on both days, however, these differences are largest on Day 2 owing primarily to the weaker energy observed in controls on Day 2. Day 2 differences are largest in one cluster of frontal and another cluster of occipitoparietal electrodes for fast spindles and only in occipital and right parietal electrodes for slow spindles. Day 3 shows smaller increases in fast and slow spindle energy in mostly frontal electrodes with slow spindles also showing some significant increases in right parietal and left occipital sites. Differences at starred electrode locations are significant after adjustment for false discovery rate (see Table 5). However, only mean fast spindle energy in the starred frontal cluster  is correlated with Day 2 navigation performance . There were no significant correlations between spindle energy in the occipitoparietal cluster  and behavior at any interval. Similarly, there were no significant correlations between slow spindle energy in either cluster and navigation performance.In the current study, we developed a system that allowed closed-loop delivery of TMR protocols during sleep. CL-TMR enhanced navigation skills and increased spindle energy compared with non-stimulated sleep. Our results showed that sleeping with the closed-loop TMR (CL-TMR) system is feasible, can be successfully used during a daytime nap, and, importantly, did not negatively impact sleep architecture. Previous studies with TMR have demonstrated its success in a range of reasonably well-controlled laboratory tasks e.g., . AlthougUsing the large magnitude negative potentials (-80 μV) to reliably detect SOs during NREM3 has been demonstrated previously , 2015. HSD = ±54 ms) or at the ∼300° phase of the slow oscillation and on average 100 ms prior to the zero crossing into the next cortical up-state. A recent study into the relationship between the phase of the slow oscillation at the time of cue delivery and its impact on memory found that this phase bin (270–360°) represented a relatively suboptimal time for memory enhancement in a cued spatial association task, whereas the optimal phase bin was 180–270° degrees . This result is in line with recent studies in which cues delivered after the negative peak of the SO led to increases in fast spindle power  locked to DUPTs , and no correlation was revealed between time spent in either sleep stage and performance. One caveat to this is that not all participants spent a significant amount of time in NREM3 due to the duration of the nap. Applying this approach during overnight sleep would be useful in elucidating the relative contributions of cues delivered during NREM2 vs. NREM3 sleep.Since our approach targets large amplitude slow oscillations in both NREM2 and NREM3 sleep it is possible that there are differences in the response to cues delivered during k-complexes vs. slow oscillations in SWS, and that these may explain some of the behavioral variance. However, analysis of the spectral power density in the spindle band found no significant differences between cues delivered during NREM2 and NREM3 on any channel  based on parts of the described research. RS, PC, RE, MA, and SS were employed by Teledyne Scientific & Imaging. DA and MW were employed by Rio Grande Neurosciences. The other 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.


Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. Catheter ablation of atrial fibrillation plays an important role in the management of AF. Radiofrequency ablation is widely used in practice all over the world. Cryoablation has emerged as an alternative method for AF ablation. The FIRE and ICE trial was a non inferiority, multicentre, randomized trial that compared between the two modalities and proved cryoablation to be non inferior to radiofrequency in terms of efficacy and safety. However, the rate of AF recurrence was markedly high in both arms of the study. Since its introduction in the late 1990’s, catheter ablation has gained an increasingly important role in the management of AF. The fundamental concept of AF ablation is based on recognizing that electrical foci arising from the myocardial sleeves of pulmonary veins trigger and maintain AF2. Therefore, pulmonary vein isolation (PVI) by different ablation techniques may help in maintaining sinus rhythm. Over the years, numerous mapping, imaging, and ablation technologies have been developed to improve the outcomes of radiofrequency (RF) AF ablation. Recently, cryoablation had been introduced as an alternative method to RF with increasing popularity among electrophysiologists3. Many studies have compared between the RF and cryo AF ablation modalities, however, these studies either were nonrandomized or included small number of patients5. The FIRE AND ICE trial, which has been recently published in the New England Journal of Medicine, represents the largest-to-date prospective randomized multicentre trial comparing the efficacy and safety of RF and cryo ablation in patients with paroxysmal AF6.Atrial fibrillation (AF) is the most common arrhythmia worldwide and represents a significant burden on global healthcareThe FIRE and ICE study was a multicenter, randomized trial to compare the efficacy and safety of pulmonary vein isolation either by cryoablation or radiofrequency ablation. The study was designed to test whether cryoballoon ablation was non-inferior to RF ablation, assuming event-free 1-year survival rates of 70% with 10% non-inferiority margin, corresponding to a hazard ratio of 1.43. Inclusion criteria were symptomatic paroxysmal AF, prior antiarrhythmic drugs failure, and age from 18 to 75 years. Exclusion criteria were previous left atrial ablation or surgery, left atrium diameter larger than 55 cm, myocardial infarction or percutaneous coronary intervention with 3 months of enrollment, stroke or transient ischemic attack within 6 months of enrollment, and left ventricular ejection fracture less than 35%. The primary efficacy end point in a time-to-event analysis was the first documented clinical failure. The authors defined clinical failure as recurrence of atrial fibrillation, occurrence of atrial flutter or atrial tachycardia, use of antiarrhythmic drugs, or repeat ablation, following a 90-day period after the index ablation. The primary safety end point was a composite of death, cerebrovascular events, or serious treatment-related adverse events .A total of 762 patients underwent randomization . The mean duration of follow up was 1.5 years. The primary efficacy end point occurred in 138 patients in the cryoballoon group and in 143 in the radiofrequency group . Subanalysis of each of the components defining clinical failure showed similar results in both arms. However, the mean total procedure time was shorter in the cryo group than in the RF group , as was the left atrial dwell time (the length of time the catheter was present in the left atrium during the procedure), . The mean total fluoroscopy time was shorter in the radiofrequency group than in the cryoballoon group .6.Regarding safety, the primary safety end point occurred in 40 patients in the cryoballoon group and in 51 patients in the radiofrequency group . However, there were no tracheosophageal fistulae in the RF group and only 10 phrenic nerve injuries in the cryo group, with only one persisting as a clinical problem after 2 years follow up6. PVI using radiofrequency ablation necessitates creating continuous lesion line around the ostia of the pulmonary veins which will electrically isolate the veins from left atrium7. RF uses high frequency alternating current (in the range of 300 kHz to 1 MHz) to create a thermal lesion. Permanent tissue necrosis occurs by direct resistive heating and indirect, passive conductive heating, when tissue temperature reaches around 55°C , ganglionic plexi, and rotor ablations20. However results from Substrate and Trigger Ablation for Reduction of Atrial Fibrillation Trial (STAR AF II) have shown the PVI only was as efficient as PVI plus CAFÉ or additional line ablations in patients with permanent AF21. Initial results on rotor-targeted ablation in persistent AF were promising20; however much more extensive studies are needed before adopting this strategy as part of the routine of persistent AF ablation22.The FIRE and ICE represents one of the largest prospective randomized controlled trials not only in comparing RF versus cryo technologies, but regarding results of AF ablation in general. The investigators recruited patients with paroxysmal symptomatic drug-refractory AF23.Regarding results, an important finding was that the mean total procedure time was shorter in the cryo group than in the RF group , as was the left atrial dwell time (the length of time the catheter was present in the left atrium during the procedure), . In addition, the mean total fluoroscopy time was shorter in the radiofrequency group than in the cryoballoon group . The procedures in FIRE AND ICE were done in high volume centres, with more than 100 cases of AF ablation per year. RF ablation and creating a complete line of ablation around the pulmonary veins is a technically demanding procedure and requires a long learning curve. This may be in contrast to the relatively easier cryo ablation which is less operator-dependent. These points must be considered when extrapolating FIRE and ICE results to the real world practice where, for example, more than 80% of AF ablation cases in the United States are performed by operators with less than 25 cases per year and in centres with less than 50 cases per year8. Even with such low rates, one must acknowledge that AF ablation carries a significantly higher risk of complications relative to other invasive electrophysiology procedures7. In a large analysis of 93,801 cases of AF ablation done in United States, in-hospital complications were inversely related to the experience of the operator23.Another significant result was the lower rate of major complications in both study arms. There were no tracheosophageal fistulae in the RF group and only 10 phrenic nerve injuries in the cryo group, with only one persisting as a clinical problem after 2 years follow up8. Silent AF episodes missed in between those recordings would further increase the rate of recurrence24. Moreover, the study population may be more favourable than AF patients ablated in real life as this group of patients were relatively young, with small left atria, and paroxysmal AF. The recurrence rates would be expected to be even higher in older patients with bigger atria and more persistent AF26.The most striking point regarding FIRE and ICE results was the high recurrence rate in both arms, which is about 35% . Recurre31. The Atrial Fibrillation Follow-up Investigation of Rhythm Managament (AFFIRM) trial which compared rate control versus rhythm control in patients with AF have showed that rhythm and rate control strategies do not differ in long term morbidity and mortality in AF patients32. In addition, no large trials have investigated AF ablation effect on mortality in comparison to antiarrhythmic medications. To answer this fundamental question, we will need to wait 2 more years before the long anticipated CABANA trial results are out. The CABANA  (NCT00911508) is multicenter trial, with planned recruitment of 3,000 patients with paroxysmal, persistent or “chronic” AF in North America, Europe, Australia, and Asia, who will be randomly assigned to either antiarrhythmics or catheter ablation. The primary end point of the CABANA trial is all-cause mortality. Finally, the “favourable” reported improvement in symptoms of patients who have undergone AF ablation was never tested against a sham control trial. The SIMPLICITY 3 trial have emphasized the importance of such studies in evaluating the futility of invasive risky procedures33.This high percentage of recurrence during the follow up in this study and in many other large studies should direct the leaders in the electrophysiology community to question the futility of PVI as an effective management strategy in patients with AFAF ablation still carries suboptimal results regardless of type of technology with significant complication rates. This notion is true even when AF ablation is performed in highly experienced centres and with optimal patient selection. Thorough understanding of the electrophysiological and pathological basis that cause and maintain AF remains the missing element in our quest for optimal management of AF.


S. cerevisiae yeast, we here used hybrid promoter engineering to construct tightly-controlled, externally-inducible promoters that only express in haploid mother cells that have contributed a daughter cell to the population. This is achieved by combining elements from the native HO promoter and from a TetR-repressible synthetic promoter, with the performance of these promoters characterized by both flow cytometry and microfluidics-based fluorescence microscopy. These new engineered promoters are provided as an enabling tool for future synthetic biology applications that seek to exploit differentiation within a yeast population.Engineered promoters with predefined regulation are a key tool for synthetic biology that enable expression on demand and provide the logic for genetic circuits. To expand the availability of synthetic biology tools for  Saccharomyces cerevisiae yeast has been one of the most popular chassis for synthetic biology studies as it is a well-studied model organism that enables many applications in biotechnology  [Synthetic biology seeks to expand on existing biotechnology principles and develop technologies that simplify the design and construction of engineered biological devices and systems that carry out tasks , 2. As aroteins) –6.et al. created a series of repressible promoters by taking the native galactose-inducible GAL1 promoter and adding tetO operator sites to the core promoter region downstream of the TATA-box thus creating a synthetic promoter that is also regulated by TetR [GAL1-based TetR-repressible promoters of various strengths and a similar GAL1-based LacI-repressible promoter Iibrary inducible via addition of IPTG [PFY1 promoter) was also demonstrated to convert this normally constitutive promoter into an externally-inducible one [The availability of well-characterized gene expression control tools is essential for all potential synthetic biology applications. The most popular target for gene expression control in yeast synthetic biology has always been via promoter regulation, as control of transcription is best understood in eukaryotes. Initially, research focused on the characterization and use of native constitutive and inducible promoters, such as the constitutive TEF1, the GDP and the ADH1 promoters, and inducible FIG1 and GAL promoters , 8. In t by TetR . This st of IPTG . Additioible one .GAL10-CYC1 hybrid promoter helped identify the role of the GAL4 regulatory region upstream of the GAL10 gene [S. cerevisiae promoters with the core elements of others, Blazeck et al. were able to make new promoters stronger than any seen naturally in yeast and to change the regulation of different existing promoters [While these efforts focused on engineering orthogonal regulation into the core of yeast promoters, others have instead looked at engineering regulation by changing the upstream sites that activate expression. Regulated promoters found naturally in the yeast genome typically have upstream enhancer elements that control the rate of transcription in response to inputs, while the core promoter elements serve more to determine the start site and direction of transcription . RecentlL10 gene . HoweverL10 gene –17. Inderomoters . This woromoters .vice versa. The HO promoter is a classic example of this kind of promoter and the HO gene it expresses encodes an endonuclease that causes mating-type switching between MATa and MATα cells [So far, all promoter engineering in yeast has focused on constructing promoters to respond to external inputs or internal metabolites and give a reliable, uniform output across the population of cells. However, some natural yeast promoters respond differentially within a population of growing yeast, expressing in mother cells but not in newly-budded daughter cells, or Tα cells , 19. It Tα cells , 21.S. cerevisiae cells, we here set out to engineer hybrid promoters that combine the mother-specific expression of the HO promoter with externally-inducible control. The goal was to create promoters that can enable genes of interest, such as cytosolic enzymes, to be activated when desired but restrict their expression to mother cells within the population. Here we show that this can be achieved by the construction of synthetic hybrid promoters that combine core elements from TetR-regulated GAL1-based promoters and upstream elements of the HO promoter.In order to expand the toolset of yeast synthetic biology, so that differential regulation can be controlled within a population of TX promoter, while for the mother-specific regulatory region, we took upstream sequence from the native HO promoter [To produce externally-inducible mother-specific promoters, we employed the hybrid promoter engineering strategy to combine the core promoter region of a synthetic repressible promoter with native upstream regulatory sites known to be bound differently in mother cells versus daughter cells. For the core promoter region, we took sequence from the TetR-regulated promoter , 21.HO promoter is a tightly-regulated, relatively large promoter that is bound by several activators and repressors in order to control transcription to only occur in that right type of the cell and at specific points during the cell cycle [HO transcription [HO promoter and prevents its expression [HO gene, but it does shift HO expression from late G1 to the anaphase stage, leads to loss of the mother cell specificity and results in almost a third of the daughter cells to switch mating types [The ll cycle –22. The ll cycle , 23. Thecription . In the pression . Deletiong types , 26–28. TX promoter is an engineered variant of the extensively-used GAL1 promoter, designed to include a tandem pair of TET operator sites immediately downstream of its TATA-box in order to enable repression of transcription in the presence of constitutively expressed TetR  [The  . It has  , 29, 30. , 31–33.HO promoter also has a distinct core promoter which is paired with the two upstream regulatory regions, URS1 and URS2, which ensure HO expression is only in haploid mother cells [The er cells . In conter cells . The staTX promoter TATA-box was removed and replaced with equivalent HO promoter upstream sequence. This meant that the PGAL upstream activation sequence (UAS) and the Mig1 repressor sites were both removed, eliminating galactose/glucose regulation. This is particularly important since galactose-induced expression of the TX promoter is very strong and would likely mask activation via URS1 or URS2.For the hybrid promoters constructed here, all sequence upstream of the HO-TX hybrid promoters were designed, constructed and tested. The first configuration (TXC) added both HO promoter regulatory sites (URS1 and URS2) upstream of the TX promoter core, whereas the second (TXC2) added only the URS1 site. Both promoters were designed to lack galactose-based regulation but maintain the ability to be repressed by constitutive expression of TetR, which can itself be blocked by addition of ATc or doxycycline to the culture media . To test these promoters for their expression output, their regulation and cell-cycle performance, they were both placed upstream of a gene encoding a fast-degrading Green Fluorescent Protein (FD-GFP). This GFP variant has a fused N-terminal ubiquitin tag that promotes fast degradation in the S. cerevisiae cytoplasm via deubiquitination, allowing expression dynamics to be monitored over time [tetR gene under the control of a TEF1 promoter was also included in both plasmid constructs.Two different configurations of ver time , 35. Thiver time . The proFD-GFP gene were first assessed using flow cytometry. Expression from both promoters was induced by relieving TetR-repression by addition of ATc to the glucose-based growth media. As the PGAL UAS and Mig1 sites are not present in the TXC and TXC2 promoters, their expression should not be regulated by carbon source. Green fluorescence per cell was measured 5 hours post-ATc induction for a minimum of ten thousand cells. For both yeast strains the distribution of fluorescence per cell was plotted and the overall mean fluorescence of the population was calculated . This revealed that strains with the TXC and TXC2 promoters exhibit minimal expression in glucose media, but expression is activated when ATc is added with fluorescence increasing by 8.18x for the TXC promoter and 5.12x for the TXC2 promoter, when only the “ON” populations are considered. The bimodal distribution is an indication of differential expression in the population, which we attribute to differences in expression between mother and daughter cells, assuming that no spontaneous mutations in the have arisen in the integrated constructs. When the cells analysed from our sample are gated into “ON” and “OFF” populations (OFF defined by the fluorescence range for the 98th percentile of the uninduced sample), we see approximately 40% of the population in the “OFF” state, and the remaining 60% “ON”, albeit at different levels of expression. This is close to the ratio one would expect to see for a growing population of yeast cells expressing with the standard HO promoter, whose differential expression has been shown to be strict, with around 98% of the mother cells expressing from this promoter and 98% of daughter cells not showing any expression [To characterize expression from the constructed promoters, the two strains carrying the new hybrid promoters with the pression . This wopression . The ratFD-GFP expression per cell ( 2.5-fold for the TXC promoter and 3.5-fold for the TXC2 promoter over the integrated version when only the “ON” cell populations are considered). While this represents increased expression per cell, the increase is less than would be expected and may partially be attributed to the use of the LEU2 marker which is known to lower the copy number of 2-micron plasmids in cells [Both hybrid promoters were also assessed for their expression when placed on 2-micron plasmids, rather than when integrated into the yeast genome. As these are high-copy plasmids  they have the potential to greatly increase overall protein expression from the hybrid promoters . Flow cyin cells . Notablyin cells .S1 Video (TXC strain) and S2 Video (TXC2 strain).To verify that the promoters show mother-specific expression and to characterize the stringency of this, both of the strains with genomically-integrated constructs were further inspected using inverted fluorescence microscopy. To be able to track differential expression of the yeast, microscope images were captured over several hours as the yeast cells divide in a colony. These images were used to generate time-lapse movies showing colony growth and cell fluorescence. These are provided as TXC and TXC2 promoter characterization respectively were cultured in an ONIX microfluidics platform designed for continual perfusion of growth media to cells captured in fixed positions suitable for reliable time-lapse microscopy. Growth of the strains started in glucose media without ATc and cells were then induced after 2 hours (time = 0 min) and left to grow for another 10 hours with images captured every 10 min. Fig 4A shows selected time-points from a time-lapse of growth for the two strains, with further images provided in S1 and . Comparing the strength of the green signal of the fluorescence cells confirms the flow cytometry result that the TXC strain expresses more protein per cell than the TXC2 strain. Most importantly, it is quite clear that at least for the TXC strain only a fraction of the cells in the colonies produce fluorescent signal at any given moment.The Y02569-GPY022 and Y02569-GPY020 strains for TXC promoter has consistently shown the more desirable expression characteristics, this strain was the focus of the image analysis . All frames were analyzed for their average green signal per cell (corrected for background) in order to quantify the fluorescent protein expression in each cell over time. These values were then used to generate green-scale heat maps produced for all cells in the colony that become mother cells during the 10-hour induction period. To enable easy tracking of each cell, all cells were numbered based on the order that they separate from their mother cell at the end of mitosis. A family tree of cell growth in this experiment s provided in S3 Fig.To confirm that fluorescent protein expression is indeed mother-cell specific the time-lapse microscopy images were analyzed further. As the strain with the FD-GFP. Only one cell (No.13) appears not to follow the anticipated expression pattern and never fluoresces despite becoming a mother, perhaps due to accumulation of a mutation that inactivates the FD-GFP expression. Another cell (cell 14) also shows strong fluorescence at the beginning of its first cell cycle, however we attribute this to carryover of a strong FD-GFP signal when it divided from its mother cell No 2, rather than incorrect promoter expression. In all other cases only a faint green fluorescent signal is seen in daughter cells during their first cell cycle which we attribute to the seeping of some FD-GFP molecules from the mother cell cytoplasm into the bud. Furthermore, no fluorescence above background was seen in any of the cells in the colony that did not divide during the experiment, and all cells at the start of the experiment show no fluorescence, indicating that the TetR-based repression prevents leaky expression from the promoter when inducer is absent.Analysis of cell fluorescence in the growing colony revealed that all cells except one that become mother cells exhibit waves of green fluorescent protein expression every cell cycle, except for their first cell cycle. During this first cell cycle they are classed as daughter cells, having not produced any buds themselves and so should not be expected to express Figs  and 2 and the time-lapse microscopy  analysis of the TXC strain, confirm that the constructed hybrid TXC promoter is a mother-specific promoter that can be externally controlled by ATc induction. Upon induction, it produces a biomodal distribution of expression in the population, with only mother cells expressing the downstream reporter gene and doing so late in their second and all subsequent cell cycles. This therefore represents a successfully designed, constructed and tested hybrid promoter that gives mother-specific expression and can be externally induced. In order to ensure precise differential expression, we recommend that this promoter is used within genome-integrated constructs, as expression heterogeneity is seen when placed on a 2-micron plasmid.Taken together, the results of the flow cytometry TXC strain and this is likely due to TXC2 promoter expression before cell separation at mitotic division. This is likely to be explained by the lack of the URS2 regulation site in the TXC2 promoter, which would otherwise delay FD-GFP expression until after cell separation (G1 phase of the cell cycle in activated cells). Without URS2, the HO promoter is known to become independent of SBF control and starts production of gene transcripts upon reaching the cdc15 block stage of late anaphase [A further observation from the microscopy images is that the TXC2 strain shows some fluorescence in daughter cells, but that this is less apparent for the anaphase , 27. As HO promoter is activated only in mother cells from the second half of the G1 phase up to a point during S phase [TXC promoter should be expected as the cells reach the late G1 phase of their second cell cycle. In the image analysis , the peak of green fluorescence coincides with (or is just after) the very end of the second cell cycle as the cells move into S phase and a further bud is formed. We attribute this expression delay due to the time required to go from activating mRNA transcription from the TXC promoter to translating, maturing and accumulating significant levels of the green fluorescent protein per cell.It is known that the  S phase . This meHO promoter for experiments and applications that seek to control expression between mother and daughter cells. Differential expression between mother and daughter cells has in the past been used for generating mother-rich ‘older’ populations for ageing studies, as in the previously described mother-enrichment program [The promoter developed here offers a new alternative to the normal  program , 41. SinTXC promoter is via the orthogonal TetR protein, induction or repression of its mother-specific expression can be linked to other gene regulatory systems by having the expression of the TETR gene change in response to environmental conditions or other inputs. Thus, mother-specific expression could be an outcome or a component of a wide variety of future genetic circuits that exploit differential expression between S. cerevisiae cells.In future studies, it would also be interesting to see if the concentration of inducer (ATc or doxycycline) given in the culture media could be used to tune the relative fraction of the population that is dedicated to production versus growth. Finally, because the ultimate control of expression from the FD-GFP gene downstream of the TXC and TXC2 promoter respectively, are derived from the pTVGI plasmid (both based on pRS4D1 plasmid) provided by Dr Tom Ellis  and described in Ellis et al. 2009 [TETR gene expressed from the constitutive TEF1 promoter, have a LEU2 selectable marker, and integrate into the URA3 locus of the BY4741 genome via homology to a short region included in the plasmid. The FD-GFP gene containing a ubiquitin tag was developed and kindly provided by Dr Felix Jonas. The pGPY055 and pGPY054 2-micron plasmids were assembled using the MoClo method of modular assembly in combination with the Yeast ToolKit created by the Dueber lab [FD-GFP sequence is downstream of either the TXC or TXC2 hybrid promoter. Plasmids were constructed to also include a LEU2 marker and the TETR gene with TEF1 promoter. Annotated sequences for the promoters used in this study and for FD-GFP are provided in the S1 Text along with plasmid maps . Plasmids from this study will be made available via Addgene.org.Both the pGPY022 and pGPY020 integrating plasmids carrying the al. 2009 . Both pleber lab . All necS. cerevisiae Y02569  strain provided by EUROSCARF. Cells are cultured in synthetic complete drop-out glucose media (SC-Glu) for transformation and general proliferation and in synthetic complete drop-out glucose media (SC-Glu) media with 200 ng/μl ATc for hybrid promoter induction. During the time-lapse and ONIX experiments, cells were grown at 30°C inside the microscope. For all other cultures, cells were grown in liquid cultures at 30°C with shaking at 225 rpm.All engineered strains were derived from the haploid Images were taken through a 60x CPI60 objective mounted on a Nikon Eclipse Ti inverted microscope with live cells imaged using the CellASIC ONIX Microfluidic platform (Merck Millipore). Cells were grown in Y04C-02-5PK plates and flow rate was adjusted at Psi = 4 which was determined to be good for yeast cells. To visualize the samples a Phase filter 3 is used to enhance contrast and Brightfield illumination. For fluorescence capture, excitation, emission filters, and exposures were respectively 480 nm, 535 nm, 1000 ms for the GFP channel. During time-lapse experiments, the software autofocus function of the microscope is used to adjust for any potential movement of the cells during growth in order to keep clear track of the samples. NIS-Elements Microscope Imaging Software (Nikon) is used for capturing and ImageJ  is used for image presentation.3 A.U. was applied to the forward (FSC) scatter to minimize non-yeast events. Data analysis was performed using FlowJo software (Tree Star), gating samples for forward scatter and side scatter to exclude non-yeast events and obtaining fluorescence values from BL1-H (height) channels.Flow cytometry assays were performed using the Attune NxT flow cytometer with the Attune NxT autosampler attachment from ThermoFisher Scientific. A 488 nm laser was used for excitation of green fluorescence detecting through a 530 nm band-pass filter (BL1). The voltages of the FSC, SSC and BL1 channels for the promoter characterization experiments were 200, 320 and 480 respectively. A threshold of 3.0 x 10S1 Text(DOCX)Click here for additional data file.S1 FigtetR gene under the TEF1 promoter, the leucine (LEU2) yeast selection marker, a bacterial ColE1 origin and either the ampicillin (ampR) or the kanamycin (kanR)resistance marker. Genes are represented by coloured arrow-shaped boxes and key promoters by grey boxes upstream of the genes. Plasmid sizes are given in base pairs (bp).All plasmids carry the (TIFF)Click here for additional data file.S2 FigCells are grown in synthetic complete glucose media and induced with 200 ng/μl ATc for 10 h. The first frame corresponds to the moment when induction started and after that, frames at 30 minute intervals are shown. Both brightfield and green fluorescence images were captured using a 60x objective. Brightfield images are captured using a Phase 3 contrast filter. Each frame is a combination of the brightfield and green fluorescence channels. On each frame, cells are numbered based on the moment they appear to separate from the mother cell (end of mitosis). White lines represent scale bars with a length of 10 nm.(TIFF)Click here for additional data file.S3 FigCells are grown in synthetic complete glucose media and induced with 200 ng/μl ATc for 10 h. The first frame corresponds to the moment when induction started and after that, frames at 30 minute intervals are shown. White arrows are pointing to some of the daughter cells that exhibit fluorescence due to leakage from the mother cells. Both brightfield and green fluorescence images were captured using a 60x objective. Brightfield images are captured using a Phase 3 contrast filter. Each frame is a combination of the brightfield and green fluorescence channels. White lines represent scale bars with a length of 10 nm.(TIFF)Click here for additional data file.S4 FigOne tree for each one of the starting cells was created. Numbers in parenthesis shown in green represent the frame numbers when the cells are about to separate from the mother cells (end of mitosis). Numbers in parenthesis shown in orange represent the frame numbers when the cells appear as buds since they don’t achieve separation before the end of the experiment.(TIFF)Click here for additional data file.S1 Video(AVI)Click here for additional data file.S2 Video(AVI)Click here for additional data file.