Nearly 20% of night shift nurses will cause shift work disorder (SWD) due to the disruption of sleep-wake cycle, which not only affects the daily work efficiency, but also brings a huge burden on physical and mental health. Acupuncture is a safe and effective intervention on SWD. This trial will combine with functional magnetic resonance imaging (fMRI) to study the clinical effects and potential mechanism of acupuncture in the treatment of SWD.This is a randomized controlled neuroimaging trial, with enrolled participants, outcome assessors, and data statisticians blinded. 60 patients with SWD and 30 healthy controls who sleep regularly will be recruited and divided into divided into a control group, a true acupoints treatment group (TATG) and a sham acupoints treatment group (SATG) by the ratio of 1:1:1. The TATG and SATG will receive 8 sessions of acupuncture treatment in 4 weeks. Cognitive function scales and MRI scanning will be performed before and after 4-week acupuncture treatment. The control group will receive no intervention. Functional connectivity of intra-network and inter-network will be the primary outcome and effect indicator. The secondary outcomes included Repeatable Battery for the Assessment of Neuropsychological Status, Attentional Network Test, Pittsburgh Sleep Quality Index scale and needle sensation assessment scale. Neuroimage indicators will be correlated with clinical data and scores of cognitive function assessment to explore the possible mechanisms underlying the changes of brain activity caused by acupuncture treatment.The results of this study will enable us to verify the therapeutic effect of acupuncture on SWD and explore the potential central mechanism of acupuncture on SWD from the change of brain activity. Nurses make up the largest proportion of shift workers (15%–20%). Evidence suggests that long-term shift work will not only affect work efficiency and patient satisfaction, but also lead to physical and mental health problems.,4 In hence, given enough time, this may lead to more severe disorders, such as cardiovascular disease, cerebrovascular events, metabolic disorders, gastrointestinal complaints and multiple forms of cancer.–7 Meanwhile, SWD also impairs cognition, such as attention and memory, which will directly affect work efficiency or even cause safety consequences not only for the individuals concerned, but also for society. Therefore, it is essential to explore an effective intervention on impairments of cognitive abilities for patients with SWD.Shift work disorder (SWD) is a condition defined by excessive sleepiness or insomnia accompanied by total sleep time reduction. Such as benzodiazepines, nonbenzodiazepines and antihistamines. These drugs have a definite effect to a certain degree, while side effects may occur following a long time medication. The most common symptoms are headache, dizziness, nausea and anxiety. Clinical studies have confirmed that long -term taking benzodiazepines and non-benzodiazepines could impair attention, memory, response speed and mental activity,,12 even increasing the risk of Alzheimer's disease. Furthermore, with the accumulation of drugs will lead to addiction. In hence, it is necessary to explore a safe and effective method to treat SWD.Medication is the main effective method to treat shift work sleep disorder, Abundant randomized controlled trials have confirmed that acupuncture treatment on insomnia has the advantages of stable effect and less side effects.–19 A recent study also confirmed that manual acupuncture could improve memory function in chronic insomniacs. In hence, it is essential to definite efficacy of acupuncture treatment on cognitive decline. However, to our knowledge, there is no study focusing on acupuncture therapy on SWD.Acupuncture, as one of the traditional treatment methods in China, has a long history in the treatment of sleep disorder.In this study, we will conduct a randomized controlled trial to study the effect of acupuncture on cognitive abilities of SWD and reveal the underlying neuroimaging mechanism. The results will provide evidence and promote acupuncture therapy on SWD during clinical practice.22.1This is a randomized controlled, paralleled neuroimaging study, with patients, assessors and data statisticians blinded to the group assignment. A total of 60 patients with SWD and 30 healthy subjects will be recruited from October 2019 to December 2020 at Beijing Anding Hospital of Capital Medical University. They were divided into a control group, a true acupoints treatment group (TATG) and a sham acupoints treatment group (SATG) by the ratio of 1:1:1. The TATG and SATG will receive 8 sessions of acupuncture treatment in 4 weeks (2 sessions every week) by a trained acupuncture operator. Cognitive function scales and MRI scanning will be performed before and after 4-week acupuncture treatment. Meanwhile, the needle sensation will be evaluated by assessment scale after each session. The control group will receive no intervention.The study procedures are detailed in Figure 2.2A total of 90 participants will be recruited from October 2019 to December 2020 at Beijing Anding Hospital of Capital Medical University through posters and other publicity methods. Patients with SWD or healthy subjects who match the inclusion criteria will be considered potential participants. If they are eligible and interested in participating in the study, they will be fully informed of the entire study procedures, benefits and risks. Written informed consent will be obtained from the patient or their legally authorised representative before the allocation. Each participant has the right to withdraw from the study unconditionally.2.3 female nurses working at Beijing Anding Hospital; aged from 18 to 40 years, right-handed; continuous regular night shift for at least 1 year and work for 5 to10 years, at least 2 shift work per week; with no history of prophylactic or therapeutic medicine in the past 3 months; with no history of long-term use of analgesics.Patients will be enrolled if they meet the following criteria: meeting the diagnostic criteria for SWD according to International Classification of Sleep Disorders (2nd Edition) by the American Sleep Disorders Association;2.4Healthy subjects will be recruited following inclusion criteria: relative regularity of sleep in the past 12 months; aged 18 to 40 years, right-handed; sleeping less than 3 times per month after 23 o’clock in the latest 1 year and night shift less than 3 times a month in the latest 1 year.2.5Participants will be ruled out if they meet any criteria below: pregnant or lactation; history of neurologic or psychiatric disorders; participating in such cognitive experiments within 1 year; any other health problems or poor physical conditions that may influence participation; any brain structure damage or abnormalites identified by MRI examinations; any history of alcohol or drug dependency; any MRI contraindications.2.6 In the present study, we plan to recruit 60 patients with SWD and 30 healthy subjects with regular sleep.When calculating the sample size during the acupuncture-neuroimaging study, the number in published literature and previous studies is often considered. On the one hand, in fMRI studies, millions of voxels are used to estimate the blood oxygen level dependent (BOLD) signal indirectly, and conventional power calculations often make no sense. On the other hand, the number of participants is often constrained by scanning time and costs. In hence, considering the published acupuncture-neuroimaging literature focusing on sleep disorder, the sample size range of 16 to 32 cases show enough statistical power for brain functional analysis.2.7Randomization will be carried out in 2 steps. Firstly, according to the inclusion exclusion criteria, the 90 participants will be divided into experimental group and control group by the ratio of 2:1; secondly, the patient with SWD will be divided into TATG and SATG according to the computer-generated random sequence using Excel's rand function (1:1 ratio). The code will be sealed in opaque envelopes and opened by researchers not involved in the recruitment following informed consent procedures and baseline testing, with direct responsibility for treatment allocation. The researchers and acupuncturists will know the allocation. Meanwhile, the participants, outcome assessors and data analysts will be blinded.2.8Participants in this study will not be involved in the design, recruitment or conduction of the study. After the MRI scanning, all participants will be informed of the results of neuroimaging data in the form of pictures.3Patients with SWD will receive true or sham acupuncture treatments for 4 weeks, while healthy subjects will receive no intervention.3.1Patients in this group will receive 4-week manual acupuncture treatments at 10 verum acupoints (VA) , Benshen (GB15), Shenting (GV24), Shenmen (HT7), and Sanyinjiao (SP6)) with disposable sterile filiform needles . Needles will be perpendicularly inserted into acupoints by clamping after skin disinfection using 75% alcohol. The depth of acupoint insertion was 0.5 to 1 inch, needles will be retained at the acupoints for 20 minutes after the deqi sensation is attained through the manipulation of lifting, thrusting and thrusting mild reinforcing-reducing method.Two acupuncturist with over 5 years of clinical experience in acupuncture will be trained together and conduct the acupuncture treatments. After the first MRI scanning, the participants will receive a total of 8 sessions of acupuncture treatments in 4 weeks.3.2 the SA should be set aside with 1 inch beside the true point as the sham acupoint. In hence, the deqi experience of the participants in SATG will not be overemphasized. In spite of the locations of acupoints, the treatment procedures and course will be the same with TATG.Patients in sham acupuncture group will receive 4-week manual acupuncture treatments at 10 sham acupoints (SA). According to the previous literature on setting locations of SA,4The measurements consist of 3 parts including neuroimaging scanning assessments, cognitive function assessments, and needle sensation assessment. The study schedule is shown in Figure 4.1The demographic information , and the history of other concomitant diseases will be obtained at the baseline. Vital signs  will be measured before each scanning.4.24.2.1MRI examinations, including resting-state blood oxygenation level-dependent imaging (RS-BOLD fMRI) and high-resolution anatomical T1-weighted imaging (T1W1), are designed for detecting cerebral function changes.MRI Examination will be acquired applying a 3.0 Tesla MRI scanner  at Anding Hospital, Beijing, China. Prior to scanning, all participants were asked to rest for 30 minutes before scanning. They were instructed to stay still, think of nothing in particular, keep eyes closed, and not to fall asleep during scanning. Earplugs were worn to attenuate scanner noise. The foam head holders were immobilized to minimize head movements during scanning.Prior to the functional scanning, we collected high-resolution structural information for anatomical localization by using 3D MRI sequences. The resting-state fMRI data were collected using a single-shot, gradient-recalled echo-planar imaging sequence with the following parameters: repetition time = 2000 ms, echo time = 30 ms, flip angle = 90°, matrix = 64 × 64, field of view = 225 mm × 225 mm, slice thickness = 3.5 mm, gap = 1 mm, 32 interleaved axial slices, and 180 volumes.4.2.2,24 In hence, FC could reflect whether specific brain regions or resting-state brain networks are involved in improvements of cognitive functions after 4-week acupuncture treatments.The neuroimaging outcomes will be acquired after the MRI scanning and analysis, mainly including functional connectivity (FC) within and between resting-state brain networks. FC will be taken as the primary outcome and effect indicator, which is an optimal parameter to reflect the functional connections within and between resting-state brain networks. Numerous studies revealed changes of the brain's intrinsic functional connectivity stemming from cognitive impairments on patients with sleep disorder.4.3The following cognitive function assessment scales, including Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and Attention Network Test (ANT), will be conducted at the baseline and after the 4-week acupuncture treatment by a trained assessor blinded to the random sequence. The results of RBANS and ANT will be taken as the secondary outcome in this trial.4.3.1 The test undergoes about 30 minutes. A trained neuropsychologist performed the test according to standardized procedures.RBANS, a cognitive screening test including 12 subtests, was utilized as the measure of global cognition. The RBANS generates 5 domain-specific index scores applied to evaluate 5 cognitive abilities: immediate and delayed memory, language, visuospatial/constructional ability and attention. In spite of delayed memory, a component based on 4 subtests, the other 4 are based on 2 subtests. The immediate memory index comprises the story memory and list learning subtests; the visuospatial/constructional index comprises the line orientation and figure copy subtests; the language index comprises of the photo naming and semantic fluency subtests. The attention index comprises the digit span and coding subtests; the delayed memory index comprises the list recall, story recall, list recognition and figure recall subtests. The Chinese version of the RBANS translated by Cheng et al was adopted in the current study.4.3.2 was used to evaluate the efficiency of the alerting, orienting, and executive control. Subjects were required to click the mouse as quickly and accurately as possible to define the direction of the target, which was a rightward or leftward arrow in the center of screen and flanked on either side by 2 arrows in the same direction (congruent condition), or the opposite direction (incongruent condition). Each subject performed 6 blocks during this test, while each block last 5 minutes 42 seconds and included 36 trials plus 2 buffer trials. During each block, the 6 trial types were displayed in a predetermined counterbalanced order. All participants would be trained before the formal test. The entire test would be conducted on an experimental control computer.ANT, a cognitive task designed by Fan et al,4.4 This scale could measure the quality and patterns of sleep during the last 1 month. It included 7 domains: subjective sleep quality, use of sleep medication, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances and daytime dysfunction. Each items varied from 0 to 3 scale. An overall score of 5 or greater indicates poor sleep.Pittsburgh Sleep Quality Index (PSQI), was used to evaluate the quality of sleep.4.5The needle sensation assessment scale will be conducted after each acupuncture treatment by the acupuncturists. The result may be a prognostic marker associated with the effectiveness of acupuncture and improvements of cognitive function, which will be taken as the additional outcomes.Deqi” is a needle response describing how the patients feel when they receive the needle insertion. The self-designed Needle Sensation Assessment Scale has been applied in previous studies, including “Deqi” features  and rating of “Deqi”. Patients will describe the intensity of needle sensation by visual analogue scale (VAS) .“4.6Any adverse events during the intervention period will be reported, such as bleeding, fainting, pain, infection or other severe events will be recorded with details such as the date of occurrence, degree and causality with the acupuncture treatment. If the situation worsens, the patients will be discontinued from the study and will be sent for further treatment.4.7A series of training sessions will be held before the beginning of this study, which ensuring all researches get fully understood the standard procedures of this study. The medical imaging technicians will monitor the qualities of neuroimaging data after every scanning. The clinical and cognitive function data are required to be recorded on printed Case Report Forms (CRF) at each visit point. The completed CRFs will be undergone the double-entry verification in EpiData Entry software to ensure the accuracy of the data. All the MRI data will be stored in discs after finishing every scanning. After the data entry and verification as required, the CRF should be kept in the order of number, and filled with the search catalogue for reference. All original files shall be kept within the time limit specified.55.1The measurement data of each visit in different groups will be described by means of mean ± standard deviation. Clinical data including scores of cognitive function assessments from the 3 groups will be compared using one-way ANOVA, and further statistical analysis will be conducted between 2 groups.2 test or nonparametric test were used for the changes of the 3 groups before and after treatment.The counting data of each visit in different treatment groups were described by frequency (composition ratio). χ5.25.2.1http://rfmri.org/DPARSF). The first 10 volumes were discarded to allow the adaption of the subjects and the stabilization of the magnetization. The remaining volumes were slice-timing corrected for different acquisition in slice times and realigned to the first volume for head-motion correction. Based on the head motion data, the subjects were excluded according to the criteria of maximum translation as 1.5 mm and rotational parameters as 1.5 degrees in any direction. After that, the image data will be under the spatial normalization based on MNI space and resampled to 3 × 3 × 3 mm3. Finally, the data will be smoothed with a Gaussian kernel of 6 × 6 × 6 mm3 full width at half-maximum. Then, the data were temporally band-pass filtered (0.01–0.08 Hz) to reduce the low-frequency drift and high-frequency physiological noise.The fMRI data were preprocessed with Data Processing Assistant for Resting-Sate fMRI (DPARSF) software  of each network component and other network time series, namely functional network connectivity (FNC). The resulting r values were normalized into Z values by Fisher-Z transformation. For group-level analyses, the functional connectivity of Intra-network and inter-network was performed by one-way ANOVA using SPM12 software  for multiple comparisons). Finally, BrainNet Viewer was used to display the result onto a 3D brain surface.Independent component analysis was applied to extract the resting-state brain networks by GIFT software . The number of independent components in all data was calculated by method of the minimum description length (MDL) technique. 30 components were estimated. Randlnit and Bootstrap operations were applied to evaluate the independent components. According to research objectives, independent components were selected based on the largest spatial correlation comparing with previous resting-state brain network templates. Then, resting-state brain networks were calculated by the way of goodness of-fit after removing the components not related to the spectrum of resting-state networks. The best goodness-of-fit score of components were normalized to 6 Systematic reviews and a large number of trials have confirmed that acupuncture is safe and effective in the treatment of insomnia.,31 However, there are few studies on acupuncture treatment of SWD. Previous studies and literatures have shown that acupuncture can effectively treat insomnia and sleepiness of shift work disorder, and improve attention decline. In the current research, we will focus on the effect of acupuncture on cognitive functions of patients with SWD, and explore the neural mechanism of acupuncture on SWD through fMRI.Acupuncture is a common therapy for insomnia in China, and great progress has been made in the study of its pathogenesis.,34 Meanwhile, the fMRI technology provides a direct tool for the study of brain response to acupuncture intervention. Numerous studies revealed changes of the brain's intrinsic functional connectivity stemming from cognitive impairments on patients with sleep disorder.,24 A previous study also reported that patients with SWD showed brain perfusion changes in multiple brain areas significantly correlated with insomnia severity. In this study, the relationship between acupuncture sensation and fMRI is preliminarily discussed. fMRI can infer that regional changes in brain activity reflect whether specific brain regions or resting-state brain networks are involved in improvements of cognitive functions after 4-week acupuncture treatments.A large number of studies showed that, the activity of cerebral cortex and the internal and external functional connectivity of neural network were closely related to the occurrence of shift work disorder.However, there are few studies on acupuncture in the treatment of SWD, which need to be further studied through larger, more rigorous and good randomized clinical trials. At the same time, the purpose of setting up the false acupuncture group in this study is to eliminate the placebo effect. However, it is difficult for Chinese patients to choose the sham acupoint as the acupuncture placebo. As patients may have their own acupuncture treatment experience and know the location of common acupoints. In hence, the blinding of patients in this study has a certain impact.In conclusion, the purpose of this study is to evaluate effect of acupuncture on cognitive impairments of SWD and to interpret the neural mechanism of acupuncture on SWD through fMRI.YZN and XYL contributed equally in drafting this manuscript. YZN and XYL prepared the informed consent and finished trial registration. HY and XY are in charge of acupuncture treatment. PC is in charge of the motor assessment of participants. XYL and HY are in charge of screening and recruitment. YZN is in charge of neuroimage data processing and analysis. HXJ conceived and designed the study and is the corresponding author of the manuscript. All authors discussed, revised and approved of the final manuscript.


The year 2017 has been filled with news, controversy, and turmoil on many stages, in the realms of politics, entertainment, and economics—both around the world and locally. While original investigation regarding atrial fibrillation (AF) may not be one of these areas, nevertheless, this year was still filled with very significant contributions to our field.AF is still the largest area of investigation in the field of clinical cardiac electrophysiology. At the time of this writing, if one enters the term “atrial fibrillation” into the search field of PubMed and limits their search to publications in the year 2017, there are 4,867 results. This is compared with only 1,394 entries that appear when using the term “sudden cardiac death” and the same search criteria. Even if one limits their interest to data from clinical trials, there is a mountain of literature published annually. Thus, the following should be viewed as only a limited representation rather than a comprehensive review of the literature published surrounding AF.1 The number of individuals with AF in the United States is expected to double by the year 2050.2 “AF begets AF” is a long-heralded mantra reflective of the idea that AF is a progressive diease.3 Padfield et al., in a manuscript published in June of this year, sought to determine the rate of progression from paroxysmal to persistent AF over a 10-year period.4 Analyzing data from the Canadian Registry of Atrial Fibrillation, they demonstrated that more than 50% of patients with paroxysmal AF over 10 years will either have progressed to having persistent AF, or have died. The rate of progression was 36.3% at 10 years. This sobering result suggests that we have a long way to effectively halt the progression of this disease despite early recognition.It has long been recognized that AF is an arrhythmia of pandemic proportions, with the lifetime risk of developing the disease after the age of 40 hovering at around 25%.5 Cherian and colleagues examined the effects of AF on patients with heart failure within the ORBIT-AF registry.6 They report that, among patients with AF, those with heart failure had a similar rate of stroke but a higher risk of death or hospitalization [hazard ratio (HR): 1.69 and 1.31, respectively) as compared with those without heart failure. In patients with a left ventricular ejection fraction < 40%, the HRs were 2.06 and 1.38, respectively. This was despite a good background of heart failure therapy. These results, while equally concerning, are notably both the result of detrimental hemodynamic effects, which may support the notion that the treatment of AF in this population should include a rhythm control strategy versus methods aimed solely at preventing thromboembolism.AF also remains intertwined with other disease epidemics that continue to plague our health care system. The association between AF and congestive heart failure remains an incredibly common finding, with roughly 40% of patients having one of these conditions being likely to develop the other.7 Balla and collaborators examined the relationship of body mass index (BMI) and the risk of stroke in patients with AF8 in patients treated in the ROCKET AF trial.9 They found that individuals with a BMI ≥ 35 kg/m2 had a lower risk of stroke when compared with their normal-weight counterparts, whether on rivaroxaban or warfarin . While the mechanism of this effect is secondary to increased time in the therapeutic range in the warfarin group or is related with favorable effects on the potential prothrombotic state, it remains a fact that despite this obesity paradox, there is research linking obesity with an increased risk of the development of AF10 and the progression of paroxysmal AF to persistent AF.11Another growing health problem is the obesity epidemic. Interestingly, there are reports that detail a protective effect of obesity regarding AF, termed the “obesity paradox.”™ device .12 This trial included more than 1,000 patients who met the criteria for LAA occlusion according to the European Society of Cardiology guidelines13 with or without a contraindication for anticoagulation. In the trial, 73% of patients were deemed unsuitable for anticoagulation. Thus, a mix of patients taking warfarin (16%), novel oral anticoagulants (11%), dual antiplatelet therapy (60%), single antiplatelet therapy (7%), and no therapy (6%) in the immediate peri-procedure period were included in this study. Subsequent findings indicated a high level of procedural success (98.5%) and low residual leaks > 5mm (99.5%), with device thrombus present in 3.7% of participants. Notably, the presence of device thrombus did not correlate with anticoagulant regimen. Furthermore, the annual stroke rate was 1.1% despite a high baseline CHA2DS2-VASc score of 4.5. Thus, LAA occlusion with the WATCHMAN™ device  was deemed effective at preventing stroke even without peri-procedural anticoagulation. These data also pave the way for the ASAP-TOO trial, a randomized prospective trial investigating the effectiveness of LAA occlusion with the same device in patients who are labeled unsuitable for oral anticoagulation.14The last several years has seen an increase in a focus on the prevention of thromboembolism secondary to AF corresponding with the growing number of treatment options beyond warfarin. This year was no exception. Boersma et al., on behalf of the EWOLUTION investigators, reported on their one-year follow-up data of left atrial appendage (LAA) occlusion with the WATCHMAN™ device  versus long-term warfarin therapy in patients with nonvalvular AF.15 Reddy and colleagues report on findings from more than 1,100 patients who were followed for five years, and demonstrate noninferiority in the composite endpoint of stroke, systemic embolism, or cardiovascular/unexplained death when compared with warfarin. However, much like the previous trials, there were significant decreases in hemorrhagic stroke, disabling stroke, cardiovascular/unexplained death, total mortality, and post-procedure bleeding. The most significant decrease was in all-cause mortality, with a demonstrated HR of 0.73 for the device as compared with warfarin. These results are extremely encouraging and, although we still do not yet have a head-to-head comparison between the WATCHMAN™ device  and novel oral anticoagulation therapy, it appears that LAA occlusion therapy should be included in our arsenal for the prevention of stroke in patients with nonvalvular AF.Hot off the press are the five-year outcomes data from the PROTECT and PREVAIL trials, which examined again the WATCHMAN16 This underscores the importance of identifying patients at risk for developing AF in a predictive manner. The PREDATE AF study presented by Nasir et al. prospectively evaluated the use of the CHA2DS2-VASc score to predict new-onset AF using implantable loop recorders  and determined whether this led to the initiation of anticoagulant therapy.17 In almost 250 patients with a CHA2DS2-VASc score ≥ 2, 22.4% were noted to have new F, defined as an episode lasting longer than six minutes, over the 18-month study period. Of these individuals, 76.4% were subsequently started on oral anticoagulation. There was no demonstrated increase in incidence of AF correlated with an increase in CHA2DS2-VASc score, but the study was likely underpowered to detect such a finding. These data, taken together with those of trials presented previously, such as the ASSERT-II trial,18 support the use of implantable monitors to detect AF in high-risk patients.While our therapy choices for stroke prevention continue to grow, many patients will only present for the first time with a significant thromboembolic event.19 Increased stellate ganglia sympathetic nerve activity has been demonstrated to be associated with atrial arrhythmias20 but requires invasive measurements to elucidate. Skin sympathetic nerve activity is a potential marker of stellate ganglia activity. Interestingly, a group of researchers from Indiana University has elegantly demonstrated simultaneous measurements of skin sympathetic nerve activity and electrocardiogram recordings.21 From this group, Uradu and colleagues further reported based on findings from a small group of patients that skin sympathetic nerve activity increases prior to the onset and termination of atrial tachycardia and AF.22 This technology, though only in its infancy, has a significant potential to both predict AF noninvasively and further our understanding of atrial arrhythmias. These findings may lead to the development of treatment algorithms that help to modulate autonomic activity to ensure better control of arrhythmias.An exciting development in the ability to predict AF is utilizing our knowledge of mechanisms or arrhythmia onset or initiation. It has been demonstrated that the autonomic nervous system plays a vital role in the modulation of cardiac arrhythmias, from sudden cardiac death to AF.The past year has had no shortage of literature regarding the use of catheter ablation as a treatment for AF. While we all eagerly await the next “big thing” with regards to this area, 2017 still provided us with some important data to refine the technology and techniques for AF ablation.23 Adding to this literature, Fink et al. published the results of the Alster-LOST-AF trial.24 One hundred twenty-four patients with persistent or longstanding persistent AF were randomized in a 1:1 fashion to undergo PVI alone or PVI with substrate modification. They reported a one-year tachyarrhythmia-free incidence of 54% in the PVI-alone group versus 57% in the group who underwent PVI with substrate ablation. Notably, the substrate ablation followed the stepwise approach.25 As such, while these results suggest that targeted substrate ablation may not add value to PVI, the low single-procedure success rates underscore the need for further investigation regarding what is the ideal approach for catheter ablation of AF.The last several years have solidified pulmonary vein isolation (PVI) as the cornerstone of AF ablation, while suggesting that additional ablation procedures including lines or substrate targeting do not confer an additional benefit. Trials such as the STAR-AF study have suggested that this is true even in patients with persistent AF.26 In the CAMERA-MRI trial, Prabhu and colleagues report their results of catheter ablation versus rate control for AF in a population of patients with idiopathic cardiomyopathy.27 Specifically, they demonstrate very significant improvements in ejection fraction in the ablation group as compared with in the rate control group . Moreover, they indicate that the degree of improvement correlates with a lack of late gadolinium enhancement on cardiac magnetic resonance imaging. The single-procedure success rate in the trial was 56% off antiarrhythmics and 75% on medications. This important finding supports the idea that AF itself is an important cause of left ventricular dysfunction even when there is adequate rate control, and the lack of significant fibrosis predicts recovery. Catheter ablation for AF in this population would expect to lead to significant improvements in overall mortality given the improvements in systolic function.Despite success rates that leave room for much improvement, catheter ablation for AF still may be the best treatment for patients with advanced disease, especially those with an increased risk for poor outcomes, such as those with heart failure or cardiomyopathy. Previous trials such as the AATAC trial have demonstrated the superiority of catheter ablation over pharmacologic therapy and associated improvements in left ventricular ejection fraction.28 They suggest that this approach has similar results to the conventional approach while decreasing the total amount of cryoablation time, as a time-to-isolation on first application of < 60 seconds only requires a single freeze. Also, Cardosos et al. published an important meta-analysis of ablation with uninterrupted novel oral anticoagulant use29 demonstrating equivalency to uninterrupted warfarin but with a significantly lower risk of major bleeding.In regards to ablation protocol, there were multiple reports in 2017 refining current techniques. Aryana et al. report using a time-to-isolation parameter as a means of dosing cryoablation for AF.30 The authors suggest that using temperature control allows for an appropriate modulation of power so as to achieve transmurality without the risk of steam pops or char formation. Temperature feedback, of course, is used in traditional nonirrigated ablation and has long been understood to have the greatest correlation with lesion depth in standard radiofrequency (RF) ablation.31 Temperature control capabilities have also been used previously in an internally cooled ablation catheter during AF ablation.32 The authors of this current study demonstrate a 70% decreased RF time in comparison with that seen with conventional contact force ablation, but a 100% rate of acute procedural success. This catheter design may prove to greatly increase the efficiency of point-by-point catheter ablation.Finally, the one interesting development in catheter technology in 2017 was the first reported human studies involving a unique diamond-tip catheter with six thermocouples that allows for irrigated ablation with temperature control. Iwasawa and colleagues reported that the use of this catheter is feasible in a single-center series.33 First, the guidelines make a distinction in the treatment of paroxysmal versus persistent AF, that while patients with paroxysmal AF should have symptoms and fail a medication in order to reach a point of consideration for ablation, that it is reasonable to consider ablation as a first-line therapy in patients with persistent AF whether or not they have been tried on an antiarrhythmic drug. The guidelines also clearly state that ablation is reasonable in patients with heart failure or hypertrophic cardiomyopathy. These statements reflect the importance of recognizing that these individuals are at a higher risk of morbidity and mortality associated with AF. Third, the guidelines note clearly that there is strong evidence that ablation improves symptoms and left ventricular function. Lastly, they indicate that the decision to discontinue anticoagulant therapy should be based on the individual risk of thromboembolism and not the clinical outcome of the procedure.Last but certainly not least, 2017 gave us an updated consensus document for the treatment of AF. The 2017 Heart Rhythm Society/European Heart Rhythm Assocation/European Cardiac Arrhythmia Society/Asia Pacific Heart Rhythm Society/ Sociedad Latinoamericana de Estimulación Cardíaca y Electrofisiología expert consensus statement on catheter and surgical ablation of AF, chaired by Dr. Hugh Calkins, gives us several important new takeaways.I hope that this review is useful in summarizing some of the key literature published in 2017. Again, this is simply representative and not all-inclusive of the available topics, and clearly reflects my own biases. I anxiously look forward to new discoveries and innovations for the treatment of AF sure to come in 2018.


Many adolescents in special education are affected by anxiety in addition to their behavioral problems. Anxiety leads to substantial long-term problems and may underlie disruptive behaviors in the classroom as a result of the individual’s inability to tolerate anxiety-provoking situations. Thus, interventions in special needs schools that help adolescents cope with anxiety and, in turn, diminish disruptive classroom behaviors are needed.This study aimed to evaluate the effect of a virtual reality biofeedback game, DEEP, on daily levels of state-anxiety and disruptive classroom behavior in a clinical sample. In addition, the study also aimed to examine the duration of the calm or relaxed state after playing DEEP.A total of 8 adolescents attending a special secondary school for students with behavioral and psychiatric problems participated in a single-case experimental ABAB study. Over a 4-week period, participants completed 6 DEEP sessions. In addition, momentary assessments  of self-reported state-anxiety and teacher-reported classroom behavior were collected throughout all A and B phases.d=–0.29) and a small, nonsignificant reduction of disruptive classroom behavior (d=−0.16) on days when participants played DEEP. Moreover, it was found that the calm or relaxed state of participants after playing DEEP lasted for about 2 hours on average.From analyzing the individual profiles, it was found that 6 participants showed reductions in anxiety, and 5 participants showed reductions in disruptive classroom behaviors after the introduction of DEEP. On a group level, results showed a small but significant reduction of anxiety (This study demonstrates the potential of the game, DEEP, as an intervention for anxiety and disruptive classroom behavior in a special school setting. Future research is needed to fully optimize and personalize DEEP as an intervention for the heterogeneous special school population. Adolescents attending schools for special education in the Netherlands  are characterized by profound behavioral and psychiatric problems , such asConventional school-based interventions for anxiety are usually based on cognitive behavioral therapy (CBT) . HoweverA promising alternative approach to enhance mental health among children and adolescents is the use of video games. A recent review showed that video games provide youths with immersive emotional experiences, teaching them new forms of emotional, cognitive, and behavioral strategies . Video gphysiological reactivity. Anxious youths tend to experience hyperarousal  in response to stressors [Recently, a virtual reality biofeedback game (DEEP) was developed as a potential intervention to reduce anxiety in youths . In DEEPtressors . The bodtressors ) to prodtressors ,26. ThusThe mechanism through which DEEP teaches players how to regulate physiological responses is diaphragmatic breathing . DiaphraThe breathing exercises incorporated in DEEP are based on biofeedback, which is defined as the process of feeding information back to the individual about one’s physiological state to gain awareness and control over physiological processes . BiofeedPreliminary evidence for the efficacy of DEEP as an intervention for anxiety has been demonstrated in a recent pilot study . In a saNext to the potential of DEEP as an intervention for anxiety, an additional yet uncertain beneficial effect of DEEP could be an improvement in the ability to regulate disruptive classroom behaviors. Previous research has shown that traditionally delivered deep-breathing exercises reduced rates of disruptive classroom behaviors (assessed by blind independent observers) among youths attending a special school for students with behavioral problems . MoreoveSecond, playing DEEP could potentially reduce disruptive behaviors through its effect on interoceptive awareness: the ability to recognize internal physiological states . PlayingOur primary aim in this study was to investigate the effect of DEEP on daily levels of state-anxiety and disruptive classroom behavior in a clinical sample. It was expected that playing DEEP would reduce both participants’ state-anxiety and disruptive classroom behavior. Second, we explored the duration of the calm or relaxed state of participants after playing DEEP. No specific hypotheses were formed as this is the first study examining the duration of the effect of playing DEEP. To meet the research aims, we conducted a single-case experimental design (SCED) study in a special school setting. The SCED methodology was particularly appropriate because it allowed us to evaluate the efficacy of an intervention for individuals with heterogeneous characteristics . ThiDisruptive classroom behavior outcomes were personalized per adolescent as difficulties to regulate oneself may manifest differently in each individual. This idiographic assessment approach may provide insight into whether the intervention is impacting the problems that teachers frequently observe and consider most important . The SinParticipants were 8 adolescents  attending a secondary special school for students with behavioral and psychiatric problems in the northern part of the Netherlands. Adolescents were considered eligible for the study if teachers had the impression that the adolescent showed symptoms of anxiety, displayed disruptive behaviors in the classroom, and could handle the burden of completing momentary questionnaires . In total, teachers put forward 10 eligible adolescents. A school clinician contacted the parents of those 10 adolescents by phone to inform them about the study goals and to invite their child for participation. A total of 8 adolescents and their parents expressed their interest to participate in the study. The research team contacted these parents by phone to provide detailed information about the study’s procedure. All adolescents and their parents provided initial verbal consent and received an information and consent letter. Parental written informed consent was sent by mail. Individual demographic characteristics including participants’ age, gender, educational level, pretest trait-anxiety score, diagnoses, current medication, and treatment are provided in 0), intervention (B), and withdrawal or no intervention (A1) phases. A and B phases were alternated several times over a period of 4 weeks . All participants started with the A0 baseline phase, which lasted for 5 or 6 days. After the baseline phase, the first intervention period  began. The B phases usually lasted for 1 day, in which participants completed 1 DEEP session in the morning. However, some participants played DEEP on 2 subsequent days because the planned DEEP sessions did not synchronize with the participants’ schedule. The intervention days  were alternated with withdrawal days, in which participants did not play DEEP . All participants completed 6 DEEP sessions throughout the study period, except for participant 8 who completed 5 DEEP sessions because of illness. Another exception to this design was participant 5, who was doing an internship outside of the school for 2 days a week. Therefore, he participated in the study 3 days a week for 8 subsequent weeks .This study followed an ABAB withdrawal or reversal design, with baseline . Teachers reported what they had observed about participants’ disruptive classroom behavior in the past 2 hours around 10:20 AM, 12:25 PM, and 2:30 PM. During B phases, teachers reported what they had observed about participants’ behavior Participant 4 was observed by 3 teachers: 1 teacher observed for 4 days a week, 1 teacher observed for 1 day a week, and 1 teacher observed for 2 days in total when one of the other 2 teachers was ill. Participants 6 and 7 were classmates and were also observed by 2 teachers: 1 teacher observed for 4 days a week, and the other observed for 1 day a week. The remaining participants were observed by a single teacher. Participants 1 and 2 were classmates, so their behavior was observed by the same teacher.At the start of the study, interviews with the teachers were held to discuss specific disruptive classroom behaviors of each participant. On the basis of their input, a personalized questionnaire about each participant’s disruptive behavior was developed. Afterward, participants filled in questionnaires regarding their demographics and trait-anxiety at pretest. Then, participants completed the ABAB study that lasted for 4 weeks. The study procedure was followed twice; in the first block, participants 1 to 4 participated, and in the second block, participants 6 to 8 participated. Participant 5 participated in both blocks. After participation, participants and their teachers both received a monetary compensation of €25.00. A total of 2 teachers received a monetary compensation of €37.50 because they had observed 2 adolescents for 4 or 5 days a week. Ethical approval for this study was obtained from the Radboud University Ethics Committee Social Sciences (ECSW-2017-038R1).Adolescents’ state-anxiety was assessed with the 6-item short form of the state scale of the Dutch State-Trait Anxiety Inventory (STAI) ,51. The never, 1=rarely, 2=sometimes, 3=often, 4=very often, and 5=almost always. Items 1 and 2 from participant 8 were recoded as the desired effect for this participant would be an increase in behavior rather than a decrease.On the basis of the interviews with the teachers, 2 or 3 disruptive behaviors were defined per participant . During Participants completed DEEP sessions in a separate room at school. Upon arrival, participants first sat in a turnaround desk chair after which the DEEP breathing belt was placed around the abdomen. The DEEP belt contains an Arduino-compatible FLORA wearable electronic platform  that ope0, B, and A1 phase assessments of state-anxiety were calculated for each participant. In addition, the percentage of data points exceeding the median  was comt review  was used0 baseline phase only), variability, and level [0 baseline phase; reduction of the level of anxiety over the course of the intervention [0 baseline phase, the median absolute deviation (MAD) was computed for participants’ A0 baseline phase and the B phases. To calculate the MAD of the B phases, data points of all B phases were merged together for each participant. The MAD was calculated on the 6 STAI items instead of participants’ mean anxiety to optimally represent participants’ variability.To gain a general impression of the data, a visual analysis was performed involving the examination of trend . To assrvention  were calrvention . Lane anrvention  proposed1) phases [To assess changes in level, a visual analysis usually focuses on mean score differences between phases . However) phases . Especia) phases . Recursi) phases ) in R [6) phases .0 baseline phase only with the data points in the B phases.After the visual analysis, nonoverlap of all pairs (NAP ) was calFinally, the between-case standardized mean difference (BC-SMD ) was comThe same procedure, as described earlier, was repeated for classroom behavior. As NAP scores revealed the same pattern of results for all observed behaviors within the majority of participants, it was decided to only report the results of behavior 1 of each participant. However, for participants 2 and 3, behavior 2 was reported, and for participant 5, behavior 3 was reported because these were the only behaviors that showed medium effects for these participants; all other behaviors of these participants showed no effect.In terms of missingness, 133 (27.0%) out of the 492 state-anxiety assessments were missing for reasons such as practical lessons, national holidays, or illness. Furthermore, 161 (32.7%) out of the 492 disruptive classroom behavior assessments were missing because of the absence of the adolescent  or lack of time. We decided not to use imputation strategies, as these percentages of missing data may not affect the quality of statistical inferences . Moreove0, B, and A1 phase assessments of anxiety and PEM scores for each participant. Overall, participants reported a mean anxiety of 3.32 (SD 1.81) in the A0 baseline phase, 2.35 (SD 1.48) in the B phases, and 2.30 (SD 1.31) in the A1 phases. Thus, participants’ mean anxiety scores fell within one-third of the full range of the scale in all phases. The findings indicate that participants’ anxiety scores may have declined over the course of the intervention. The minor difference between the participant’s anxiety in A1 and B phases suggests that carryover effects may have occurred. PEM scores ranged between 61% (11/18) and 100% (17/17), indicating that most assessments of anxiety were lower on the days of DEEP sessions compared with the A0 baseline phase.0 baseline phase seemed to decrease for half of the participants . This d statistic indicates a small but significant reduction of anxiety with the introduction of DEEP [d=−0.43  for the data measured around 10:00 AM, a value of d=−0.34  for the data measured around 12:00 PM, and a value of d=−0.02  for the data measured around 2:00 PM. These d statistics indicate a medium significant reduction of anxiety directly after gameplay, a small but significant reduction 2 hours after gameplay, and no reduction 4 hours after gameplay  in the A0 baseline phase, 2.11 (SD 1.53) in the B phases, and 2.21 (SD 1.49) in the A1 phases. These findings indicate a small decrease in participants’ disruptive classroom behavior during the intervention. The minor difference between participants’ classroom behavior in A1 and B phases suggests that carryover effects may have occurred. PEM scores ranged between 0% (0/11) and 77% (10/13), indicating that the effect of DEEP on classroom behavior varied between participants.0 baseline phase decreased only for participant 6. However, this finding should be interpreted cautiously as the level of disruptive behavior for this participant seemed to increase over the course of the intervention. The data pattern of all other participants did not correspond to what is expected from an effective intervention as variability in disruptive behaviors in the B phases compared with the A0 baseline phase increased or remained the same , indicative of a small, nonsignificant reduction of participants’ disruptive behavior with the introduction of DEEP [The assumptions of normality and absence of clear baseline trends were tested and met. The overall A vs B comparison of the BC-SMD analysis yielded a value of  of DEEP .This SCED study evaluated the efficacy of the virtual reality biofeedback game, DEEP, as an intervention to reduce anxiety and disruptive classroom behaviors in adolescents in a special school setting. The primary aim of the study was to test the effect of playing DEEP on daily levels of state-anxiety and disruptive classroom behavior. On a group level, results indicated a small-sized reduction of state-anxiety after the introduction of DEEP. On the individual level, strong evidence was found for 5 out of 8 participants as their NAP scores indicated a medium-sized reduction and their level of anxiety seemed to decrease over the course of the intervention. Moderate evidence was found for 1 participant, as NAP scores indicated a medium-sized reduction, but no change was found in the level of anxiety. In terms of disruptive classroom behavior, a small, nonsignificant reduction was found on the group level. On the individual level, strong evidence was found for 4 out of 8 participants as their NAP scores indicated a medium-sized reduction and their level of disruptive behavior seemed to decrease. Moderate evidence was found for 1 participant as the NAP score indicated a medium change, but no change was found in the level of disruptive behavior. The secondary aim of our study was to investigate the duration of the calm or relaxed state of participants after playing DEEP. Results indicated that, on average, the effect of playing DEEP lasted for 2 hours.In line with our hypothesis, the current findings indicate that, on a group level, DEEP reduces daily levels of state-anxiety. These results corroborate the previous pilot study conducted by Van Rooij et al , who fouThis study is the first study examining the duration of the effect of DEEP on state-anxiety and found that, on average, the calm or relaxed state of participants after playing DEEP lasted for 2 hours. This duration indicates that the effect of playing DEEP does not persist through the whole school day but may be particularly valuable to use in specific anxiety-provoking situations in class, such as during exams or when giving a speech. Although this study provides insight into the duration of the effect of DEEP on a group level, insight into individual variability in the duration of the effect is lacking. The NAP and visual analysis strategies used in this study are not suitable to analyze the duration of an effect on an individual level. It is possible that some individuals mainly reported reduced levels of state-anxiety directly after gameplay, whereas others still felt calmer or more relaxed at the end of a day playing DEEP. In addition, it is unknown if there are differences in the duration of the effect of DEEP between the 6 sessions. Participant 6, for example, showed a steep increase in anxiety during the day after the first and second DEEP session but seemed to feel calm or relaxed for a longer period after the last couple of DEEP sessions . Future Analyses yielded mixed results for the efficacy of DEEP to reduce levels of disruptive classroom behavior. The results partly confirmed our hypothesis as 5 out of 8 individuals showed a reduction of disruptive classroom behavior after the introduction of DEEP. DEEP may have affected participants’ behavior in various ways. First, as high levels of anxiety may underlie escape-driven disruptive behaviors ,38, partSecond, DEEP may have reduced participants’ disruptive behavior through its effect on interoceptive awareness. Although empirical evidence is yet lacking, it is likely that participants’ interoceptive awareness improved over the course of the intervention because participants were continuously informed about their stage of breathing while playing DEEP. It has been theorized that increased interoceptive awareness may enable individuals to mobilize self-regulation resources , which aContrary to our hypothesis, we found a small, nonsignificant effect of DEEP on disruptive behavior on a group level. The 3 participants that did not seem to benefit from DEEP in terms of their behavior may have cancelled out the effect of the participants that did, leading to an unobservable effect on a group level. There are several possible explanations why DEEP did not affect the behaviors of those participants. First, anxiety may not have been the cause of disruptive behaviors in the classroom for these individuals. Rather, neurological deficits in individuals with ADHD or ASD that are associated with attention-related problems -79 may hN-of-1 Trials .Regarding the RoBiNt recommendations for internal validity, not all recommendations were carried out. First of all, we did not randomize the beginning of the study phases. We explicitly chose not to randomize the conditions because of constraints of the school setting  and characteristics of the target group . In terms of sampling of the target behaviors, the required minimum of 3 data points in each study phase  was not Although this study provided initial insight into the efficacy of DEEP in a special school setting, future research is needed to fully optimize DEEP as an intervention for this heterogeneous population. The intervention effects of DEEP could be optimized using a parametric analysis of the optimal amount of play time for a given individual . MoreoveThe aforementioned limitations notwithstanding, this study has important implications for clinical practice. Although it should be noted that findings need to be interpreted cautiously in a study of this type, this study demonstrated the potential of DEEP as an intervention to reduce daily levels of state-anxiety and disruptive classroom behavior in a special school setting. The results implicate the clinical techniques of diaphragmatic breathing and biofeedback in treating individuals with anxiety. Moreover, the results demonstrate that an applied game incorporating those techniques can be used either in isolation or as an add-on to existing interventions in a clinical sample. On overage, our results showed that the calm or relaxed state of participants after playing DEEP lasted for 2 hours. Therefore, school clinicians are recommended to tailor implementation strategies of DEEP to the individual with different needs early or later in the day, considering individual variation in the duration of the effect of DEEP. The implementation of game-based interventions might be a promising avenue for the special school setting, as video games can be tailored to the diverse needs and learning paces of the heterogeneous special school population.This study also has strong implications for future research on behavioral health interventions. This study demonstrated that there are individual differences in the extent to which an applied game to enhance mental health is effective. Therefore, we need to tailor behavioral interventions to the personal needs of different individuals. Although the randomized controlled trial may be the golden standard in behavioral health intervention research, this costly and time-invasive method is limited because of a lack of attention paid to individual differences ,83. We a


PgiC1 and “foreign” PgiC2 in the perennial grass Festuca ovina. Both PgiC1 and PgiC2 encode cytosolic phosphoglucose isomerase, a dimeric enzyme whose proper binding is functionally essential. Here, we use biophysical simulations to explore the inter-monomer binding of the two homodimers and the heterodimer that can be produced by PgiC1 and PgiC2 in F. ovina. Using simulated native-state ensembles, we examine the structural properties and binding tightness of the dimers. In addition, we investigate their ability to withstand dissociation when pulled by a force. Our results suggest that the inter-monomer binding is tighter in the PgiC2 than the PgiC1 homodimer, which could explain the more frequent occurrence of the foreign PgiC2 homodimer in dry habitats. We further find that the PgiC1 and PgiC2 monomers are compatible with heterodimer formation; the computed binding tightness is comparable to that of the PgiC1 homodimer. Enhanced homodimer stability and capability of heterodimer formation with PgiC1 are properties of PgiC2 that may contribute to the retaining of the otherwise redundant PgiC2 gene.Duplicative horizontal gene transfer may bring two previously separated homologous genes together, which may raise questions about the interplay between the gene products. One such gene pair is the “native”  Among non-parasitic flowering plants, HGT of functional nuclear genes has been shown to be uncommon3, but frequent in grasses6, one example being the PgiC2 gene in the perennial grass Festuca ovina8. PgiC2 has been suggested to be horizontally transferred into F. ovina from another grass genus Poa, based on the incongruent phylogenetic relationship between PgiC2 and the corresponding genes in several different Festuca species7 as well as the close sequence similarity between the down- and upstream (containing a transposition associated factor) regions of PgiC2 and those of the corresponding gene in one potential Poa donor species8. The horizontal transfer of PgiC2 from Poa to F. ovina has been estimated to have occurred ca.<600000 years ago4.Horizontal gene transfer (HGT) is a phenomenon in which organisms acquire genetic material from a different species, via unconventional means (e.g. via vectors such as a shared virusF. ovinaPgiC2) have native counterparts in the recipient species (i.e. duplicative HGT)5. The fate of a gene acquired via duplicative HGT is like that of a gene duplicate9, which could be nonfunctionalization10, neofunctionalization11, subfunctionalization12, or redundancy13. The last possibility may be immediately beneficial when the native gene copy accidentally loses its function due to reasons like lethal mutations9. In the grass F. ovina, the predominant PgiC2 gene product has been shown to more frequently occur in dry habitat3, implying that the foreign PgiC2 gene products may have some superior properties (over the native PgiC1 gene products) that hold the potential to make its presence beneficial to F. ovina under drought stress. Thus our first goal in the current study is to look for such properties.Most of the horizontally transferred functional nuclear genes between grasses identified so far (including F. ovinaPgiC2 gene and its native counterpart PgiC1 code for the cytosolic phosphoglucose isomerase (PgiC) enzyme, which is a metabolic enzyme that catalyses the reversible isomerization between glucose-6-phosphate and fructose-6-phosphate in glycolysis at a metabolic intersection spot that is also connected with several other metabolic pathways, e.g. starch/sucrose synthesis and gluconeogenesis16. The functional unit of a PgiC protein is a dimer formed by two polypeptides17, and the catalytic centres of a PgiC protein are composed of residues contributed by both monomers. Therefore, the proper association of the two monomers within a dimeric PgiC protein is essential not only for structural stability but also for the function18. In F. ovina, it has been shown that, in addition to the PgiC2 homodimer, the polypeptide product of PgiC2 is able to form a PgiC1–PgiC2 heterodimer with the polypeptide encoded by PgiC119. Therefore, the second goal of the current study is to investigate how the two genes, originally separated by speciation, structurally “get along” with each other when forming a heterodimer at their abrupt encounter after the duplicative HGT.In addition, both the F. ovina. Using reference dimer structures based on homology modelling, two sets of biophysical MC simulations were conducted. The aim of the first set was to explore native-state conformational fluctuations of the dimers. Based on the resulting native-state ensembles, structural and inter-monomer binding properties of the dimers were investigated. The second set of simulations explored the mechanical response of the dimers to a stretching force. The ability of the dimers to withstand dissociation when pulled by a force depends on the inter-monomer binding tightness. We find that these two different approaches lead to very similar conclusions regarding the relative binding tightnesses among the three dimers. Both methods suggest that the binding is tighter in the PgiC2 than the PgiC1 homodimer, and the binding strength of the PgiC1–PgiC2 heterodimer is comparable to that of the native PgiC1 homodimer.In this paper, to address these goals, we present a Monte Carlo (MC)-based computational investigation of all these three PgiC dimers in 23. Compared to previously studied systems, the PgiC dimer is unusually large, with >500 residues per monomer unit.There have been many previous molecular simulation-based studies of the role of dimer stability in biological processesF. ovina PgiC1 sequence (GenBank accession no. AED99454) and one representative F. ovina PgiC2 sequence (AED99455) for our study. These protein sequences both contain 567 residues, and differ at 20 of the residue positions . The comparison reveals that the difference between the three systems in general is small, but some potential exceptions exist. To precisely assess the significance of these potential differences would require more statistics. Nevertheless, we note that there are four residue positions , all of which sit on or near the dimer interface, where the 30. Averaging over the native-state ensembles, we obtained 31. In perfect agreement with the contact analysis, the binding energy was lower for the PgiC2 homodimer . Therefore, focusing on the two homodimers, we also counted inter-monomer interactions involving these four specific positions. It turns out that these positions explain very little of the overall differences between the PgiC1 and PgiC2 homodimers in inter-monomer hydrogen bonds and hydrophobic interactions, respectively  at which the PgiC2 homodimer displays a notably higher In summary, the above analysis of the binding strength of the dimers, based on our simulated native-state ensembles, suggests that the binding is strongest for the PgiC2 homodimer. Hydrogen bonding, hydrophobic interactions and ionic interactions between the monomers were all found to be more abundant in the PgiC2 homodimer than in the other two dimers. These differences, in general, cannot be attributed to a small set of specific residue positions. However, the more abundant ionic interactions of the PgiC2 homodimer are largely explained by two variable residue positions located at the dimer interface.22. Taking this approach, we simulated the three PgiC dimers when subject to equal and oppositely directed forces acting on the Another computational approach that can shed light on the binding tightness of a dimer is by pulling simulations, where the response to external mechanical forces is probedThe inset in Fig. The dissociation time, mentclass2pt{minimP(t), in which the dimer has dissociated at a given MC time t. The P(t) data, plotted against t, support the above conclusion that the dissociation occurs more rapidly for the PgiC1 homodimer than for the PgiC2 homodimer  data are quite well described by an exponential saturation curve, Alternatively, the mechanical resistance of the dimers can be quantified by computing the fraction of runs, mer Fig. . Again, ess Fig. . In drawThe relatively strong binding tightness of the PgiC2 homodimer in the native-state simulations described earlier matches very well with the enhanced mechanical resistance of this dimer seen in the pulling simulations.PgiC2) and its native counterpart (PgiC1) in the grass F. ovina8. Both genes encode a PgiC protein, the functional unit of which is a dimer comprising two homological monomers. The catalytic centres of a PgiC protein are composed of residues contributed by both monomers18. Therefore, the proper association of the two monomers within a PgiC protein is essential not only for PgiC’s structural stability but also for its function18.This study focuses on one foreign gene (PgiC1 gene), the PgiC2 homodimer (coded for completely by the foreign PgiC2 gene) and the PgiC1–PgiC2 heterodimer. Starting from these modelled structures, we conducted biophysical simulations of the three dimers, to explore their native-state conformational ensembles and their mechanical resistance when pulled by an external force. All simulations were carried out under a constraint on the global structure of the monomers, to ensure that the monomers stayed native-like throughout the runs. No constraints were imposed on the relative position or orientation of the monomers in the simulated dimers. While artificial, this constraint still permits local structural fluctuations to occur anywhere along the polypeptide chain. Therefore, the generated conformational ensembles should offer a less biased understanding of the biophysical properties of the dimers, compared to examining single rigid structures.In this study, we have modelled the 3-D structures of the three types of PgiC proteins that are produced by the above-mentioned foreign-native gene pair: the PgiC1 homodimer , but indicated an elevated 36, indicating that stress-induced protein denaturation is common. This is probably what happens to the PgiC1 homodimer under extreme water deficit conditions in the Alvar grassland on Öland (Sweden), where the ecological genetic studies of the F. ovina PgiC proteins were carried out37. This biological environment is characterised by a thin layer of (or no) soil on a limestone plain, so the soil in some area there can easily dry up during summer, leading to intense desiccation. A loss of function of the PgiC1 homodimer may be especially problematic when stress-induced, because the glycolysis pathway that the PgiC protein is involved in plays an important role in providing both ATP and biosynthetic precursors for stress acclimation38.Could the presence of a more stable foreign gene product have any ecological significance? In plants, severe stresses usually induce the production of protein-stabilizing molecules (such as dehydrins and chaperones)PgiC2 gene product has been found to be significantly associated with dry environmental conditions in the Alvar populations of F. ovina on Öland3.In such adverse circumstances, the presence of a more stable PgiC2 homodimer that is more likely to stand the stress and stay functional may compensate for the denaturation-induced functional loss of the PgiC1 homodimer. This could be why the presence of the principal 39. This is, however, not likely to be the case in the current system, because the foreign PgiC2 gene is not fixed in the F. ovina populations (at least not yet). Instead, it can only be found in some individuals40, and even when it does occur, its presence can be very complex and cryptic. For example, silenced PgiC2 (pseudogene) is very common for individuals with PgiC240, and the PgiC2 gene may also exist as tandem duplicate and/or hemizygote41. All of these facts may suggest that under normal rather than extreme water-limited conditions, the presence of the foreign PgiC2 gene is inessential.The occurrence of a functional heteromer between a paralogous gene pair (like the PgiC1–PgiC2 heterodimer), may lead to a situation where the two genes require each other’s presence for maintaining their functionsPgiC1 and PgiC2 genes to be capable of heterodimer formation? Beside the cytoplasmic PgiC protein, plants also have a plastid-specific phosphoglucose isomerase, hereafter referred to as PgiP, which has been suggested to have a bacterial origin (maybe from cyanobacteria)43. Consistent with this, plant PgiP has a much higher protein sequence identity with cyanobacteria PgiC  than with plant PgiC . From the low protein sequence similarity between PgiP and PgiC in plants, we can see that these two phosphoglucose isomerases diverged very long ago. As a result, lots of differences have accumulated between them at the dimer interface, so that no heterodimer can be formed between them anymore44.Should one expect the products of the two previously separated F. ovinaPgiC1-PgiC2 system, the two previously separated genes are suddenly brought together by one or more recent inter-genus HGT events between F. ovina and a Poa species8. In contrast to the PgiC-PgiP pair, the PgiC1 and PgiC2 polypeptides can still form a heterodimer that functions properly19. The results presented here indicate an inter-monomer binding that is at least as strong in the PgiC1–PgiC2 heterodimer as it is in the native PgiC1 homodimer. This may not be surprising considering that PgiC heterodimers can form even between monomers from different plant families 44. In addition, inter-family PgiP heterodimers are possible 42. That all these inter-taxon PgiC or PgiP heterodimers can occur probably reflects the essential functions of the PgiC or PgiP proteins. The need to ensure their proper function may explain why their 3-D structures are relatively conserved among (at least not too distant) organisms .In the F. ovinaPgiC2 gene represents an interesting example of exchanges of functional nuclear genes between distantly related non-parasitic flowering plant species. Ecological significance has been suggested for the presence of this foreign PgiC2 gene in F. ovina under stressful environmental conditions on Öland, Sweden3. In support of this suggestion, our computational analysis has found a significantly stronger inter-monomer binding for the PgiC2 homodimer than for the PgiC1 homodimer, suggesting that a higher stability of PgiC2 (than PgiC1) that may help the host handle extreme stresses. This finding is supported by evidence derived both from equilibrium simulations of the native dimers, and from simulations probing the ability of the dimers to withstand dissociation when pulled by an external force. A second aim of this study was to find out how the foreign PgiC2 gene and its native counterpart PgiC1 get along with each other when forming a heterodimeric protein product, at their abrupt encounter after the horizontal gene transfer event. The PgiC1–PgiC2 heterodimer was found to show inter-monomer binding properties consistent with a proper functioning of the hybrid protein, with a binding strength comparable to that of the native PgiC1 homodimer. The results presented here thus support a picture where the foreign PgiC2 gene in F. ovina is conditionally advantageous over the native PgiC1 gene, and at the same time able to “collaborate” with the native gene in a non-harmful manner (through the formation of a hybrid protein). These factors may contribute to the retaining of this otherwise redundant foreign gene.The F. ovina PgiC1 (GenBank accession no. AED99454) and PgiC2 (AED99455) were identified and downloaded from the GenBank database (the only two full-length sequences available). Differences between the two sequences are summarised in Supplementary Table 49. The crystal structure of Toxoplasma gondii PgiC  served as template. This template has a high sequence identity with the input PgiC1 and PgiC2 sequences . The modelled structures were relaxed using Rosetta (v. 3.7) all-atom refinement50, to relieve steric clashes. For the PgiC1–PgiC2 heterodimer, a rigid-body docking strategy was adopted, using the ZDOCK (v. 3.0.2) server52 with the structures of the PgiC1 and PgiC2 monomers as input. The docking calculation was repeated three times, with consistent results. The docked hybrid complex was relaxed using Rosetta all-atom refinement.Representative protein sequences for 54 with the ProSA web server55. The computed Z-scores  if the assumptions of homogeneous variances and/or normality for the ANOVA test were not fulfilledUsing the biophysical model described above and MC sampling, the native-state ensembles of the dimers were explored. To gather statistics, a set of 24 independent runs was generated for each of the three dimers. Although no inter-monomer constraint was imposed, the dimers stayed intact throughout the runs. Each run comprised 500,000 MC cycles, which translates to about F denotes the strength of the force. The strength was set to As an independent way of assessing the tightness of the inter-monomer binding, an additional set of runs was performed in which the monomers were pulled apart by an external force. The external force was taken to act on the Supplementary information 1.Supplementary information 2.