Patients with end-stage kidney disease (ESKD) treated with hemodialysis (HD) experience many distressing symptoms. One frequently reported symptom is insomnia. There are unique issues about HD treatments and schedules that disrupt regular sleep/wake routines and possibly contribute to the high severity of insomnia. Despite evidence for broad-ranging health effects of insomnia, very few clinical trials have tested the efficacy of treatments for HD patients. Cognitive-behavioral therapy for insomnia (CBT-I) is a recommended first-line therapy but largely inaccessible to HD patients in the United States, partly because they commit considerable amounts of time to thrice-weekly dialysis treatments. Another important reason could be the logistical and reimbursement challenges associated with providing behavioral health care at the dialysis center. CBT-I delivered by telehealth can overcome barriers to access, but its efficacy has never been rigorously tested for these patients. Pharmacotherapy is the most widely used treatment for insomnia; however, some drugs presently used are unsafe as they are associated with a higher risk for death for HD patients (benzodiazepines and zolpidem-like drugs). The efficacy and safety of other medications (trazodone) for the treatment of insomnia has never been tested for patients treated with HD.This trial tests the short- and long-term comparative effectiveness of 6-week treatment with telehealth CBT-I, trazodone, or medication placebo. This will be accomplished with a randomized controlled trial (RCT) in which 126 participants treated with HD in community-based dialysis facilities with chronic insomnia will be assigned 1:1:1 to telehealth CBT-I, trazodone, or medication placebo, respectively; short-term effectiveness of each treatment arm will be determined at the end of 6-weeks of treatment and long-term effectiveness at 25-weeks. The primary and secondary patient-reported outcomes will be assessed with computer-based telephone interviewing by research scientists blinded to treatment assignment; additional secondary outcomes will be assessed by participant interview and actigraphy.This clinical RCT will provide the first evidence for the comparative effectiveness of two distinct approaches for treating chronic insomnia and other patient-reported outcomes for patients receiving maintenance HD.NCT03534284 May 23, 2018.SLEEP-HD Protocol Version: 1.3.4 (7/22/2020). In the United States, most adults with end-stage kidney disease (ESKD) are undergoing long-term in-center hemodialysis (HD) in community-based facilities . ApproxiAt least two factors may contribute to the high prevalence of insomnia in this patient population. First, day/night sleep reversal is a cardinal manifestation of uremia, the symptom complex associated with kidney failure. Studies have also shown that the severity of sleep problems are related to biochemical measures of kidney failure, suggesting insomnia may be the result of the accumulation of various uremic toxins . Second,Insomnia is an essential contributor to other poor health outcomes in HD patients. Insomnia in dialysis patients is strongly associated with daytime sleepiness and worse quality of life . Over 50Notwithstanding the wide-ranging adverse effects of insomnia on the health and well-being of HD patients, no clinical trial has ever been done to determine the efficacy of any treatment for insomnia in HD patients in the United States. Short and Long-term Effectiveness of Existing insomnia therapies for Patients undergoing HD (SLEEP-HD) is the first multi-center randomized controlled trial (RCT) for treatment of insomnia in patients undergoing HD and seeks to test the comparative effectiveness of two treatments for insomnia in patients undergoing HD.Specific Aim One – To compare the efficacy of 6-week treatment with telehealth CBT-I vs. trazodone vs. placebo for the treatment of chronic insomnia in patients undergoing HD. Hypothesis One - We hypothesize that the efficacy of six-week treatment with both telehealth CBT-I and trazodone will be superior to medication placebo in improving self-reported sleep.Specific Aim Two – To compare the sustained efficacy of the 6-week treatment with telehealth CBT-I vs. trazodone vs. placebo for the treatment of chronic insomnia in patients undergoing HD by examining outcomes at 25 weeks from randomization.Hypothesis Two - We hypothesize that telehealth CBT-I will be superior to both trazodone and placebo in improving self-reported sleep at 25 weeks from randomization.SLEEP-HD is a parallel-group RCT wherein 126 HD patients treated in community-based dialysis facilities in Seattle and Albuquerque will be randomized 1:1:1 over 31 months to 6-week treatment with telehealth Cognitive Behavioral Therapy for Insomnia (CBT-I), trazodone, or medication placebo Inclusion CriteriaAge 18 Years and olderUndergoing thrice-weekly maintenance hemodialysis for ≥3 monthsAble to speak EnglishInsomnia Severity Index (ISI) score ≥ 10 at pre-screening with sleep disturbances for ≥3 nights per week for ≥3 months .B)Exclusion CriteriaSevere depression assessed by Patient Health Questionnaire (PHQ)-2 and if appropriate, PHQ-9 Suicidal IdeationAlcohol abuse on alcohol assessment questionnaire (score ≥ 2) or substance abuse on Drug Abuse Screening Test (DAST)-10 questionnaire (score > 5) , 49Severe restless legs syndromeTreatment with trazodone in the past 1 monthKnown allergy to trazodone (self-reported or by chart review)Current treatment with monoamine oxidase inhibitors or in the preceding 14 daysCurrent treatment with linezolid (self-reported or by chart review)Current treatment with other drugs that are inhibitors of CYP3A4 , or known to prolong QT interval including Class 1A antiarrhythmics  or Class 3 antiarrhythmics , antipsychotic medications , and quinolone antibioticsPregnancy, or lactation, or women of childbearing potential not willing to use adequate birth controlLife Expectancy < 3 monthsExpected to receive a kidney transplant or transition to home dialysis  within 6 monthsAny other condition including cognitive impairment that, in the opinion of the investigator, should preclude patient participation in the clinical trial.Patients meeting inclusion criteria with ISI score ≥ 10 will be invited to sign an informed consent form to complete screening activities. The ISI is a 7- item instrument that measures self-reported insomnia. Each item is rated on a 0–4 scale with a total score range of 0–28. The instrument has been validated using both the classical test and the item response theory. After signing the consent form the following assessments will be completed: a) Medication Review to ascertain current use of trazodone or in the preceding 1 month; b) Patient Health Questionnaire-2 (PHQ-2); if the score on PHQ-2 is > 3, the PHQ-9 will be administered; c) Alcohol abuse on CAGE alcohol assessment questionnaire and DAST-10 for substance abuse; d) Cambridge-Hopkins RLS questionnaire; e) Medical history for co-existing illnesses; f) Pregnancy, or lactation, or women of childbearing potential.The participants’ nephrologist will be informed about their enrollment in the clinical trial and if randomized to drug therapy, with a list of medications that could prolong QTc on EKG in people treated with trazodone, as listed in the United States Food and Drug Administration package insert for the drug;Placement of a wrist actigraph on the participant and provision of instructions on wearing the device (see section 4.3 below). The actigraph will be placed on the arm opposite to the upper extremity with the functional arteriovenous access being used for HD. In participants with a lower extremity arteriovenous access or with a central venous catheter, the actigraph will be placed on the non-dominant arm.The 7-day run-in period will start as soon as possible after the completion of the screening procedures and placement of the actigraph.After a final review of inclusion/exclusion criteria, eligible participants will be provided with instructions to complete sleep diaries for the run-in period which will also include the following procedures:This will occur 7 days after the start of the run-in period and to the extent possible, on the first dialysis day of the week (Monday or Tuesday).The actigraphy devices will be collected from the participants;The online or paper sleep diaries will be reviewed and/or collected;The dialysis shift days and the dialysis shift start time for each study participant will be noted;The case-report form on cumulative weekly use of sedatives/hypnotics and opiates will be completed, assessing the use of sleep aids for the preceding 7 nights.The participants will be scheduled to receive a phone call for the baseline Computer-Assisted Telephone Interviewing (CATI).The following procedures will be completed at this visit:An interviewer blinded to study intervention allocation will call the participant to initiate the CATI to complete the ISI (primary outcome measure) and seven pre-specified secondary patient-reported outcome measures Table . CATI wiThe participants will be randomly assigned, 1:1:1 to CBT-I or trazodone or medication placebo using block randomization through a secure study web portal after the baseline CATI calls are completed, and no more than 25 days after completion of pre-screening with ISI. Participants will be randomly assigned to telehealth CBT-I, trazodone, or medication placebo, stratified by site using blocks of varying size [3 or 6] to ensure that the three groups are balanced at periodic intervals. The participants should begin treatment within 10 days from the date of randomization (first CBT-I session or provided with the first supply of medication).In this clinical trial, we will use telehealth CBT-I as a comparator and this will serve the dual purpose of testing the efficacy of CBT-I itself for HD patients, and its delivery via telehealth. Participants randomized to this arm will receive a treatment session once weekly for 6 weeks Table  51]. Ea. Ea51]. Each therapist will undergo in-person training by certified CBT-experienced study psychologists . Following the completion of training, each therapist is required to complete mock interventions prior to implementation of the intervention in the clinical trial. These mock sessions will be audio recorded and will be reviewed by a study psychologist using a structured fidelity adherence form. Once the therapists are deemed competent in the intervention, they will be ready to implement the intervention in the clinical trial.During the conduct of the trial, weekly conference calls will be scheduled with the therapists during which clinical and implementation issues will be discussed, and cases reviewed. All individual CBT sessions will be audio recorded using the zoom platform. All CBT-I sessions for the first four participants assigned to each therapist will be reviewed to ensure the therapists are consistently following the CBT-I intervention protocol, and thereafter 2 sessions will be reviewed to ensure ongoing fidelity to the intervention. Review of audio recordings will be as timely as possible to provide meaningful feedback to therapists and feedback will be provided at the ongoing supervision calls or through email. This approach will allow therapists to receive support and real-time feedback to ensure the highest standards for delivering the behavioral intervention.Adherence to CBT-I will be monitored by recording number of completed sessions, duration of each session, and completion of other behavioral homework assignments .The site investigators will prescribe trazodone (50–100 mg) or corresponding medication placebo to the study participants randomized to the respective arms. The dose range selected for the trial (50–100 mg) is guided by prior studies with trazodone . The staPrior to launching the clinical trial, the Investigational Drug Services at Harborview Medical Center in Seattle, where the PI is based, will procure 50-mg tablets of trazodone from the same manufacturing stock for both clinical sites and prepare a matching placebo. The medications will be dispensed as per the protocol by the Investigational Drug Services at each site, and the study coordinators will hand the study drug to the participants at the dialysis facility when they arrive for their routine HD treatments; this will occur once during week 1 where a 3-week supply of the drug will be provided (with capacity to increase the dose to 100 mg starting week 2), and at week 4 where a 3-week supply of the drug for the dose being taken during weeks 4, 5 and 6.The participants will be asked to return the medication bottles for study drug at week 4 and week 7, and the coordinators will perform a pill count to assess adherence with the medication. All unused drugs will be destroyed by the Investigational Drug Supply.The summary score from the ISI at weeks 7 and 25 from randomization will be the primary outcome measures for Aims One and Two, respectively.The primary ISI score and secondary patient reported outcomes Table  will be This secondary patient reported outcome measure will be ascertained during the same week as the CATI call is completed, at baseline, and weeks 4, 7, 13, and 25 from randomization. The participants will be asked to report the number of nights during the previous 7 nights that they used a prescription, or over the counter or other sleeping aid(s) . This information will be supplemented by a review of the electronic medical records. The cumulative use of prescription opioids in the previous 7 days will be recorded similarly.Actigraphy will be performed at three time-points: (a) the 1-week run-in period; (b) week 7 from randomization; and (c) week 25 from randomization. The key secondary outcome measure will be the average nighttime sleep efficiency .The actigraphy data will be used to compute the following variables for additional exploratory analyses: a) Sleep onset latency; b) Total sleep time; c) Total wake time; d) Daytime inactivity; and e) Light exposure. Each assessment will last 7 days, will be scheduled to start and end with a regularly scheduled dialysis treatment, and the study coordinator will meet with the participant in his/her HD facility. Actigraphy data collection will be scheduled such that the 7-day period starts the week prior to the CATI and ends the week CATI is performed. The actigraphy data will be downloaded and scored using the Actiware 6.0.9 software  to determine wake and sleep based on one-minute epochs. The actigraphy scorers will be blinded to the randomized treatment assignment of the participant. The validity of data from actigraphy will be increased by our approach of asking participants to maintain sleep diaries to guide scoring of bed and rising times, and to examine artifacts in data collection, e.g., removal of the device . AdditioThese visits Table  will occFor participants randomized to medication (trazodone or placebo), the visits and related procedures will be identical. Medication visits will occur at weeks 1,2,3,4 and 6.Visit one will occur, to the extent possible, on the day of the first dialysis treatment of the first week following randomization. The participants will be provided with a three-week supply of 50-mg trazodone or matching placebo Table .Visit two and three will occur on the day of the first dialysis treatment of the second and third week respectively following randomization and 1 week following the “Medication Assessment Visit One and Two”. The coordinator will ask the participant if they have been prescribed any new medications since the last visit to screen for contra-indicated drugs to prolong QTc interval on the EKG. The participants’ response to drug therapy will be assessed and they will be advised to either continue on the 50 mg dose if they are satisfied with their sleep over the previous 7 days, or to increase/decrease 50–100 mg based upon problems/tolerability with sleep in the previous 7 days.Visit four and five will occur on the day of the first dialysis treatment of the fourth and sixth week respectively following randomization.For participants that choose to terminate participation in the study, all efforts will be made to collect information about cumulative use of sedatives and hypnotics and opioids in the previous 7 days, and an early closeout CATI call will be scheduled for collecting data on primary and secondary patient reported outcome measures.Adverse Effects from CBT-I: The risk classification for this arm is “not greater than minimal.” The greatest risk with CBT-I is discomfort/fatigue associated with restricting time in bed (including reducing daytime napping); these symptoms are usually temporary but will be monitored closely given their overlap with post-dialysis symptoms. Additional potential risks include breach of confidentiality which is inherent to any telehealth intervention regardless of the participant’s location.Adverse Effects from Trazodone:With drug-drug interaction trazodone could prolong QTc interval on the EKGSide effects (> 5%) reported more frequently than with placebo are (i) drowsiness; (ii) nervousness; (iii) dizziness; (iv) fatigue; (v) dry mouth; (vi) nausea; and (vii) vomitingPriapism is another possible but extremely rare side effect3.Psychological discomfort from completing patient-reported scales4.Loss of patient confidentialityPatients undergoing HD experience many adverse events from their underlying health, co-existing illnesses, and concomitant medications. These adverse events include: (1) death; (2) fluid overload; (3) congestive heart failure; (4) vascular access events, such as thrombosis or infection or dysfunction; (5) atherosclerotic cardiovascular events; (6) infections, such as pneumonia; and (6) laboratory abnormalities such as anemia, hyperphosphatemia, and secondary hyperparathyroidism.The informed consent document will provide the name of a study staff person with a phone number to be contacted in the case of an emergency or if an adverse event occurred outside the time frame of study visits;Study staff will evaluate the participants 5–6 times during the 6-week treatment period either via tele-health, telephone, or in-person; and.Participants will be withdrawn from treatment if they experience a serious adverse event directly attributable to the study intervention and, in the opinion of the site investigator, if the treatment cannot be safely reinstituted.The participants will be monitored for the occurrence of adverse events as:All adverse events experienced by study participants from the time of registration into the study (randomization) will be recorded and summarized by random assignment group. The summary will be submitted to oversight groups at periodic intervals.All serious or unanticipated adverse events will be reported by each of the clinical sites to the Data Coordinating Center within 24 h of becoming aware of these events using a structured reporting form. Each site will also be expected to follow local reporting policies such as to the Institutional Review Board. Serious adverse events are the ones that result in death, are life-threatening, lead to prolonged hospitalization, or result in disability or incapacity. Unanticipated adverse events are unexpected adverse events, which in the opinion of the investigator, could reasonably be considered associated with participation in the research study. The Data Coordinating Center will be responsible for communicating these to the Data Safety and Monitoring Board consistent with the policies agreed upon and outlined in the Board’s charter.The primary outcome for Aim 1 is the ISI measured at 6 weeks after initiation of treatment. Given that we have three arms, a simple analysis for this aim would be an ANOVA analysis. However, to maximize the use of information and to account for potential loss to follow-up and/or intermittent missing outcome measures, we will use a comprehensive longitudinal analysis to make inference regarding group differences across time. Specifically, we will use linear mixed models for the analysis of all outcomes including the baseline, 4, 7, 13 and 25-week ISI measurements . For lonn = 126.The standard deviation for ISI in large clinical trials in other populations has ranged from 3.8–5.6 , 35. BasThe primary outcome for Aim 2 is the ISI measured 25 weeks from randomization. The analysis will leverage the same longitudinal model fit for aim 1, with a parallel primary hypothesis test and confidence interval estimation at 25-weeks as at 7-weeks. Secondary analysis will estimate the full trajectory over time for each treatment group and allow comparison of the group longitudinal profiles. A similar analytic approach will be taken for analyses of data for each of the 9 pre-specified secondary outcomes. Exploratory analyses will be undertaken to determine if there is evidence for effect modification by gender, race/ethnicity, presence of restless legs syndrome, concomitant medications, and treatment adherence.We have based our sample size on Aim 1, yet we retain strong power to detect long-term treatment effects. A power analysis using a simple pre-post analysis for 25-week outcomes controlling for baseline is a conservative estimate of the power for the longitudinal model since the simple analysis would only use 25-week complete cases. If we assume 20% loss to follow-up through 24 weeks, then we would have 100 evaluated participants. For Aim 2 we would have 90% power to detect a CBT-I treatment effect of 4.0 points relative to trazodone and placebo and within the range of moderate treatment effects defined by standardized group differences of 3.0–4.8 points.Insomnia is a common and distressing symptom for patients on HD, and there is evidence for a much larger impact on the health of patients. The SLEEP-HD study is an open-label RCT to compare two types of treatment for insomnia in participants who have ESKD on HD with chronic insomnia. The two types of treatment involved in the study are CBT-I or treatment with a drug (trazodone vs placebo).CBT-I is the first line therapy for insomnia but is largely inaccessible to dialysis patients , 41. TwoAs for pharmacotherapy, there is limited evidence to support current practices for insomnia for dialysis patients. Notwithstanding clinical practice guidelines, pharmacotherapy is widely used as a first-line therapy for insomnia including HD patients; it is also an alternative for patients unwilling to engage with CBT-I or for whom CBT-I is unavailable or inaccessible. However, the drugs used widely have either concerns about safety or efficacy, or have been insufficiently studied for patients undergoing HD. Benzodiazepines and non-benzodiazepine benzodiazepine-receptor agonists such as zolpidem are prescribed for 8–26% of HD patients , 63 evenThere is a compelling need to identify effective treatments for insomnia in HD patients, and the interventions being studied in this clinical trial  have a strong scientific premise. Most HD patients have significant impairments in quality of life, mainly from the high frequency of disabling symptoms. Insomnia is one of the most frequently reported symptoms, and studies of HD patients and other populations suggest that it is a significant contributor to other common symptoms and poor health outcomes. It is essential to know that insomnia is common in HD patients with comorbid depression but is independent of sleep apnea and restless legs syndrome , 12 . ThSLEEP-HD is the first multi-center RCT for the treatment of insomnia in patients undergoing HD and seeks to compare the efficacy of telehealth CBT-I vs. trazodone vs. medication placebo for the treatment of chronic insomnia in patients undergoing HD.Dialysis is a life-sustaining treatment for patients with kidney failure. Nevertheless, most patients experience many troubling symptoms, and disturbances in sleep are among the most reported problem. However, there has been limited research done to identify what treatments are effective for patients undergoing HD with sleep problems. Moreover, HD treatments are scheduled three times every week, making it extremely difficult for patients to have the time to receive cognitive behavioral therapy – a treatment effective in improving sleep in individuals without kidney failure. In this study, we plan to test the short- and long-term effectiveness of cognitive behavioral therapy with a therapist face-to-face delivered over the web, instead of in-person, with a drug. This study has the potential not only to benefit patients treated with HD but other patients that have difficulty in accessing treatment for sleep problems such as residents of rural and underserved areas.


The main outcome was well-being, measured by the Cohen-Mansfield Agitation Inventory (CMAI) and the Cornell scale for depression in dementia at 0, 3, and 6 months. Secondary outcomes were QUALIDEM quality of life domains and biochemical parameters of bone homeostasis. Intention-to-treat analysis with linear mixed modeling showed no significant between-group differences on agitation (p = 0.431) or depressive symptoms (p = 0.982). At six months, the UVB group showed less restless/tense behavior compared to the VD group  and lower serum 25(OH)D3 concentration . The exposure of nursing home residents with dementia to UVB light showed no positive benefits in terms of wellbeing. UVB treatment may have a positive effect on the restless/tense behavior characteristic of advanced dementia but more research is needed to confirm this finding.There are indications that ultraviolet B (UVB) exposure has beneficial effects on well-being through mechanisms other than vitamin D synthesis alone. We conducted a randomized controlled multicenter trial to compare the effects of UVB light and vitamin D supplementation (VD) in terms of the well-being of nursing home residents with dementia. Participants were randomly assigned to the intervention group (UVB group,  Observational studies indicate that low sun exposure increases the risk of cardiovascular disease ,2 and thInadequate sun exposure leads to vitamin D deficiency and insufficiency ,12. SuppInduction of cutaneous vitamin D production by using UVB exposure may be preferable to oral supplementation amongst older nursing home residents because it cannot induce toxic levels, it helps prevent polypharmacy and it is plausible that vitamin D synthesis is not the sole mechanism by which sunlight or UVB light exerts its beneficial effects on human health and well-being. Getting older is inevitably accompanied by perceiving a continuous loss in functioning, healthy state and social engagements, and this process is strongly delineated and progressive in persons with dementia . ImproviTherefore, the aim of this study is to compare the effect of UVB irradiation to oral vitamin D supplementation on well-being in nursing home residents with advanced dementia.This study had a randomized controlled multicenter trial design. Written informed consent was obtained from the legal representatives of all participants. The study was conducted in accordance with the declaration of Helsinki, and protocol was approved by the Medical Ethical Committee of Leiden University Medical Center (Registration No P16.010) on 11 April, 2016 and was registered in the Dutch Trial Register (NL5704).Participants were recruited from three nursing homes connected to the University Network for the Care sector South Holland (UNC-ZH). Team leaders of the nursing homes sent information letters with an informed consent form to all nursing home residents and their families. An independent physician with a specialty in internal medicine was assigned to answer the questions of the participants and their families.Inclusion criteria were a diagnosis of dementia and an age exceeding 70 years, while exclusion criteria were (1) actinic keratosis, (2) skin cancer, (3) porphyria, (4) sun allergy, (5) use of drugs that may induce photodermatosis, (6) hypercalcemia, (7) use of vitamin D fortified food, (8) anxiety, agitation or resistance to bodily contact. Examination of the participants for actinic and cancer skin lesions, as well as skin type according to the Fitzpatrick scale , was perThe participants were randomized in blocks of four and assigned to either receive the intervention  or standard vitamin D treatment . The group assignment files were placed in sequentially numbered opaque sealed envelopes to conceal the sequence until individual interventions were assigned. Intervention delivery and outcome assessment was not blinded. Nursing staff administered the medication, intervention and questionnaires to avoid disturbance of the daily routine.The sample size was calculated on the basis of the Cohen-Mansfield Agitation Inventory (CMAI), an instrument measuring agitation and covering 29 behavioral items, each rated on a 7-point Likert scale of frequency . Summed scores ranged from 29 to 203. Assuming a standard deviation (SD) of 13 points, an ά of 0.05 and an estimated drop-out of 40%, a sample of 80 patients would provide an 80% probability of detecting a mean between group differences of 10 points.−2, ultraviolet A (UVA)—4.650 W·m−2, ultraviolet C (UVC)—0.00001W·m−2, with UVB accounting for 54.6% of the spectrum. The exposure time was set at eight minutes a session, which was safeguarded by an electronic timer to prevent unintended longer exposure. Protective glasses were worn during treatment. The total treatment time over 6 months was 432 min. UVB treatment was discontinued when participants clearly objected or showed signs of discomfort on two consecutive sessions. They were then removed from the UVB exposure group and started again on vitamin D capsules. The control group received vitamin D capsules, 5600 IU cholecalciferol supplementation once a week, which is the standard treatment dose for all older persons >70 years according to the Dutch Health Council [The intervention consisted of half body UVB-irradiation with 1 standard erythema dose (SED). The procedure was carried out twice a week with a portable, tilting sunbed canopy  positioned at a fixed distance of 75 cm above a bed. The standard tanning lamps were replaced with UVB spectrum lamps . Lamp light emission consisted of UVB—5.013 W·m Council . All nurThe primary outcome was well-being, monitored with the CMAI and the Cornell scale for depression in dementia at 0, 3 and 6 months. Higher CMAI scores indicate a more frequent display of agitated behavior ,22. The Information on participant’s sociodemographic characteristics  and dementia severity were obtained at the baseline. Dementia severity was assessed using the Bedford Alzheimer Nursing Severity Scale (BANS-S), which is composed of 7 items, scaled 7–28, and a score of 17 or higher indicates severe dementia.p-value <0.05 was considered statistically significant. Analysis of treatment effects was conducted using linear mixed models that accounted for repeated measurements in the subjects, estimated using restricted maximum likelihood  [Statistical analyses were performed in SPSS 23.0  in accordance with the intention-to-treat principle. Descriptive statistics were used to outline the basic characteristics of the study population. The results are reported using the mean and standard deviation (SD) for normally distributed variables and median and interquartile range (IQR) for non-normally distributed variables. Pearson’s chi-square test, student’s t-test and Mann-Whitney U-tests were used to test differences between the baseline measurements in the intervention and control groups. A t, 2009) . Time waThis study was carried out between October 2016 and April 2017 in two nursing homes, and between October 2017 and April 2018 in a third nursing home. We started with the trial at the third location later because the number of persons who gave informed consent from the first two locations were not enough to reach the calculated power of the study. The legal representatives of seventy-nine nursing home residents gave informed consent to participate in the study .Twelve of the participants (30%) in the UVB group refused to adhere to the intervention procedure following initial sessions for a variety of reasons, including an unwillingness to remove clothes or to wear protective glasses, feeling cold or anxious, not understanding the purpose of the procedure or being unable to lie quietly on a bed during UVB exposure. The other participants (70%) showed variable adherence to the UVB treatment regime or died before the end of the treatment period, which resulted in the following duration of the UVB exposure: 8 of the participants (19%) completed UVB sessions of between 24 and 100 min in total, 3 (7%) between 100 and 200, 14 (34%) between 200 and 300 min and 4 (10%) between 300 and 400 min (when participants clearly objected to the UVB session it was discontinued). Eleven (28%) of the participants experienced the sessions as being pleasant and reinforcing, as observed by the nursing staff.p = 0.194) at three months and −0.2  at six months. The Cornell estimated difference was 1.3  at three months and −1.3  at six months.No significant between-group differences were observed for the primary outcome measures. With the VD group as a reference, the CMAI estimated difference between mean change scores was 4.4  at three months and 1.1  at six months. The linear mixed model analysis showed a significant time x group interaction effect (p = 0.003), with less restless/tense behavior at six months in the UVB group with the VD group as a reference, compared to the three months outcomes .Quality of life as measured by QUALIDEM showed a significant difference between groups and over time on the subscale “restless/tense behavior”. With the VD group as a reference, the estimated difference between mean change scores on restless/tense behavior was −1.1  at three months and -21.9  at six months.The 25(OH)D3 serum concentrations in the UVB group at six months was lower in comparison to the VD group, with an estimated difference between mean scores of −9.3 .Transitional redness of the skin was observed in 3 participants in the UVB group, although this disappeared after 24 h.p = 0.012. With the VD group as a reference, the estimated difference between mean change scores on restless/tense behavior was −0.6  at three months and 1.2  at six months.Because of the variability in duration of UVB exposure in the intervention group, we performed an additional analysis, keeping those who maintained any duration of UVB exposure as “UVB-exposed” and moving those who refused the intervention to the control group (12 participants) Table A1In this study, the first randomized control trial to assess the effect of UVB on agitation and depression in people with dementia, we found no significant effect of UVB light on the well-being of nursing home residents. By comparison, in a population of dermatological patients and healthy volunteers, Edstrom et al. reported a significant improvement in scores on the Montgomery Asberg Depression Rating Scale (MADRS) after six weeks (2–3 sessions weekly) of whole body UVB exposure [Our study showed an increase in restless/tense behavior in the UVB group in the first three months and less restless/tense behavior in the same group in the second three-month period compared to the control group. The additional analysis showed no difference between two groups in the first three months and the same results in the second three months. A similar effect was found in a study by Gambichler et al., where healthy volunteers reported feeling more balanced and less nervous after three weekly sessions of whole-body UVA exposure . A positSerum concentrations of 25(OH)D3 increased significantly in the VD group in the last three month period in comparison to the UVB group. This was not in line with the pilot study of Chel et al. which showed a significant increase in 25(OH)D3 in persons with dementia after 8 weeks of UVB exposure and this was not the case in our study. This could be due to differences in adherence to the prescribed regime, an inability of older skin to synthesize 25(OH)D3 over a longer period or to 25(OH)D3 reaching a plateau (81.5% of our participants in the UVB group were VD sufficient (25(OH)D >50 nmol/L ) in compA major strength of our study was the multicenter RCT design, which included a six-month follow-up period. We also carried out an intention-to-treat analysis that provides not only an estimation of the effect of treatment but also the applicability of the procedure in this specific population.The main limitations of our study were the lack of blinding and the low adherence to the intervention by nursing home residents with dementia. To partially reduce the last limitation, we performed a sensitivity analysis comparing the participants who actually were UVB exposed with participants who were not UVB exposed. The additional analysis showed the same results as the intention-to-treat analysis for the main outcomes of the study.There are strategic lessons to be learned from this study, especially for researchers dedicated to the population of people with dementia. In terms of adherence, it was really difficult to have all the participants stick with the intervention. For ethical reasons, UVB treatment was discontinued when participants clearly objected or showed signs of discomfort on two consecutive sessions. The use of sunbeds by nursing home residents with dementia also highlighted certain practical problems underlying the low adherence, including feeling cold, anxious, being unable to lie still or being unable to understand the purpose of the procedure. Future research efforts in this field should first attempt to find more comfortable approaches to administering UVB light.The effect of UVB light on wellbeing has not yet been examined in this population. In our study, the amount of the UVB light administered was calculated on the base of the UVB light needed to sustain a sufficient 25(OH)D3 serum concentration. The exposure needed to achieve any effect on agitation, depression or quality of life is not yet known. It is possible that a better adherence to the prescribed regime or a more intensive treatment than the treatment our participants actually received might present other results on the effect of UVB on wellbeing and quality of life.The exposure of nursing home residents with dementia to UVB light showed no positive benefits in terms of wellbeing. UVB treatment may have a positive effect on the restless/tense behavior characteristic of advanced dementia, but more research is needed to confirm this finding.


Cavotricuspid isthmus (CTI) ablation is a current first-line management method for typical atrial flutter. A voltage-directed technique that systematically targets points of maximal voltage has be found to reduce procedure and fluoroscopy times without increasing recurrence. We hypothesized that this technique’s efficiency would be enhanced by using signals from radial minielectrodes of a novel catheter . Prospectively, atrial flutter patients underwent voltage-directed ablation with a nonirrigated 8-mm-tip catheter. Ablation was either directed by conventional bipolar electrodes  or mini-electrodes  with the goal of achieving bidirectional block at the CTI and a subsequent observation time of 30 minutes. Total radiofrequency application time and lesion numbers were not significantly different. Group B had a lower mean power  and a tendency for longer fluoroscopy and procedure times. In three of the cases in group B, a switch to an irrigated catheter was required in order to achieve bidirectional block. In group A, bidirectional block was obtained in all patients using the nonirrigated catheter with no significant increase in reconnection. Differences in the catheter performance between the two groups were driven by poorer performance of the MiFi™ catheter  in patients presenting in atrial flutter. Electroanatomical mapping revealed a more proximal localization of the maximal voltage by the minielectrodes as compared with the conventional bipolar electrodes, resulting in less efficient identification and ablation of the conducting muscle bundles. Final results indicated CTI ablation using minielectrodes is not superior to conventional bipolar electrodes in the use of 8-mm, nonirrigated electrodes. Radiofrequency ablation by a voltage-direction strategy, which systematically targets points of maximal voltage across the CTI, has been shown to reduce procedure and fluoroscopy times without increasing recurrence rates.Figure 1), with three mini electrodes measuring 0.8 mm in diameter that are arranged radially 1.3 mm from the tip, has been shown in animal studies to demonstrate enhanced detection of electrical signals4 and, in humans, to improve the detection of viable tissue in redo flutter procedures5 and in the ablation of atriofascicular pathways, respectively.6A novel ablation catheter, the IntellaTip MiFi™ , all subsequent typical atrial flutter ablations were performed with their involvement until all of the provided supplies were utilized. The subsequent typical flutter ablations were performed using an identical catheter without the minielectrodes, ie, the 8-mm Prime Blazer™ . Data in all ablations were collected prospectively. The cohorts were separated by catheter used. In group A , ablation was guided by the conventional electrodes in the Prime Blazer™ catheter . In group B , ablation was guided by the signals from the minielectrodes located 2 mm from the tip of the catheter in the IntellaTip MiFi™ catheter , whose platform is identical to that of the Prime Blazer™ catheter  used in group A. In both groups, ablation was temperature-limited with an initial power of 60 watts (W) and a temperature of 60°C. Three experienced operators from two separate centers carried out the ablation procedures in both groups.In two large, tertiary centers, data were collected prospectively in consecutive atrial flutter patients undergoing ablation. The patients underwent voltage-directed CTI ablation using an 8-mm-tip, nonirrigated ablation catheters without the assistance of electroanatomical mapping as per the standard technique. This technique has been previously describedPatient demographics were collected from medical records and ablation, fluoroscopy, and procedure details were collated locally in each department. Data from all procedures across the two centers were then combined and statistical analysis was performed by unpaired t-test within Excel  and Fisher’s exact test. Data are presented in the format of mean ± standard error.Table 1, total radiofrequency (RF) ablation time and number of applications were not significantly different between the two groups. The MiFi™ catheter  was associated with significantly lower powers achieved  and a tendency towards longer fluoroscopy (21.5 minutes ± 3.0 minutes versus 15.8 minutes ± 2.0 minutes) and procedure times (100.6 minutes ± 11.8 minutes versus 82.0 minutes ± 4.8 minutes) in group B. In three cases, bidirectional block was not obtained with the MiFi™ catheter  and a switch to a 4-mm irrigated tip  had to be made in order to achieve this. No such switches were required with use of the conventional 8-mm catheter in group A.As detailed in Table 2, patients who were in flutter at the start of the procedure had statistically lower powers in group B as compared with in group A , as well as longer fluoroscopy times  and longer procedure times . Those patients in flutter also showed significantly higher temperatures in group B as compared with in group A . By comparison, there were no statistical differences in any of the parameters between the groups for patients presenting in sinus rhythm .An analysis of outcomes according to presenting rhythm appears to show that the differences in ablation were driven by poorer performance of the MiFi™ catheter  in patients presenting in atrial flutter. As shown in Figures  and 2B versus the minielectrodes Figures  and 2D. The first panel in Figure 2 shows a cut-away view of the ablation catheter on the CTI during pullback with the catheter at the point of maximal voltage signal. This showed a more caval location of the catheter on mapping with minielectrodes.Electroanatomical mapping (EAM)  was performed in a single case to compare the location of the catheter on mapping signals according to conventional electrodes 7 Recurrence of CTI-dependent flutters postablation are due to reconduction through the CTI.8 One potential mechanism for this conduction recovery is insufficient effective ablation of the conducting fibers and with lack of appreciation of this due to attendant tissue edema associated with ablation. In an attempt to avoid unnecessary edema, a voltage-dependent technique was developed and verified in London, Ontario, Canada.3Catheter ablation of the CTI is a well-established and curative first-line therapy for patients with typical atrial flutter with long-term success rates of ~90%.1 With conventional bipolar 8-mm-tip ablation catheters, the highest voltage from the distal bipole is measured over a comparatively large surface area, the midpoint of which is towards the proximal end of the ablation area . Theoretically, this will result in the large portion of the ablation lesion being applied slightly ahead of the highest signal. By comparison, the MiFi™ recording electrode is situated at the distal tip of the 8-mm electrode .The concept of the voltage-directed technique is to target conducting bundles of the CTI whilst avoiding intervening nonconducting fibrous tissue.9 We found that careful mapping of voltages in the absence of an EAM was surprisingly associated with longer procedural times and, in three occasions, a switch to an irrigated catheter was necessary. Similarly, Iwasawa et al. found that the use of the minielectrode catheter was associated with the need for a significantly higher number of RF energy applications and longer fluoroscopic and procedure times as compared with results in cohorts in which an 8-mm dumbbell catheter or 8-mm cryocatheter were used.9 In both linear drag and targeted CTI ablation, the operator is guided by local bipolar signals, positioning the catheter at the optimal candidate signal with the assumption that this is also the point of maximal ablation delivery. This is highly dependent on the catheter orientation and design, positioning of recording electrodes, and the electrodes’ relationship with the ablation tip. In this study, we show a lack of benefit of use of an 8-mm-tip in CTI ablation; the explanation for this is illustrated by the EAM use shown in Figure 2. Mapping with minielectrodes resulted in a more caval location of the catheter tip, so the RF energy would be delivered somewhat proximal to the optimal signal, whereas, in a conventional bipole configuration, the ablation is slightly distal in a typical catheter orientation on the CTI, wherein the electrodes are parallel rather than perpendicular to the tissue. Such a location mismatch, particularly in a targeted, voltage-guided procedure, would deliver less-sustained tissue contact, as the catheter would intermittently be opposed and unopposed on the “muscle bundle” Figures  and 2D, whereas, by comparison, the conventional bipole mapping would result in a catheter that rocks across the “muscle bundle” with cardiac motion Figures  and 2B. This mechanism is supported by the lower powers and lower efficiency of the ablation strategy in cases in which the voltage was mapped using the radial minielectrodes together with the details provided by the use of EAM.In this study, we report a lack of benefit with the use of minielectrodes in voltage-directed CTI ablation and thereby provide some insights into the lack of efficacy, in keeping with other research that found no benefit of the IntellaTip MiFi™ catheter  in linear drag lesion technique ablation of atrial flutter.10 Here, the authors demonstrated that sinus rhythm at the start of ablation was independently associated with short ablation times . While this initially appears counterintuitive, as one would expect the bundles employed during atrial flutter to be readily identified during the arrhythmia, there are a number of confounding variables: (1) the direction of propagation during coronary sinus pacing, sinus rhythm, and atrial flutter are very different and thus affect the voltage profile of the isthmus . Furthermore, the initial target in CTI ablation is clockwise conduction, as manifested during pacing from the coronary sinus. This conduction vector is in the opposite direction to that seen during typical, counterclockwise atrial flutter and may employ different muscle bundles or concealed conduction. If the voltage is altered and this is employed to target muscle bundles, then the “less-forgiving” MiFi™ catheter , already centered away from the putative bundle, would do particularly badly given the location mismatch explained. Additionally, (2) catheter stability/contact during the rapid atrial contractions of flutter may be less than that during sinus rhythm. The mean temperature and variability in power between lesions might also be an indicator of this.The disparity of procedure success in patients presenting for ablation in atrial flutter has previously been observed by Subbiah et al.4 Moreover, in a traditionally perpendicular orientation, the 8-mm MiFi™ catheter  performed well as found by ourselves in the ablation of an atriofascicular pathway.11 These data presented illustrate the importance of the location of the recording electrodes and the catheter’s orientation. A similar distal electrode would reduce the mismatch and one might anticipate a more optimal result; however, this requires prospective study.The intent of the minielectrodes is to provide a measure of contact and accuracy, and there are studies supporting this.While IntellaTip MiFi™ technology  is highly effective at detecting the small voltage signals in redo atrial flutter ablation and atriofascicular pathway ablation, the 8-mm nonirrigated-tip catheter does not enhance voltage-directed CTI ablation. The same may not be true for the 4-mm irrigated platform. Further studies on this will be required.


The its mutation appeared to be a hypomorphic mutation compared to sycp2 knockout mutations generated by TALEN mutagenesis. Taking advantage of these sycp2 hypomorphic and knockout mutant lines, we demonstrated that Sycp2 is required for the assembly of the synaptonemal complex that is initiated in the vicinity of telomeres in wild-type zebrafish spermatocytes. Accordingly, homologous pairing, the foci of the meiotic recombinases Dmc1/Rad51 and RPA, and γH2AX signals were largely diminished in sycp2 knockout spermatocytes. Taken together, our data indicate that Sycp2 plays a critical role in not only the assembly of the synaptonemal complex, but also early meiotic recombination and homologous pairing, in vertebrates.Meiotic recombination is essential for faithful segregation of homologous chromosomes during gametogenesis. The progression of recombination is associated with dynamic changes in meiotic chromatin structures. However, whether Sycp2, a key structural component of meiotic chromatin, is required for the initiation of meiotic recombination is still unclear in vertebrates. Here, we describe that Sycp2 is required for assembly of the synaptonemal complex and early meiotic events in zebrafish spermatocytes. Our genetic screening by N-ethyl-N-nitrosourea mutagenesis revealed that  S. cerevisiae Red1.Meiosis is an essential type of cell division whose purpose is to produce gametes, such as eggs and sperm, for sexually reproducing eukaryotes. Most cells in the human body are diploid cells containing two homologous copies of each chromosome. Meiosis halves the genetic contents of diploid cells and produces haploid gametes with a single copy of each homologous chromosome pair. The faithful transmission of chromosomes is one of the main tasks in meiosis, since extra or missing chromosomes are common causes of genetic disorders, birth defects and infertility. Prior to proper segregation, homologs must pair up and be physically connected. This is achieved by homologous recombination initiated by programmed DNA double-strand breaks. Since it can be deleterious to cells, this initiation step is highly regulated by factors that are not fully understood. In this study, we found that Sycp2, a structural component of meiotic chromatin in metazoans, is essential for the initiation of homologous recombination, meiotic chromatin organization, and homologous pairing in zebrafish. These findings support recent biochemical studies that Sycp2 and Sycp3 serve as a vertebrate counterpart of  Meiotic recombination plays a key role in the faithful segregation of homologous chromosomes in the gametogenesis of most sexually reproducing organisms. The key challenge of meiotic recombination is coordination between the processing of recombinant molecules and changes in chromosomal organization to ensure homologous pairing and the formation of at least one crossover per homologous pair. This process is initiated with programmed DNA double-strand breaks (DSBs) catalyzed by the meiotic endonuclease SPO11. Meiotic DSB formation is tightly regulated by several factors, such as the spatiotemporal regulation of chromosome structure and of proteins essential for DSBs.Caenorhabditis elegans suggest that axial elements are required for DSB formation. In budding yeast, the axis proteins Hop1 and Red1 are required for efficient DSB formation, since DSB levels are drastically reduced in mutants of these wild-type proteins We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Paula E. CohenAssociate EditorPLOS GeneticsGregory P. CopenhaverEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The the zebrafish Ietsugu (its) mutant was previously identified by the authors in a forward genetic screen for male sterility mutants and was found to be defective in generating the meiotic chromosome axis. In this study, the researchers map the its mutation to the sycp2 locus and show it is a T to A substitution that causes aberrant splice site usage. The its mutation behaves as a hypomorphic allele with expression levels ~26% of wild type. The authors created a TALEN generated knockout of the sycp2 gene and find it is required for axis formation (i.e. Sycp3 staining) and DSB formation (Dmc1 staining), but that some SC (Sycp1) forms ectopically. The zebrafish sycp2-/- mutant phenotypes are comparable to a SYCP2-/- mouse. Several new resources were developed including antibodies raised to zfDmc1 and zfSycp2 that could be useful for the burgeoning zebrafish meiosis field.The work describing the sycp2 phenotypes is solid and the immunofluorescence figures are very nice and support the novel conclusions of the paper. In addition, having a hypomorphic allele in hand adds to the study. As zebrafish is an emerging model for meiosis, understanding the evolutionary conservation of function of potential orthologs is important so identifying the its mutation as an allele of Scyp2 is significant.There are several issues that need to be addressed before publication. Several conclusions made by the authors are either not supported by the data or are not based on novel observations . The comments below suggest ways that the paper can be better focused to highlight the novel aspects of the work.Comments:1. The authors suggest that they have discovered Sycp2 as a functional ortholog of yeast Red1 protein since pairing, DSB formation, axis formation and SC assembly are defective in the sycp2-/- mutant. Reporting these data and discussing its relevance to Red1 is important, but arguing that they have made the novel discovery that Sycp2 is the functional Red1 ortholog is a bit of an overstatement. The link between Red1 and Sycp2 has been explored previously in mouse studies and recent structural analysis has revealed commonalities and differences between the two proteins . Red1 protein forms a homotetramer while mouse SYCP2/SYCP3 forms an analogous heterotetrameric structure. One of the main arguments the authors make supporting the notion that Scyp2 is a potential ortholog of Red1 stems from the failure to see DSBs in the zebrafish sycp2 mutant and previous reports that DSBs form in the mouse SYCP3-/- mutant. This is rather circumstantial evidence. To really nail this down, the authors would need to create the zebrafish sycp3-/- mutant which seems beyond the scope of the current paper. The authors should also be aware that yeast red1 mutant does in fact form DSBs, but they are repaired with the sister chromatid . Most of the discussion on pages 16 and 17 could be cut so that the authors say briefly that their findings are consistent with the notion that Scyp2 is the functional ortholog of Red1.2. The authors suggest that Sycp2 is required for ovarian development in zebrafish based on a sex reversal phenotype described in a previous publication (pages 15-16). Granted, other mutations affecting oogenesis have been shown to cause sex reversal, but these studies have also shown that sex reversal is suppressed by tp53-/- mutation, further suggesting a defect in meiotic chromosome dynamics during oogenesis. To make the claim that sex reversal is due to a check point arrest, the scyp2-/- tp53-/- double mutant should be made.3. In addition, there should be some documentation on how many fish were analyzed for a sex reversal phenotype and the criteria used for sexing fish. Without directly scoring gonads with arrested oocytes, it is a bit of an overstatement to conclude that Scyp2 is required for ovarian development. It would be important to know if some small fraction of females escape sex reversal. Perhaps females arise more frequently in the its mutant? If so then analysis of ovarian development could be carried out on those females. If the tp53-/- mutation suppresses the sex reversal phenotype then there should be a population of females where ovarian development could be analyzed. Exploring this phenotype in more detail would be interesting since the phenotype is markedly different from the spo11 mutant, which also fails to pair or synapsis chromosomes, but produces fertile oocytes.4. The authors also elaborate on their observations that DSBs form near telomeres in males (pages 14-15). This is not a novel observation and has been reported in other zebrafish studies, including a paper by the author  and Blokhina et al, 2019). This section should be shortened to focus the discussion on the role of Sycp2 in DSB formation.5. The formation of SC stretches in the absence an axis is interesting. Perhaps the authors could speculate how the SC is forming without an axis. Is there a precedence for this? Could they be forming between sister chromatids?Minor points:Line 173: “Sycp1 began to be observed as short fragments followed by the appearance of Sycp3 and Sycp2 signals”. Is this correct? The figure looks like Sycp1 forms fragments after Scyp3 and Scyp2.Reviewer #2: The Manuscript of Takemoto et al presents the characterization of SYCP2 in zebrafish through the cloning of the its locus as a splice-site mutation in the sycp2 gene. The creation of additional alleles with similar phenotypes and the development of spreading techniques allow the authors to demonstrate defects in pairing, synapsis, and DSB formation/early repair intermediate processing and the interdependencies with SYCP3 and SYCP1.The experiments are clearly presented and well controlled and the elucidation of SYCP2 functions is novel. A number of the details of wild type meiosis and the characterization of pairing at telomeres are partially redundant with the recent work from Sean Burgess’ lab, although some of the approaches are complementary rather than redundant.Overall the paper could benefit from a bit of reorganization. Since SYCP2 is an axis proteins, it seems that it would make sense to present the role of mutation in pairing and then synapsis and DSB formation.Given the Burgess paper has recently described staging of the events of meiosis with SYCP3 staining and telomere probes, it would behoove the authors to spend less time on the naming of the stages rather than the differences seen in the sycp2 mutants.In the discussion of sex reversal, the authors should consider the possibility that the SYCP2 and may activate checkpoints exclusively in the female that eliminate oocytes , allowing for male development. Differences in meiotic checkpoint activation is a feature of numerous species.Related to this, the difference between spo11 and sycp2 mutants vis-à-vis sex reversal suggests either 1) that DSBs are made in sycp2 but are incapable of recruiting DMC1 and H2AX  or 2) that a synapsis checkpoint is killing off female cells. Given the uniqueness of these phenotypes, it would be valuable to add these details to the main text and perhaps examine checkpoints in the fish, if possible. Indeed no mention is made of a female phenotype even in the weak loss of functions. Is there one?Minor comments:In leptonema in Fig. 3A, it is unclear what the upper right corner is. Is this a different cell? A zoom in of a part of the image?Line 173, it is unclear what is meant by “followed by” since they are fixed images and the stains are superimposable.For continuity, in Figure 4, SYCP3 should be on top and SYCP1 below (similar to figure 3).It would be helpful to label Figure 4A as wild type.Figure S6 B should indicate in labels colocalization with telomere probes.Reviewer #3: The synaptonemal complex is a meiosis-specific chromosome structure that is critical for the execution of recombination during gamete development. While previous studies in mammals have focused on the effects of chromosome axis component Sycp2 on synaptonemal complex assembly and synapsis, its role in DSB formation has not yet been investigated in mammals. As evidenced by recent papers , the use of zebrafish as a model system for studying meiosis has proven powerful for understanding meiotic processes in organisms that initiate SC formation in subtelomeric regions (e.g. humans). In the following manuscript by Takemoto et al., the authors have identified an allele of the conserved synaptonemal complex protein Sycp2 through a previously published forward genetic screen for zebrafish sterility phenotypes . The authors convincingly demonstrate that this allele acts as a hypomorph of variable penetrance arising from a splicing defect due to a mutation within exon 8 of sycp2. Further, the authors utilized TALEN mutagenesis to generate a null allele of sycp2 and characterized its meiotic phenotypes. Using the combination of these two alleles in zebrafish, the authors clearly show that Sycp2 is required for sperm production in males. The lack of sperm production appears to be due to defects that occur during meiotic prophase I: assembly of the synaptonemal complex, pairing of homologs, and meiotic DSB formation. Based on these phenotypes and the fact Sycp2 is a meiotic chromosome structure component, the authors provide support for the conclusion from West et al., eLife 2019 that Sycp2 serves as an axis component analogous to the well-studied Red1 in S. cerevisiae and HTP-3 in C. elegans. Further, similar to spo11 and other meiotic null mutations, the authors find that loss of sycp2 prevents the development of viable female zebrafish. Overall, the evidence that the authors provide for the role of Sycp2 in assembly of the SC and DSB formation is convincing. Taken together, the authors provide a good characterization of two alleles of a known meiotic chromosome axis protein homolog.Comments to improve the conclusions and impact of the manuscript:1. The authors qualitatively show a decrease in meiotic DNA damage by immunofluorescence using Dmc1 and gamma-H2AX; however, the amount of DNA damage they observe in sycp2 mutants appears to be lower than that of wild-type, but not entirely eliminated . The extent of DNA damage (via number of Dmc1 foci) in wild-type, sycp2, and spo11 mutants should be quantified to indicate the exact severity of the mutant phenotypes relative to each other and wild-type.2. Using quantification of telomere foci in fixed cells, the authors suggest that chromosome pairing at the telomeres is partially impaired in the sycp2 hypomorph. As mutants for various meiotic chromosome structures have been shown in several systems to lead to nonhomologous pairing, the authors should definitively demonstrate whether homologous pairing is occurring at all in this mutant by using fluorescence in situ hybridization targeting an autosomal locus, as performed in a recent PLoS Genetics publication . This result would provide important, strong validation of their conclusion and eliminate the possibility that nonhomologous pairing is occurring in this mutant, as occurs in chromosome structure mutants in other systems.3. In the present study, the authors find an effect on meiotic recombination initiation by looking at Dmc1 staining in sycp2 mutants. Previous studies by Doug Bishop’s lab and Akira Shinohara’s lab have established that in S. cerevisiae, Rad51 is required for Dmc1 foci formation. To both further validate the finding of this manuscript that recombination initiation is inhibited and add additional mechanism to the action of Sycp2 in the early stages of meiotic recombination, the authors to can evaluate Rad51 foci localization  together with Dmc1 localization in wild type, spo11, and sycp2 mutants. Further, this co-localization experiment would be useful to determine whether Dmc1 and Rad51 colocalize  in zebrafish.4. In the discussion, the authors draw comparisons between Sycp2 homologs in mice, yeast, nematodes, humans, and zebrafish. Supplementing this discussion with a specific bioinformatic analysis of conserved domains and sequence of zebrafish Sycp2 and Red1  would strengthen their conclusion as well as possibly provide interesting insights into the components of synaptonemal axis proteins required for performing diverse roles in meiotic prophase I.5. For Figure 3B, please indicate in both the image and figure legend what part of the image the inset panel is for the leptotene images.6. For Figure 5B, several changes would increase the clarity of this figure. First, please include the genotypes and stages of the images in the Figure itself. Second, the labels for the three- and four-color images are confusing, especially the label for Sycp3 . Third, include better zoom-in panels of these images similar to Blokhina et al., PLoS Genetics 2019, they are zoomed in at a higher level with clear colors that enable the reader to see individual DSB sites along the axis near the telomeres.7. The ladder for the RT-qPCR in Figure S4B appears to be from a different gel than the result of the lanes in the gel; however, there is no indication either the presentation of the image or the figure legend that this is the case. This panel as well as the figure legend should be amended appropriately.8. The western blot in Figure S4C appears very overexposed as well as extremely adjusted with post-processing methods outside of the dynamic range. Inclusion of panels from under/over exposed images of the blot as well as without any image adjustment should be included for this figure.9. To improve clarity of the figure, Figure S5 should have the different meiotic stages indicated for the images.10. In Figure S7, the DAPI levels for the spo11-/- mutant appears to be decreased significantly relative to the wild type images. Please check the exposures of the channel for these images.11. Several of the findings of the authors have already been found in an earlier published study . For example, the authors include in their abstract that they demonstrate SC assembly occurs in the vicinity of telomeric regions (p. 1 line 12) and that DSB formation occurs adjacent to chromosome ends (p. 4 lines 87-88). As these results has already been shown by Blokhina et al., PLoS Genetics 2019, I suggest the authors remove these statements in the abstract and last paragraph of the intro where they present them as novel findings, and instead add stronger definition/emphasis of their own novel results from those results that were also found in the recent Burgess lab study.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: No: There is no documentation of the sex-reversal phenotypeReviewer #2: YesReviewer #3: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  and your responses to reviewer comments. If eligible, we will contact you to opt in or out. *Dear Dr Sakai,Thank you very much for submitting your Research Article entitled 'Sycp2 is essential for synaptonemal complex assembly, early meiotic recombination and homologous pairing in zebrafish spermatocytes' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved. In particular, Reviewers 2 and 3 were particularly adamant that clearly defined and simple experiments have been omitted from this work, and this substantially weakens the impact of what otherwise could be a strong story.We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.In addition we ask that you:1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). 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Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.While revising your submission, please upload your figure files to the data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.Please be aware that our Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.PLOS has incorporated To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.[LINK]Please let us know if you have any questions while making these revisions.Yours sincerely,Paula E. CohenAssociate EditorPLOS GeneticsGregory P. CopenhaverEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The authors did a wonderful job addressing the comments and making the story richer with more data. Overall is a very nice paper.Minor comments:The first section of the discussion on the hypomorphic allele of sycp2its is redundant with the results. I think it can be left out completely since much of the discussion is already in the results section. It takes away from the important meiotic phenotypes the authors want to highlight.Seeing 3 and 4 BAC probe spots in the sycp2 mutant indicates there may be a sister chromatid cohesion defect as the authors say. The foci are really far away from each other. Do you think the sister chromatids are completely separated from each other since there is not axis for them to anchor? It will take more experiments with multiple BAC probes to figure this out, but I was surprised. What does an sycp3 mouse mutant look like? Is that informative?Reviewer #2: Re-review: Sycp2 is essential for synaptonemal complex assembly, early meiotic recombinationand homologous pairing in zebrafish spermatocytesOverall manuscript is much tighter and easier to read and the authors have addressed a number of the issues raised by the reviewers.1) The main issue they do NOT address is the role of the checkpoint in the loss of female embryos. In multiple places they said “in the interest of time” However, the paper was submitted initially in July and returned. If they had done the experiment it would have delayed another 3 months, true, but this is one of the novel aspects of the work and would indeed answer a critical question.In the description of this arrest, the text is unclear for non-zebrafish readers. On p.8, in the observation that there are no female offspring and that it is required for ovarian development, the link between ovarian development and sex determination needs to be better described. One suggested is to add the text that was provided as a response to the reviewers:“We speculate that the checkpoint is rather active in both oogenesis andspermatogenesis in sycp2-/- zebrafish. In zebrafish, all juveniles develop gonads withimmature oocytes regardless of their definitive sex. The individual in which theseimmature oocytes degenerate becomes male. Therefore, zebrafish mutants depleted ofoocytes develop as males that are mostly infertile. In sycp2-/- juvenile gonads, weobserved previtellogenic dictyate oocytes in four among five individual fish .Therefore, the absence of females in sycp2 mutant fish (Table 1) implies that oocyteswere eliminated at the juvenile age, possibly due to checkpoint activation in oogenesis.As a result, all sycp2 mutant fish develop as males.”2) In response to comment #4 about distinguishing whether the lack of DMC1 foci is a defect in DSBs or early DSB processing, the authors claim they did not have time to find the right dose of IR. This is actually a poor excuse: if the endpoint is sterility, this is an issue, but they could easily have looked for RAD51 and DMC1 foci within 30 minutes of irradiation to check whether these were detectable. This is still a do-able and important experiment.Text:Rpa foci should be RPA foci.Line 362: I would say that in the fanc1 and brca2 mutants that they became “sterile males”Reviewer #3: The authors have made a good effort to address reviewer concerns. Their work is strengthened by their removal and deemphasis of their identical results to that already published in Blokhina et al PLoS Genetics 2019. Further, their addition of data in Figure 5 to demonstrate pairing defects in the sycp2 mutant alleles also strengthens the paper. It is unfortunate that many of the comments of all three reviewers were unable to be addressed due to lack of time. The addition of several of the requested experiments, such as assessing rescue of DSBs and fertility by irradiation of sycp2 mutants. With regards to some of their responses to reviewers, I have two major comments/concerns that decrease the impact of their work and strength of their conclusions:1) The authors mention in their response to Reviewer#3 comment #3 that their Dmc1 “antiserum was raised against a part of the Dmc1 protein that is 43% identical to the zebrafish Rad51. Thus, our anti-Dmc1 antiserum also potentially recognized Rad51.” This fact regarding their Dmc1 antibody is troubling because it may not be specific to Dmc1. The authors should either 1) amend their discussion of these results and their figures to reflect the fact their Dmc1 antibody may actually be a Dmc1/Rad51 antibody ; or, 2) they should directly determine whether their antibody is specific to Dmc1 by looking at localization in a dmc1 null mutant by IF and/or Western.2) In response to Reviewer #1 comment #5, the authors look at Rad21l1l localization with Sycp1 stretch to determine whether the SC is forming between sisters. Although they see colocalization of Rad21l1l and Scyp1, this is not a definitive determinant of whether Sycp1 is forming between sisters because Sycp1 could be forming along a single sister chromatid axis. In order to determine if Sycp1 is forming between sisters, the two sister chromatid axes need to be resolved and Sycp1 needs to be see forming between those two axes. The use of DeltaVision deconvolution microscopy is not at a sufficient resolution level to resolve two axes. The authors need to use SIM microscopy or EM to determine the formation of SC between two sister chromatid axes . Alternatively, the authors could remove this data and simply postulate in the discussion that Sycp1 forming between sister chromatids in this context.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  20 Feb 2020PGENETICS-D-19-00970R2 Sycp2 is essential for synaptonemal complex assembly, early meiotic recombination and homologous pairing in zebrafish spermatocytes Dear Dr Sakai, We are pleased to inform you that your manuscript entitled "Sycp2 is essential for synaptonemal complex assembly, early meiotic recombination and homologous pairing in zebrafish spermatocytes" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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A single HfZrOx layer deposited by sputtering exhibits polarization switching after annealing at a temperature of 850 °C. However, the achieved ferroelectric properties are vulnerable to voltage stress and higher annealing temperature, resulting in switching instability. Therefore, we introduce an ultrathin 1-nm-thick Al2O3 layer at both interfaces of the HfZrOx. The trilayer Al2O3/HfZrOx/Al2O3 structure allows switching parameters such as remnant and saturation polarizations to be immune to sweeping voltage and pulse cycling. Our results reveal that the trilayer not only makes the ferroelectric phase involved in the switching free from pinning, but also preserves the phase even at high annealing temperature. Simultaneously, the ferroelectric switching can be improved by preventing leakage charge.Since ferroelectricity has been observed in simple binary oxide material systems, it has attracted great interest in semiconductor research fields such as advanced logic transistors, non-volatile memories, and neuromorphic devices. The location in which the ferroelectric devices are implemented depends on the specific application, so the process constraints required for device fabrication may be different. In this study, we investigate the ferroelectric characteristics of Zr doped HfO Interestingly, the polarization characteristics were observed in aggressively scaled one-nanometer-thick HfO2 films [Ferroelectricity based on spontaneous polarization of dipoles in complex ternary oxides  materials after annealing at high temperatures that drive phase transformation into the orthorhombic crystalline phase. Note that the fabrication constraints of the ferroelectric devices must be considered differently for each application. When the doped HfO2 is used for the memory fabricated on top of the drain of the transistor or stacked vertically in three dimensions, the annealing temperature should be limited as low as possible to avoid malfunction of the metal electrode. Considering the back-end-of-line process, utilizing pressure, which is an alternative driving force, has been considered to demonstrate ferroelectricity at relatively low annealing temperatures [2 material can be implemented as a gate dielectric during the front-end-of-line process, where high temperature annealing is performed to activate source and drain of the transistor [In addition to the physical investigations to identify root causes ,23,24, a the ALD ,26,27. Ieratures ,29. At aansistor . In the 2O3 (AlO) layer to the HZO-based ferroelectric device systems exhibiting a trilayer AlO/HZO/AlO structure for reliable polarization operations. Compared to the properties obtained from the single HZO, we investigated what ferroelectric behaviors were affected by introducing the AlO layers. Therefore, in this work, we introduced a thin Al0.5Zr0.5O2 target with an Ar plasma at room temperature. After a Pt was deposited by an electron beam evaporator as a top electrode (TE), annealing was performed through rapid thermal annealing at a temperature of 850 °C. The high temperature was raised up for 20 s, and the target temperature was maintained for the next 50 s during annealing. Note that the specific annealing conditions mentioned above constituted the optimum process to demonstrate the ferroelectricity of the sputtered HZO in our preliminary study. The detailed experimental results were discussed in [A 17-nm-thick HZO was deposited on TiN metal serving as a bottom electrode (BE) by RF sputtering from a single stoichiometric Hfussed in . For theFirst, the physical properties of as-deposited 17-nm-thick HZO layer were analyzed. As shown in 3 cycles of positive-up-negative-down (PUND) pulses with the same width of 10 μs were used. The subsequent square pulses were used to wake up the ferroelectric domains in the HZO and evaluate the fatigue behavior. The pulse set consisting of two consecutive positive pulses followed by two negative pulses called positive-up-negative-down technique was introduced to identify the charges solely induced by polarization [The devices were evaluated by applying a triangular pulse with a width of 10 μs corresponding to 100 kHz, as shown in rization . Generalc). A remnant polarization (Pr) at 0 V greater than 10 μC/cm2 was obtained due to spontaneously polarized dipoles in the HZO. When the single layer HZO was annealed at a slightly higher temperature of 900 °C, the P–V curve became rounded at larger voltages. On the other hand, the trilayer system showed the similar Pr compared to the single layer and was operated successfully without the breakdown by PUND pulses with a larger voltage of 4 V, as shown in 3, the Pr in the trilayer remained almost constant when sweeping the voltages larger than 6 V, no matter what annealing temperature was used, as shown in r in the single layer was proportionally increased as a function of the voltage amplitude. Similar trends were observed not only in the Pr but also in the saturation polarization (Ps), defined as the polarization measured at 4 V, as shown in 3 PUND pulses were thus used to wake-up the inactivated phases. The enlarged voltage application also eliminated the defects away from the phases. These caused more phases to be involved in the ferroelectric switching. As shown in r and Ps in the trilayer were less sensitively affected by the electrical stimulation. This means that more active ferroelectric phases could be formed, which is in good agreement with the physical results obtained from the XRD. Meanwhile, the single layer HZO annealed at 900 °C seemed to be more vulnerable to the voltage stress, worsening the switching stability. Although the transition of the polarization at the Vc in the HZO occurred, the P–V curve was incompletely saturated in the large voltage range . When the AlO layer was inserted only between the HZO and BE, the ferroelectric switching began to be observed in 2 serving as a seed layer were introduced to promote the orthorhombic phase in the doped HfO2 film [2, but their role and impacts on the polarization and reliability characteristics have not been elucidated. Further research to clarify these aspects is currently underway based on the fabricated multilayer ferroelectric devices. Here, we focused on the polarization behavior in a simple capacitor structure that provided a good understanding of intrinsic ferroelectric properties. When the ferroelectric switching layer was integrated into the transistor structure called ferroelectric FET (FeFET), the achieved polarization was somewhat degraded, resulting in a smaller on/off ratio for non-volatile memory applications. Thus, it remains a challenge to identify how the switching parameters such as the Pr and Vc of the ferroelectric layer are linked to reliability characteristics such as data retention and cycling endurance in the FeFET. Similar to this work, bilayer systems with HZO/AlO ferroelectric/dielectric structures were recently reported ,35. UnlifO2 film ,37. Addi2 layer has led to great interest for next-generation non-volatile memory and neuromorphic applications. It is worth noting that the fabrication process to realize the polarization in the layer needs to be appropriately designed depending on the device configuration aimed at the target application. In this regard, we investigated the ferroelectric characteristics of the sputtered HZO film under higher temperature applications. The ferroelectric properties of the single layer HZO were vulnerable to external environments such as higher annealing temperature, causing the unwanted phase transition. The sweeping voltages accompanied by electrical stress also made the switching parameters such as Pr and Ps be disturbed by the leakage components. On the other hand, the trilayer AlO/HZO/AlO systems allowed the ferroelectric phase to be preserved even at the high annealing temperature. Rather, the temperature led the non-ferroelectric phases to be released quickly from the pinning, requiring only 10 PUND cycles for stabilization. The recent discovery of the ferroelectricity in the doped HfO