There is limited documentation about pain and side effects associated with dilation and evacuation (D&E) abortion, yet, pain and side effects are important factors that can affect a client's abortion experience. In 2016, Hope Clinic for Women, an independent abortion clinic in Illinois, altered its cervical preparation protocols before D&E to reduce the total time of the abortion process and improve the client experience. This analysis addresses the gap in data on client experience of abortion in the later second trimester by evaluating pain, side effects, and acceptability by gestational age.Abortion clients obtaining services at the clinic between March 2017 and June 2018 were eligible to participate if they had viable singleton pregnancies of 16–23.6 weeks' gestation, spoke English, and were at least 18 years old. Eligible participants completed a two-part survey about their abortion experience.We found that respondents seeking abortion care at later gestations in the second trimester were more likely to report pain during their abortions. We did not find any association between side effects and gestational age.Although most respondents were prepared for the pain they experienced, some reported experiencing more pain than they expected, and more effective pain relief was commonly reported as a way to improve the service. More research on patient experiences of later abortion is needed, particularly on experiences of pain and options for pain management. If dilation was determined to be insufficient by the provider, a second set of osmotic dilators were administered and the D&E was completed on day 3.For pregnancies 21–23.6 weeks, mifepristone 200 mg (an antiprogestin) was added as an adjunct to osmotic dilators on day 1, which providers report may make D&E procedures easier.The protocol for pregnancies 18–20.6 weeks did not change—osmotic dilators are placed day 1, and on day 2, the D&E procedure was completed 0–180 minutes after misoprostol 400 μg.18 Intrafetal or intra-amniotic injection of 500 μg to 1.5 mg of digoxin was administered to everyone with pregnancies of 18 weeks and more.These protocols are in line with the Society of Family Planning's recommendations for cervical preparation before second trimester abortion up to 24 weeks gestational age.20 People who undergo D&E procedures report pain with dilator insertion, cervical dilation, injections to administer paracervical block and digoxin, uterine contractions, and postprocedure recovery.16 Digoxin is associated with vomiting,21 and common side effects of misoprostol, if used for cervical preparation, include gastrointestinal upset as well as fever and chills. Misoprostol-associated side effects are transient and can be managed with gastrointestinal treatments such as antiemetics and antidiarrheals. Pain and side effect management, therefore, is a meaningful component of quality of care.22There is limited documentation about pain and side effects associated with D&E abortion, yet, pain and side effects are important factors that can affect a client's abortion experience.23 Given the limited data, Hope Clinic aimed to reduce overall pain and improve client experience by reducing the need for a second set of osmotic dilators.5Currently, there is no standardized approach nor clear recommendations for pain management during later abortion procedures.At Hope Clinic, a combination of preoperative oral analgesia with ibuprofen, anxiolysis with alprazolam, and intraoperative moderate sedation with fentanyl and versed was used for pain management during the D&E procedure. Oral analgesia with ibuprofen alone was administered for osmotic dilator insertion. Patients were given the oral analgesia medications such as ibuprofen and an acetaminophen combination with opioid at discharge on day 1 for self-administered use throughout their abortion process outside of the facility.24 Pain and side effect management did not change with the altered cervical preparation protocol.Other intravenous or intramuscular analgesia medications such as ketorolac and antiemetic medications such as ondansetron were available as needed for in-facility side effect management. Verbal anesthesia, heat, and uterine massage were also used for pain management. The pain management strategies in use at Hope Clinic reflect Society of Family Planning recommendations for using multiple interventions to address pain and improve patient experience.The D&E procedure is used across a broad gestational age range in the second trimester, but client experiences with pain and side effects may vary within that range based on the duration of the pregnancy. This analysis attempts to address the information gap in client experience of abortion in the later second trimester by analyzing data on pain, side effects, and acceptability by gestational age. A better understanding of client experiences among different gestational age cohorts will highlight potential ways to offer a higher quality and client-centered approach to abortion care.We conducted an analysis on pain, acceptability, and side effects using data collected in a cross-sectional self-administered survey of later abortion clients at Hope Clinic with pregnancies of 16–23.6 weeks to compare the experiences of people who underwent original and modified cervical preparation protocols before D&E procedures. This article explores self-reported pain, side effects, and acceptability of the D&E procedure by gestational age group . The study was approved by Allendale Institutional Review Board.Abortion clients obtaining services at the clinic between March 2017 and June 2018 were eligible to participate if they had viable singleton pregnancies of 16–23.6 weeks' gestation, spoke English, and were at least 18 years old. Clinic staff did not recruit clients in emotional distress or those reporting safety concerns.Clients learned about the study from a printed information sheet and/or verbally from clinic staff after undergoing abdominal ultrasound to confirm intrauterine pregnancy and estimate gestational age before initiating their abortions. Those interested in participating were given a packet with study information and informed consent procedures, a paper version of the self-administered, structured anonymous survey, and instructions on how to respond to the survey online  with a unique personalized link on a tablet at the clinic or on a personal device. Participants could instead opt to complete the paper survey and return in a preaddressed and stamped envelope.Clients consented to participating in the study before proceeding to the survey questions. Those respondents who opted to use a paper survey could submit the completed survey to clinic staff in-person or in a self-addressed stamped envelope included in the packet.The survey was completed in two sections. Respondents filled out the first section of 37 questions, including demographic information, before initiating their abortion procedures. Participants responded to an additional 32 questions postprocedure about the experience with the abortion procedure, including pain, side effects, and acceptability, as well as about clinic interactions. Participants who completed the first part of the survey received a $5 gift card and those who completed the remainder of the survey received an additional $20 gift card. Participants who completed the entire survey were entered into a raffle for one additional $100 gift card. The data used for this analysis were from the postprocedure portion of the survey.Respondents rated the pain they experienced at different moments of the abortion process: cervical preparation in the clinic on day 1, cervical preparation after leaving the clinic on day 1, cervical preparation in the clinic on day 2 (if applicable), cervical preparation after leaving the clinic on day 2 (if applicable), digoxin administration on procedure day (if applicable), and the overall abortion process. They selected a number from 1 (no pain) to 10 (worst pain possible) on a visual analog pain scale, with corresponding circles of increasing size, that best reflected their pain.Respondents also classified their perceptions about actual versus expected pain, actual versus expected side effects, actual versus expected reaction to abortion experience, the overall abortion experience, and care received at the clinic by Likert scales, and they were invited to offer recommendations to improve the abortion process.p < 0.05 considered significant. We used Pearson's chi square and Fisher's exact test as appropriate to evaluate group differences for outcomes based on categorical variables, the Kruskal-Wallis nonparametric test for multiple comparisons, and the Dunn test with Bonferroni correction as appropriate for post hoc adjusted pairwise comparisons.For this analysis, we grouped all data by gestational age cohort , regardless of cervical preparation protocol used. Data were analyzed with SPSS 20 , with A total of 161 partcipants enrolled in the study. Two participants were excluded due to ineligibility: one was pregnant with a multiple gestation and another decided not to continue with the abortion procedure. Two additional were enrolled but eligibility details were unavailable and hence were excluded. Fifty-five (38.5%) of the 143 included respondents who initiated a survey did not return to the survey postprocedure. A total of 88 respondents who responded to the postprocedure survey portion were included in this analysis: 22 with pregnancies of 16–17.6 weeks, 42 with pregnancies of 18–20.6 weeks, and 24 with pregnancies 21–23.6 weeks .Enrolled participants ranged from 18 to 42 years old; median pregnancy duration was 19 (interquartile range 17–21) weeks. Respondents identified primarily as black or African American (60.0%), followed by white (30.6%), Asian/Native Hawaiian or other Pacific Islander (2.4%), or other/mixed race (7.1%). No one in the sample identified ethnically as Hispanic, Latin, or Spanish, while 6.0% reported other ethnicity without specifying. Participants with pregnancies 21–23.6 weeks were more likely to have private insurance than those with pregnancies of <21 weeks. They were also more likely to be pregnant for the first time and be nulliparous. Nearly all (94.2%) respondents reported pain at some point during the abortion process, regardless of gestational age. Pain differed by gestational age after day 1, on day 2, after day 2, and overall. All respondents in the 21–23.6 weeks group reported pain on day 2, after day 2, and overall .p = 0.002) and after day 2, the median pain score was significantly higher in the 21- to 23.6-week group than in the 16- to 17.6-week group  and the 18- to 20.6-week group   at any of the time points during the abortion that were asked about on the survey. However, when disaggregated by gestational age, a median pain score of 7 was assessed in the 18.0- to 20.6-week group for cervical preparation in clinic on day 1 and in the 21- to 23.6-week cohort for cervical preparation after leaving the clinic on day 2. After day 1, the median pain score was significantly higher in the 18- to 20.6-week group versus 16- to 17.6-week group  .When asked to qualitatively rate their pain, most participants reported moderate (47%) or severe (20%) overall pain and discomfort during the abortion procedure, with 7% reporting extreme pain and discomfort and 12% reporting no pain and discomfort. Characterization of pain in this way did not vary by gestational age group .Respondents most commonly reported that the pain experienced was less than or same as expected, however, more than one-quarter of the sample reported that it exceeded expectations. When broken down by gestational age group, it held that participants primarily felt that the pain experienced was less than what they expected .Nearly all (89%) respondents reported experiencing at least one side effect from the overall abortion experience, most commonly cramping (74%) and bleeding (65%). Approximately 11% of respondents reported no side effects . Most paRespondents' assessments of their overall abortion experience were mostly positive. All respondents in the 21- to 23.6-week group rated their overall abortion experiences as positive or very positive, except for one person who was unsure. Only three participants rated their experiences as somewhat negative, and all were in the 18- to 20.6-week group . More thShorter wait time was cited at a similar rate as less pain across gestational ages to improve patient experience. Fewer visits to the clinic was rarely reported as a way to improve experience, even in the group that more frequently required three visits .Our findings support that longer duration of pregnancy at the time of abortion is associated with reporting higher pain scores with the procedure. Pregnancies at later gestational ages require more cervical dilation and larger uteruses may produce stronger contractions induced by misoprostol. It is not unexpected that pain was less frequent after day 1 for respondents with 16- to 17.6-week pregnancies, especially since 11/22 people in that cohort underwent quicker cervical preparation processes and likely completed their abortion procedures in 1 day.25–27Although pain frequency increased with gestational age, pain intensity was similar across gestational age cohorts. Reponses to the survey question about the severity of overall pain and discomfort  suggeste28It is interesting to note that even though frequency of pain was higher in the 21- to 23.6-week cohort, report of a positive overall abortion experience was nonetheless common. It is not unusual in abortion studies to observe high rates of satisfaction and acceptability despite undesirable pain and side effects since participants have accessed needed care.26It is not possible to determine from our data whether the clinic's pain management strategies effectively reduced pain, but our findings do suggest that there is a desire for additional pain relief strategies during the abortion procedure, regardless of gestational age. There is a dearth of research on pain management during later abortion—a recent review has noted that there is limited evidence on pain management strategies for abortion generally, and even a bigger lack of evidence for later abortion specifically.29 American College of Obstetrics and Gynecology,30 and the National Abortion Federation (NAF)31 identify various approaches to pain management, such as analgesia, anxiolytics, local anesthetic, conscious sedation, and general anesthesia, particularly after dilation is achieved, in their respective clinical guidance, but none provides evidence-based recommendations for specific protocols especially for less resource intensive approaches, which might be preferred and are important for clinics without the infrastructure for intravenous sedation. Less complicated and more accessible pain management strategies should be prioritized for future research.The World Health Organization (WHO),We did not find any association between side effects and gestational age, which is not necessarily unexpected. Misoprostol was used for cervical preparation in all gestational age groups and its known side effects, particularly cramping and nausea, were consistently reported. The routine use of digoxin at 18 weeks and beyond could have increased the frequency of nausea, but the group sizes are small and it is possible that additional doses of misoprostol in these later cohorts contributed to reports of nausea; we cannot separate out the effect of digoxin on nausea.32–34Survey respondents with pregnancies in the highest gestational age group were more likely to be pregnant for the first time and to be nulliparous compared to those in the lowest gestational age group. The published literature reflects that primary delays to care include difficulty recognizing or confirming pregnancy.35In our survey sample, participants in the 21- to 23.6-week group were not younger than those in the other gestational age groups even though delays in pregnancy recognition can be more acute at younger ages.32 however, there was no statistically significant difference in the proportions of survey respondents with children when grouped by gestational age.Other research have reported that people with children are also more likely to take longer to get an abortion, possibly due to arranging childcare and making appointments around familial commitments;Our study has a few limitations. Stratifying results by cervical preparation procedure protocol was not meaningful because group sizes were so small. Another limitation is that gestational age was based on self-report and was not confirmed with clinic records. We did identify some gestational age-related inconsistencies before data cleaning, such as respondents in the earliest gestational age cohort who reported receiving digoxin when neither the original or modified cervical preparation protocols called for it, but it is unlikely that any gestational age reporting errors were widespread, as the clinic uses ultrasound to date gestational age and provides patients with the gestational age of the pregnancy before patient education.In addition, it is possible that respondents misunderstood the lowest rating of the visual analog pain scale, where one was meant to indicate no pain and corresponded to a small circle that could have been misconstrued to represent minimal pain. Finally, statistical analyses conducted on these small group sizes cannot be generalized to the larger population.In this study, people seeking abortion care at later gestations in the second trimester were more likely to report pain during their abortions. Although most respondents were prepared for the pain they experienced, some reported experiencing more pain than they expected, and more effective pain relief was commonly reported as a way to improve the service.The findings suggest both that the clinic staff could provide more information about expected pain during counseling and that more research is needed to establish evidence-based pain management strategies, especially for cervical preparation. As pain generally increases with gestational age, future research should be powered to assess outcomes by gestational age to yield more concrete conclusions about differences in pain, side effects, and acceptability.


IntroductionElectrical burn injuries are very common in the pediatric population and are usually accidental and sometimes occupational. The objective of our study was to evaluate the epidemiology of electrical burn injuries and prospectively evaluate the long-term psychosocial impact of electrical burn injuries in children.Materials and methodsA qualitative interview study was conducted prospectively among sixty patients presented to Holy Family Hospital, Rawalpindi, Pakistan. Demographic details, mode of presentation, detail of injury, total body surface area, initial condition, and surgical interventions were noted during their stay at the hospital. Their physical and psychological outcomes were evaluated by administering the Strength and Difficulties Questionnaire (SDQ) and Body Dysmorphic Disorder Questionnaire (BDDQ) via telephonic interviews after six weeks of discharge from the hospital. The study was conducted over a span of 1 year from January 2021 to January 2022.ResultsThere were 60 patients who presented to the department of pediatric surgery during the span of the study. The mean age was 9.9 years ± 3.133 years (SD) and 80% of the patients were above 8 years of age with a male-to-female percentage of 86.67% to 13.33%, respectively. Forty-two (70%) patients incurred high-voltage electrical burns while 18 (30%) suffered low-voltage electrical burns. Mortality was 13.33% (n=8). Out of 52 patients who survived, 35 (67.30%) were labeled as having Body Dysmorphic Disorder upon administration of BDDQ through a six-week follow-up. The majority of the patients had abnormal or borderline results in different scales of SDQ.ConclusionThe long-term psychological stress and the varied spectrum of psychiatric disorders in electrical burn patients are profound. The prevention of burn injuries can be effectively achieved by educating parents about safety measures and improving health infrastructure. Implementation of a dedicated national program for psychological support of burn patients should be made accessible to all patients. Burn has been declared a preventable global health problem according to WHO's latest census. While the mortality associated with burns is reaching an alarming high of 180,000 deaths per year, the more concerning aspect is that the majority of these burns occur in lower and middle socioeconomic countries, especially in African and South Asian regions. In Bangladesh, Colombia, Egypt, and Pakistan, 17% of the children with burns have a temporary disability and 18% have a permanent disability [Burn of all sorts and its effective management proposes a grave challenge for emergency physicians as well as surgeons everywhere across the globe. According to the latest statistics, among all the types of burns, pediatric burn constitutes the largest single group admitted and treated at any tertiary care unit in developed as well as developing countries. Pediatric burns can further be classified into two subgroups that are non-electrical and electrical burns  ElectricElectrical burn injury (EBI) constitutes low-voltage and high-voltage injuries. Low- and high-voltage injuries constitute the injuries incurred by sources less and more than 1000V, respectively. High-voltage electrical burns are associated with poor surgical, medical and psychological outcomes and higher mortality compared to low-voltage electrical burns  Low voltAlthough much data has been published around the globe regarding the spectrum of injuries caused by electric burns and the economic and disability strain it puts on caregivers and patients, not many studies have been conducted on the psychosocial impact of this preventable health problem. Moreover, in clinical set-ups of developing countries, the psychosocial impact is the least concerning aspect of any illness.The objective of our study was to prospectively evaluate electrical burn patients who were being managed in the ward of pediatric surgery at Holy Family Hospital  to establish the common mode of injuries, timing of presentation, and different management strategies.one year from January 2021 to January 2022.After approval from the Ethical Review Board of Rawalpindi Medical University, we conducted a descriptive cross-sectional study of all the pediatric patients of electric burns presenting to the department of pediatric surgery, Holy Family Hospital. Data was taken regarding their demographic details, mode of presentation, detail of injury, Total body surface area, initial condition, surgical intervention, and their outcome during the period of To evaluate the psychosocial impact, a questionnaire was devised with three components: demographic details, the Strength and Difficulties Questionnaire, and the Body Dysmorphic Disorder Questionnaire. Data regarding body dysmorphia and overall strengths and difficulties was then carried out via telephonic conversation with the patient and their primary caregiver after six weeks of discharge from the hospital.Strength and Difficulties QuestionnaireThe Strengths and Difficulties Questionnaire (SDQ) is a brief measure of psychological adjustment and psychopathology of 3- to 16-year-olds and can be completed by children, adolescents, parents, and teachers. The SDQ is sensitive to change over a short period of time and is a widely used instrument in research related to mental health. Though not a diagnostic tool, SDQ efficiently measures in which areas of life, a child is facing difficulty and also appropriately gauges the impact of any such difficulty in everyday life [The SDQ covers 25 attributes grouped into five subscales: 1) emotional symptoms , 2) conduct problems , 3) hyperactivity problems , 4) peer problems , and 5) prosocial behavior (for example “I try to be nice to other people”) Psychometric properties of the SDQ have been assessed in a representative sample of 10,000 children aged 5 to 15 years, all of whom also had psychiatric assessments. Findings confirmed and extended previous reports of satisfactory reliability and validity. This measure was used for the entire age sample in this study 8-17 years) as Maskell et al inferred that children as young as 7 years can report as reliably as older children. The SDQ parent report has been examined in terms of the psychometric properties within an Australian pediatric population and has demonstrated positive use as a measure of psychopathology in young Australian children -17 yearsBody Dysmorphic Disorder QuestionnaireThe Body Dysmorphic Disorder Questionnaire (BDDQ) is a validated tool to study the effect of an imagined body defect that causes a negative effect on functioning . It alsoThere were 60 patients presented to our department of pediatric surgery, Holy Family Hospital, Rawalpindi. Among these, the mean age was found to be 9.9 years ± 3.133 years (SD), and 48 patients (80%) were above 8 years of age, with a male predominance of 86.67% (n=52) and a female percentage of 13.33% (n=8). Table The total body surface area (TBSA) involved was categorized into five sub-groups: 0 was < 10%, 1 was 10-20-%, 2 was 20-30%, 3 was 30-40%, and 4 was >40%. 20% of the patients had TBSA <10% while the majority of the patients (51.61%) had a TBSA involvement of 10-20% (Table There were unfavorable outcomes in 29 of our patients 48.33%) and the mortality rate was 13.33% (n=8), out of which one of the patients was received expired. 26 (43.33%) patients underwent amputations while 14 (23.33%) patients underwent fasciotomy and 14 (23.33%) underwent wound debridement. One patient had to undergo a suprapubic cyst-ostomy due to a severe burn of the perineal region while one (3.22%) patient underwent a diversion colostomy due to a deep abdominal wound perforating the gut viscus. The details are shown in Table 3% and thFifty-two survivors were included in the evaluation of the psychosocial impact on our patients. Interviews were conducted via telephonic calls after six weeks of discharge from the hospital and BDDQ and SDQ were administered. 35 (67.30%) out of 52 patients who responded to our telephonic interviews were labeled as having body dysmorphic disorder and were further referred to a proper psychologist for evaluation and treatment.The Strength and Difficulties Questionnaire was quite diverse in assessing the different aspects of the psychosocial health of our subjects. It was administered on parents as well as their children in order to assess the patients for the following five scales: Emotional Problem Scale, Conduct Problem Scale, Hyperactivity Scale, Peer Problem Scale, Prosocial Scale, and Total Difficulties Scale.Nineteen (36.53%) out of 52 patients were labeled as having problems dealing with and expressing their emotions . Most of our patients reported difficulties in dealing with peers, and social anxiety was also quite evident in most of the patients. A similar international study by Maskell et al., which was a multicentric study in New Zealand and Australia, reported the same finding, with patients of burn scarring having low scores on HRQoL  and SDQ with disturbed behavioral patterns .Most of our patients were labeled to be having body dysmorphic disorder and mostly they were thinking about how to avoid social gatherings mainly because of their physical appearance. These patients when interviewed told that they spent more than 4 hours every day thinking about their disfigurement and how to face peers at school and in the neighborhood. While our patients were referred for proper psychological support and therapy, the data from New Zealand and Australia by Maskell et al. suggested that the hypothesis that burn patients score low on self-esteem score was nullified as no significant association was found for them .No significant association was found between Total Difficulty Score with either mode of injury, mode of recovery, amputations, or application of prosthetics. Patients even with low voltage injuries were stressed similarly and comparatively to those with high voltage injuries. Haddadin from Jordan reported that the increasing length of hospital stay and minor burns when admitted were associated with a significantly low score on the strength and difficulties index .For a country like Pakistan, we are way behind The Burn Plan which was a 10-year program (2008-2017) devised by the World Health Organization. The Burn Plan consisted of seven components. The first one was advocacy for the prevention of burns by raising awareness, followed by effective and sustainable burn care policies. After the implementation of these policies, data needs to be maintained, according to which research-based faster trials of promising interventions can be carried out. The remaining three goals were prevention, provision of adequate services, and capacity building Needless to say, we are still at the initial step and not enough has been done because of which we are still lagging behind in every aspect of burn management. The earlier the disease is recognized as a national health problem with a major financial, psychological, and physical impact on patients and their caregivers, the sooner we will be able to counter the devastating long-term effects it leaves on the community.Our study has certain limitations. Firstly, since most patients came to our tertiary care setup through referrals from far-flung areas, in-person follow-up could not be done owing to logistical and financial constraints. We had to resort to telephonic calls for the follow-up interviews. Secondly, due to the scarcity of child psychiatrists in our setup, timely psychological support could not be provided to most of the study participants despite an evident need.Electric burn injuries are potentially life-threatening but mostly preventable, more so in the pediatric age group. The long-term psychological stress and the varied spectrum of psychiatric disorders in such patients are profound. The prevention of burn injuries can be effectively achieved by educating parents about safety measures and improving not only the health infrastructure but also through making legislation in the power supply sector. Implementation of a dedicated national program for psychological support of burn patients should be made accessible to all burn victims. Adherence to safety regulations by the people and governing bodies with respect to the placement and maintenance of power lines cannot be over-emphasized. Strict disciplinary actions against violators might help to reduce the physical and psychological morbidity and mortality associated with electrical burns. We recommend that a liaison between plastic surgeons, pediatric surgeons, and psychiatrists must be established for the proper treatment of victims of electrical burns.


Before- and during-COVID-19 comparisons showed that the average daily screen time increased by 150% from 2.04 h to 3.06 h (p < 0.05), while the average play time decreased by 12.3% . Based upon international guidelines for movement behaviours of young children, special attention and actions are needed to manage the excessive daily screen time and preserve the average daily playtime of Javanese preschool children. These results present useful benchmarking data for parents, teachers, and health authorities to initiate ameliorative interventions to better balance children’s screen time and playtime as Indonesia emerges from the COVID-19 pandemic to a COVID-19 endemic.This comparative–descriptive multi-national research examined the screen time and playtime of preschool children aged 1–6 years before and during the COVID-19 pandemic. Parents reported on the play and screen habits of preschool-aged children on the weekday and weekends using a questionnaire on the lifestyle habits of their children. Results indicated a significant difference in screen time and playtime on the weekday and weekend before the pandemic (screen time: 1.91 ± 2.40 vs. 2.16 ± 2.60 h; playtime: 3.55 ± 2.49 vs. 4.11 ± 2.58 h, both  The COVID-19 pandemic has forced families to respond to challenges, one of which is the restrictions on public activities . From prThere are strong challenges in implementing screen time and physical activity recommendations especially during the COVID-19 pandemic when schools became less accessible because of closures and children spent much time at home ,9. In thFor children, screen time and outdoor playtime are associated with sleep duration and sleep pattern, and therefore reducing screen time and increasing outdoor playtime will help improve their sleep . ChildreOther studies on screen time and playtime examined the correlation between these two variables using cross-sectional data ,15,16 anGiven these challenging circumstances for research, only a few managed to map screen time with playtime on weekdays and weekends among children before and during the COVID-19 pandemic in the Indonesian context. The purpose of the present research was to examine and elucidate the relationships between screen time and playtime on a weekday and a weekend before and during the COVID-19 pandemic in preschool children living in Java, Indonesia.The present study was a research collaboration between the universities in Java and the National Institute of Education, Nanyang Technological University in Singapore. The research was approved by the ethics committees of the Institute of Research and Community Service, Universitas Negeri Yogyakarta, Indonesia (540/UN34.21/TU/2019) and that of the Nanyang Technological University, Singapore (NTU IRB-2019-02-036). The study involved the same group of adult parent respondents living on Java island, Indonesia, as previously reported by Fauziah and her colleagues that examined the play–sleep nexus of preschool children on Java island, Indonesia [®)  after giving their informed consent. Parents were recruited using a snowball sampling technique [The research involved 618 adult parents with preschool children aged 1–6 years who answered a content-validated and internal consistency-established online questionnaire (SMALLQechnique . To gath®) is an adult-reported questionnaire on the digital and non-digital habits of children on a weekday and on a weekend with acceptable validity and internal consistency [® into the Indonesian language was based on the guidelines for cultural adaptation advocated by the World Health Organization [® consisted of 25 items that enquired about the digital media and non-digital media habits of parent and child on a weekday and on a weekend and demographic information of the caregiver and child [The Surveillance of Digital Media Habits in Early Childhood Questionnaire  and non-entertainment activities  on a weekday and weekend, respectively, were summed to provide an estimate of child total screen time over each 24 h period.Time spent on indoor play  and outdoor play  were summed to provide an estimate of the child’s total physical playtime on a weekday and weekend day, respectively.® as physical activities during which breathing is faster and harder. Parents reported the percentage of playtime during which their child breathes faster and harder. The expression to derive MVPA in duration (hr) is (indoor + outdoor playtime in hr) * (% of time during which a child breathes faster and harder ÷ 100%). The duration of MVPA is calculated on a weekday and a weekend, respectively.Moderate-to-vigorous intensity physical activity (MVPA) is described in SMALLQn = 309) and during (n = 309) the pandemic. Descriptive analysis (mean ± SD) was performed to describe the participant characteristics while t-tests (independent sample t-test and paired samples t-test) were conducted to determine whether there were significant differences in screen time and playtime on the weekday and on the weekend as well as before and during the COVID-19 pandemic. The level of statistical significance was accepted as p < 0.05.Data were analyzed using SPSS version 23.0 statistical software. The analysis included data on screen habits and playtime on weekdays and weekends before (The characteristics of the parent/ caregiver who responded to the survey are outlined in p < 0.05). Weekday and weekend screen time of preschool children increased by 150% (0.96 h for weekdays and 1.10 h for weekends) during the virus pandemic compared to before the virus pandemic.From p < 0.05), before the virus pandemic and 7.1% (0.23 h) more than the weekday, during the virus pandemic (p > 0.05). Weekday and weekend playtime of preschool children decreased by 8.5% and 15.3%, respectively, during the virus pandemic compared to before the virus pandemicPreschool children spent modest amounts of time at playtime before and during the virus pandemic. Weekend playtime in preschool children was 15.8% (0.56 h) more than the weekday (p < 0.05). Before and during the virus pandemic, entertainment-based screen time was 2.8 to 4.8 times that of non-entertainment-based screen time on the weekday and on the weekend.p < 0.05). Parent accompaniment during child screen time was higher on a weekend than on a weekday . Similarly, during child playtime parent accompaniment was higher on a weekend than on a weekday .Parent accompaniment during child screen time and child playtime were shown in The primary purpose of the present research was to examine the relationships between screen time and playtime on a weekday and on a weekend, before and during the COVID-19 pandemic in Indonesian preschool children living in Java. For children in early childhood, the American Academy of Pediatrics (AAP) stipulates no screen time at all for children until 18 to 24 months, except for video chatting, and adds that children aged 2 to 5 years should have an hour or less of screen time per day . In the From Data elsewhere support our present findings of greater screen time in young children during the virus pandemic. For instance, in a national sample of toddlers and preschool children, Jáuregui and colleagues reported higher screen time in preschool children living in Mexico, during the virus pandemic lockdown, especially in those (i) with ready access to gadgets, (ii) came from economically disadvantaged backgrounds and (iii) those who were without playmates during the virus pandemic. In the cited study, physical activity decreased by 25%, screen time doubled, and sleep quality declined by 17% .The importance of setting limits for daily screen time, especially for entertainment in preschool children is a refrain echoed by many educationalists as excessive sedentary screen time portends greater fat storage in the body and hence contributes to juvenile obesity ,27. ExceEvidence for the detrimental effects of excessive screen time in preschool children is emerging. For instance, Xie and his colleagues reported preschool children exposed to more than 60 min of screen time per day faced higher risks of behavioural problems such as inattention, temper control, and presenting symptoms of attention deficit disorder. Further, others suggest that excessive screen time in early childhood increases the likelihood of less creative play, poorer vocabulary, and delayed attainment of developmental milestones .Several explanations are offered for the increased prevalence of excessive screen use in preschool children. For instance, because of the COVID-19 pandemic and associated movement and travel restrictions, families were ‘compelled’ to go online a lot more. Additionally, with the need for education to continue, schools were encouraged to conduct online classes instead of in-person ones .Parent attitudes towards their own screen use and that of their children are mediators of overall familial screen time. For instance, Chia and his colleagues reported parents’ own screen use predicted child screen use and children ranked in the top quartile for daily screen time tended to come from households where parents had more ‘liberal’ attitudes towards screen time and tended not set time limits for child use of screens . The expThese days, many parents have built a habit or routine of prolonged screen time at home to help their children learn . HoweverOthers recognize the utility of screen time for families seeking relief and restoration from stressors during the pandemic—that children are kept occupied and entertained while eating  or when n= 2209, age 0.7–3 years; caregiver-reported), sampled from 15 laboratories across 12 countries during the COVID-19 pandemic. They explained that toddlers with no online schooling requirements, experienced increased screen exposure during the lockdown periods of the COVID-19 pandemic, with longer screen exposure going hand in hand with the severity of the lockdown. They reported further that child screen time during the virus lockdown was negatively associated with the socio-economic status of the caregivers and was positively associated with child age, caregiver screen time, and attitudes towards children’s screen time [There is support for the key finding of increased child screen use in the present study. For instance, Bergmann and his colleagues investigated children’s screen time  is enriching because it supports children’s learning and social skills development . Child hStudies elsewhere show mixed results on the impact of the virus pandemic on outdoor play in children. For example, some studies emanating from Germany  and the The present study examined two equal-sized samples of parents/caregivers in comparing the screen time and playtime of children aged between 1 and 6 years living on Java island before and during the virus pandemic using an appropriate validated and reliability-tested questionnaire. The research provided context- and situation-specific data that could be used either for further research or serve as rich data to plan for joint ameliorative programmes between parents and the schools to address the demerits of the excessive use of screen time and also to better preserve high-quality interactive outdoor play among parents and preschool children. Several limitations of the present research are noteworthy—the results obtained were from parents’ self-reports and these were subjective responses that could be threatened by recall and social desirability biases. These biases were mitigated and minimized, respectively, by having the parent recall of child behaviours limited to the last 7 days and keeping the survey anonymous. Research suggests that parent’s self-reports of children’s PA (duration and intensity) may be different from those using objective tools such as motion sensors (accelerometers) as child-proxy reports by adults tend to over-estimate physical activity and under-estimate sedentary time so interpretation of data from different methods used to estimate activity and non or low activity should be made with caution . Indeed Another limitation of the research is that as Indonesia is a very large country, the results were focused on parents and children living on Java island and cannot be generalized beyond the sample. Future studies should focus on whether these changes were altered as the restrictions from the pandemic were relieved.In summary, taken as a whole, the results of the present study on preschool children living in Java island, Indonesia showed the virus pandemic has altered people’s lives in ways that were not previously anticipated—children’s engagement with screens increased significantly while children’s playtime showed a modest dip, compared to before the virus pandemic. In view of the present findings, the authors recommend that parent education efforts coupled with parents working in concert with teachers to forge alliances where culture- and context-specific strategies, ideas, and practical tips can be exchanged and tried out in ameliorating lifestyle behaviours—namely screen time and playtime among preschool children living in Java island. The benefits and demerits of media and social screen time should be further explored in relation to the mental health outcomes of parents and preschool children during the ongoing COVID-19 pandemic. Further, to promote high-quality parent–child interactions and positive developmental outcomes during the pandemic and beyond, childcare needs and parent well-being should be supported.


Gordonia rubripertincta NRLL B-16540 strain. The results show that there is a high level of diversity of the various lysis genes and 16 different genome organizations of the putative lysis cassette, many which have never been described. Thirty-four different genes encoding holin-like proteins were identified as well as a potential holin-major capsid fusion protein. The holin-like proteins contained between 1–4 transmembrane helices, were not shared to a high degree amongst the different phages and are present in the lysis cassette in a wide range of combinations of up to 4 genes in which none are duplicated. Detailed evaluation of the transmembrane domains and predicted membrane topologies of the holin-like proteins show that many have novel structures that have not been previously characterized. These results provide compelling support that there are novel operational lysis models yet to be discovered.Holins are bacteriophage-encoded transmembrane proteins that function to control the timing of bacterial lysis event, assist with the destabilization of the membrane proton motive force and in some models, generate large “pores” in the cell membrane to allow the exit of the phage-encoded endolysin so they can access the peptidoglycan components of the cell wall. The lysis mechanism has been rigorously evaluated through biochemical and genetic studies in very few phages, and the results indicate that phages utilize endolysins, holins and accessory proteins to the outer membrane to achieve cell lysis through several distinct operational models. This observation suggests the possibility that phages may evolve novel variations of how the lysis proteins functionally interact in an effort to improve fitness or evade host defenses. To begin to address this hypothesis, the current study utilized a comprehensive bioinformatic approach to systematically identify the proteins encoded by the genes within the lysis cassettes in 16 genetically diverse phages that infect the Gram-positive  In achanisms , 5. Althchanisms , 7. Howechanisms .The lytic phage lifecycle of dsDNA phages involves infection, replication, virion assembly and genome packaging, and then cell lysis. In phages that infect Gram-negative bacteria, phages must overcome the inner membrane (IM), peptidoglycan layer (PG) and the outer membrane (OM) to achieve efficient release of phage progeny. Extensive biochemical and genetic work has established a three-step model for the lysis pathway that involves three distinct sets of proteins. The first are endolysin/s that have defined catalytic activity to lyse the PG layer . The secS gene that encodes either the 105 amino acid holin or 107 amino acid antiholin with three transmembrane domains using a dual start motif; 2) an endolysin (R gene) and 3) two overlapping spanin ORFs that generate a large protein (Rz) with an N-terminal TM domain and a small protein (Rz1) with a lipid association sequence that allows its attachment to the OM  and antiholin (S2171) produced with a dual start motif  to keep the enzyme attached to the membrane in an inactive state prior to the triggering event [The second pathway, typified by the phage 21 , 7, has rt motif  panel B.iholin S21 produceng event . The losThe model for the lysis by phages that infect Gram-positive bacteria is not as well studied and generally restricted to phages that infect actinobacteria. Phage lysis in Gram-positive bacteria must overcome the IM and PG, but Gram-positive bacteria generally lack a formal OM and instead have a much more complex PG layer and often also contain extended structural cell wall components beyond the PG later such as arabinogalactans and mycolic acids –21. PhagM. smegmatis and has a linear five gene arrangement for the putative lysis cassette that contains, 1) endolysin or lysin A (gp2), 2) lysin B (gp3), 3) gp4 that encodes a small 77 amino acid protein containing two predicted TM helices and 4) gp5 that encodes a protein containing a single TM helix [Phage Ms6 infects TM helix , 33. CroTM helix . HoweverTM helix –33. SincTM helix . Aside fTM helix , there aIt is striking that in the phages where the lysis mechanism has been rigorously evaluated through biochemical and genetic studies, that each phage would utilize endolysins, holins and accessory proteins to the OM to achieve cell lysis through three distinct operational models. This observation suggests the possibility that other phages may evolve novel variations of how the lysis proteins functionally interact in an effort to improve fitness or evade host defenses. A first step in addressing this hypothesis is to carry out a comprehensive analyses of the proteins involved in the lysis pathway of diverse phages to assess the genomic diversity of the lysis cassettes. The identification of endolysins in phages can help establish the location of the putative lysis cassette and there are numerous crystal structures and functional domains that can be utilized to identify these proteins from the various protein/DNA repositories. The holins on the other hand, do not share amino acid identity, do not have conserved functional domains and in many genomes are only annotated if a gene that encodes a protein with at least 2 TM domains is found in association with a lysA , 23–25. Gordonia rubripertincta NRLL B-16540 strain. Gordonia sp. are members of the Actinobacteria, commonly found in soil [G. terrae) [G.terrae, G.bronchalis) [G. paraffinivorans) [Gordonia sp. are from the same lineage as Mycobacterium tuberculosis [Gordonia sp. are of interest because they may also have host range to M. tuberculosis and other important pathogenic bacteria.The current study utilized a comprehensive bioinformatic approach to systematically identify the proteins encoded by the genes within the lysis cassettes in a set of 77 diverse phages that infect the Gram-positive  in soil . Various terrae) , cathetenchalis) –38 and aivorans) . Gordonirculosis , thus, pThe results show that there is a high level of diversity of the putative lysis genes within this set of 77 phages that results in 16 different genome organizations, many of which are distinct from the lysis cassettes that have been previously evaluated. The most diverse set of proteins identified in the lysis cassettes are the transmembrane holin-like proteins. In the current study, 34 different genes encoding holin-like proteins were identified as well as a potential holin-major capsid fusion protein. The holin-like proteins contained between 1–4 TM helices, the genes are not shared to a high degree amongst the 16 reference phages and are present in the lysis cassette in a wide range of combinations of up to 4 genes in which none are duplicated. Detailed evaluation of the TM domains and predicted membrane topologies of the encoded holin-like proteins show that many have novel organizations that have not been previously described. These findings suggest that the organization and diversity of genes within the lysis cassette may be important in phage fitness and provide compelling support that there are novel operational lysis models yet to be discovered.The Actinobacteriophage Database at PhagesDB  containsst iteration: mmseqs cluster–min-seq-id 0.35 -c 0.8 -e 0.001 -s 7 –cluster-steps 11nd iteration: mmseqs search–min-seq-id 0.15 -c 0.7 –cov 0.7 -e 0.001 –e-profile 0.001 -s 7 –num-iterations 32Gordonia strains are then clustered based on the percentage of genes that they have in common [Gordonia rupripertincta NRRL B-16540 of which 54 had GenBank entries. The 81 phages were found in 13 clusters  and also included the singleton phage VanLee. All phages within a single cluster were visualized by utilizing Phamerator with default settings [within each cluster were then grouped based on an analysis of the pham membership and order of the five genes flanking the putative endolysin; exact matches were required for sorting into discrete lysis cassette groups. This allowed the creation of different groups of phages within each cluster that had distinct genome organizations at the lysis cassette. This approach revealed three distinct groups within cluster CT and two within clusters DV and DQ. All other clusters either showed the same exact lysis cassette gene organization or only had a single phage within the cluster. Clusters CW and DZ did not have phages that had been annotated at the time of this analysis, therefore these clusters were omitted from this analysis. Thus, the analysis resulted in the finding of 16 different genome organizations at the putative lysis cassette that encompassed a total of 77 phages. A reference phage was then selected for each of the 16 genome organizations each to represent the similar phages within each group. Reference phages were selected based on having a complete annotation in GenBank and being isolated/annotated by students at USF or Chatham. This accounted for six of the 16 reference phages. The others were selected randomly from each group or by virtue of being the only phage in the cluster (cluster CV and DG1). The reference phage for each group is shown in Comprehensive analysis on the full dataset has shown that these thresholds assemble phams with significantly similar sequences that appear to be functionally related . Phages that infect n common . In Junesettings  to evalu−20. Any hits at or above these values were evaluated to determine the consensus of the functional hits with emphasis of observing consistency in function calls between the PDB, conserved domain (cd) and protein family (pfam) hits. This information was utilized to assign functional domains to the full query sequence. Once all domains were assigned, LysA and LysB proteins were grouped with respect to their conserved domains and predicted activities.The endolysin genes, lysin A and lysin B, were first identified through analysis of the phage genomes in GenBank, Phamerator and the Actinobacteriophage Database at PhagesDB as detailed above. The LysA and LysB protein sequences were then collected for all reference phages and organized based on their Pham membership. All query proteins were analyzed by HHpred  to identThe number and location of predicted transmembrane helices (TM) were identified within query protein sequences utilizing three different programs: SOSUI , Deep TMFor effective cell lysis, phages that infect Gram-positive bacteria require an endolysin enzyme  for digestion of the peptidoglycan (PG) layer of the cell wall and deletion of the LysA results in a non-lysis phenotype , 30, 53.lysA genes in the 16 reference Gordonia phages, each genome was manually reviewed in Phamerator and Genbank as detailed in Methods. lysA genes from each of the reference phages and they are sorted by their Pham designations. Every Gordonia phage evaluated had at least one annotated endolysin or lysA gene with Amok, CaiB, EpicDab, KappaFarmDelta, Orla, PokyPuppy and Mayweather having two distinct annotated lysA genes. Overall, there were 23 lysA genes identified within the 16 phages that grouped into 10 different Phams. To characterize the predicated domains and activities, the LysA enzymes were evaluated using HHpred as described in Methods. Five enzymatic domains were identified across the 23 proteins based on high coverage to the query, low e value scores and consistency in the activities across the PDB, conserved domain, CATH and pfam hits. E.coli [C. intestinale [Gordonia phage LysA proteins that folded with high probability and low e values to several PDBs with predicted C-terminal cell wall binding domains (CW1 and CW2) [Gordonia phages had defined CW binding regions through HHpred analysis.To identify the location of the annotated endolysin or . E.coli  and PDB estinale . This doestinale . In addiand CW2) , 61 or hGordonia phages. A single phage was selected to represent each of the combinations and the schematics are presented in Gordonia phages express a single LysA that is predicted to have multiple enzymatic domains. However, unlike the three-domain structure typically identified in the majority of the LysA proteins from mycobacteriophages, only Claws, DalanDe and ChistanaKitsune encode a LysA with more than two domains. The LysA from these phages is noteworthy as the protein is nearly 1,100 amino acids making it twice as large as any LysA enzymes described in mycobacteriophages. These LysA proteins have four predicted domains including an N-acetylmuramoyl-L-alanine amidase (Ami-2A), a glycoside hydrolase (GH43 family) [P. aeruginosa PAO1 (PDB 4BXJ_A) [lysA genes and each encodes an enzyme with a single predicted enzymatic domain. There are four different combinations of the activities across these seven phages and these are shown in Based on the predicted activities assigned to each of the reference phage LysA proteins and the genomic organization of the genes, there were 7 different domain combinations across the 16  family) , a M23 p family) , 64, and family) . Five ot 4BXJ_A)  with covGordonia phages evaluated have a lysA that can be validated bioinformatically with high probability matches to relevant PDB structures that have activities toward the PG. When all of the specific enzymatic activities for each LysA are considered in the context of the 7 different genome organizations shown in Gordonia phages (<175 amino acids) and both appear to have only activity targeted to the MurNac—L-Ala linkages between the glycan and peptide.All of the Gordonia strains have a cell wall that belongs to wall chemotype IV sensu Lechevalier and Lechevalier and contains an outer membrane consisting of 44–66 carbon mycolic acids that are covalently attached to the arabinogalactan-peptidoglycan complex [ complex –21. This complex –30. This complex , 23.lysB in each of the 16 reference Gordonia phage genomes, each was manually reviewed in GenBank and Phamerator to identify the location of the putative lysB. lysB genes from each of the reference phages and their Pham designation. Every Gordonia phage evaluated had an annotated lysB gene except Claws (cluster CP). The LysB proteins ranged from 247–287 amino acids and unlike the LysA proteins all but the LysB from EpicDab and PokyPuppy was grouped to the same Pham. To validate the lysB annotation, each protein was evaluated in HHpred as detailed in Methods. Every one of the predicted LysB proteins modeled with near full coverage to the crystal structure 3HC7_A from the LysB of mycobacteriophage D29 [−21. All proteins also had numerous other high probability hits to acetyl xylan esterases and cutinases and hit to many of the same PDB hits as the LysB from D29 . One protein is predicted to have a signal sequence and also an internal TM helix (DumpTruck gp33). Ten proteins are predicted to have a single TM helix, 15 are predicted to have two TM helices, one is predicted to have three TM helices and 18 are predicted to have four TM helices. Importantly, the 46 TM proteins group to 34 distinct Phams meaning and only nine of the genes are shared amongst the reference phage genomes. Only gp58 and gp60 from the DQ phage DalanDe appear unique as these are orphams and have no additional Pham members. However, the other 32 Phams contain between 5 to 200 members, 17 of these have members from phages that are grouped to at least two other clusters and infect strains other than G. rubripertincta  a N-in-C-in topology, 2) a TM1 helix that has a high glycine and alanine (GA) content and also a high percentage of hydrophilic residues (>30%) that allow the domain to function as a SAR and exit the membrane following loss of the PMF [2168 and has an extended C-terminal domain of 81 amino acids   the PMF , 18 and no acids , panel BM1 helix . NeitherM1 helix . The lonM1 helix , 56. AltM1 helix . Finallyix [2168 . Collect2168 pinholin. However, the TM1 helix of these proteins is not predicted to have SAR function since there is not a high level of GA or polar residues , DalanDe (gp56) and Clawz (gp66). These proteins range from 135–179 amino acids and have extended N and C terminal cytoplasmic domains of >40 amino acids . The last topology is gp10 from Secretariat (The fourth topology has is predicted to have a unique N-out-C-out structure and includes Mayweather (gp25), Orla (gp26) and Suerte (gp22). These proteins contain 148–168 amino acids and all have a very small N-terminal region of 3–6 amino acids, a large highly charged cytoplasmic loop between the two TM helices and then an extensive extracellular C-terminal region with <30% KRHDE residues , panel Dretariat , panel Eretariat . This scretariat .In summary, while the 15 proteins with two TM helices each have a few characteristics of the holins from phage 21, Ms6 and D29, they all have unique features not found in these proteins and do not show amino acid identity to these proteins. It is noteworthy that when any of the two TM genes are found in combinations with other two TM genes in the lysis cassette , all of the two TM holin-like proteins expressed are never duplicates and are always from different Phams and have distinct topologies (see schematics described below).Gordonia phages is predicted to have three TM helices (KappaFarmdelta gp26), while 18 of TM proteins are predicted to have four TM helices. KappaFarmDelta gp26 has a predicted three TM topology that is N-out-C-in with a highly charged C-terminal domain (50% KRHDE) and no dual start motif. The TM1 helix has a similar GA and hydrophilic residue content to that of the TM1 helix of S105 of phage λ [The holin proteins from phage λ (S105 and S107) and phage P2 (Y) have been classified as Type I and have 3 TM domains with an N-out-C-in topology , 7. The  phage λ , 18. ThuGordonia phages with four TM helices are grouped into 11 different Phams and all have the same N-in-C-in topology  and a single two TM protein (gp66) directly downstream. These two genes are spanned by others that don’t have known function or TM domains and the cassette itself is localized downstream of the tape measure and minor tail genes. Gp66 does not fit the same topology as the canonical Type II holin since it is larger (135 amino acids) and also does not have a highly charged C-terminal domain. The TM1 helix of Clawz does contain a high percentage of polar residues but lacks a GxxxG motif found in the S21 pinholins. It is also relevant that both TM helices are located >40 amino acids from the N and C-terminus and this is much farther spaced from the protein termini than other the validated SAR domains of S21 and other proteins [lysA and lysB (58). Since the lack of a lysB gene is not lethal to mycobacteriophages but been shown to impact lysis efficiency and overall phage fitness [lysB in this phage.The simplest lysis cassette found across all the phages in the current study was present in the CP cluster phage Clawz . This lyproteins , 18. Thu fitness , 30, it’G. rubripertincta phages and Secretariat is representative of 15 other phages that all have genes from the same phams within the lysis cassette. This cassette is simple like Clawz and also contains a single lysA and a single downstream two TM gene (Gordonia phages in this study. Secretariat has a predicted lysB (gp28), but it is not associated with the lysis cassette and is located ~15,000bp downstream from the lysA. As described previously, gp10 has a unique topology and does not fit with the canonical Type II holins since it does not have a dual start motif, is nearly twice as large as S2168 and has much more extensive N and C-terminal domains  and KappaFarmDelta (CV) have multi-gene lysis cassettes that contain both  helices . These ld the PG . Both lytopology . Thus, tGordonia phages is a lysis cassette that contains a lysA and three TM genes that are localized at a distance from a predicted lysB  and LilyPad (cluster DG1) share four of the five genes in the lysis cassette. In this regard, both of these phages express a single LysA from the same Pham and only diverge at the first of the four TM genes. In DumpTruck, the first TM protein predicted by most programs and HHpred has a signal sequence and also one internal TM helix that would render this a one TM proteins with an N-out-C-in topology. In contrast, the first gene downstream of the lysA in LilyPad encodes a larger four TM protein that is also the first TM gene found in Mayweather  is unique in that it is the only TM gene in this study that is in a reverse orientation to the lysis cassette. Thus, this gene would not be transcribed in an operon with the other genes in the cassette. However, unlike all of the other scenarios where a distally localized lysB is found 13,000–30,000bp from the lysis cassette, the lysB in PokyPuppy (gp49) is only 4,000bp from the lysis cassette and in the same reverse orientation as gp38  and LysB (gp7). EpicDab has a lysis cassette with the two lysA and lysB genes (lysB (gp7) and major capsid (gp9) revealed no proteins with TM helices. However, analysis of the major capsid protein (gp9) showed that there were two TM domains predicted in the N-terminal 62 amino acids and a putative coiled-coil that is not associated with these helices between amino acids 71–98. The two TM helices are predicted in all TM analysis programs and also by HHpred. It has been established that the major capsid protein of phage HK97 contains a cleaved N-terminal “delta” domain that functions as a scaffolding protein, and is released from the final virion capsid. The HK97 delta domain contains coiled-coils but does not have defined TM helices [The last scenario is represented by the microphage EpicDab. This is a DM cluster phage with a genome that is only 16,658bp and is significantly smaller than any of the other reference phages . EpicDabsB genes , panel A helices –76. ThuslysB gp7. However the annotated start is called at base-pair 5,522 to account for a predicted NKF reverse gene (gp8) that runs from base-pair 5,546–5,264. This results in gp9 having a 1,887bp ORF that encodes a 629 amino acid protein. HHpred analysis of gp9 reveals that there are predicted pfam serine peptidase alignments to a domain that spans amino acids 150–307, well after the N-terminal domain with the TM helices  and do not have multiple enzymatic domain that target different areas of the PG. Thus, these LysA enzymes more resemble the smaller, single domain endolysins found in T4, T5, T7 and lambda phages that all infect E. hosts [Gordonia phages with regard to the size of the LysA as these phages express a protein of >1000 amino acids that is even larger than those found in myocbacteriophages. Second, several of the Gordonia phages express two distinct LysA proteins, each that is < 300 amino acids and with a single enzymatic domain (like the lambda endolysins noted above). These split lysA genes express proteins with varying combinations of enzyme activities. The contribution of a single LysA activity to the PG or split genes with distinct activities to the lysis pathway and phage fitness is unknown. However, the split lysA genes may be an evolving feature in Gordonia since this organization has also been detected in phages that infect other Gordonia strains [All 16 reference phages contained genes encoding an endolysin equivalent protein (LysA) and is predicted to target the PG layer. The identification of this gene was used to establish the location of the putative lysis cassette in the 16 E. hosts . Only ph strains .Gordonia bacteria have an OM rich in mycolic acid [lysB gene that encodes a protein with predicted activity toward the mycolic acids in the OM was also identified in 15 of the reference phages. It is speculated that the LysB enzymes represent the third component of the proposed three-step lysis pathway [Gordonia phages has strong HHpred hits to the crystal structure of LysB that has been evaluated in wet lab experiments from mycobacteriophage D29 [lysB genes are typically found very near the lysA in a putative lysis cassette [lysB genes have been bioinformatically identified in other Gordonia phages [lysB is not expressing an enzyme involved in the lysis pathways since the temporal expression of the protein would be divergent from the genes in the putative lysis cassette. Alternatively, the distal genomic organization of the identified lysB may reflect the non-essential role of this protein in the lysis pathway [lysA and lysB in a defined lysis cassette have a fitness advantage over those that express the gene in a distal location.Since the Gram-positive lic acid –21, a pu pathway , 7, and  pathway –29. The hage D29 . Howevercassette , 23. Alta phages , these p pathway , and wetlysA in all but two of the 16 reference phages. In all, 46 genes that encode 32 distinct membrane proteins were identified with the majority of the reference phages having three or four of these holin-like genes. Thus, one of the key findings of this report is the striking diversity regarding the organization of these different genes within the lysis cassette and the identification of up to four holin-like genes within the lysis cassette. This type of organization has not been described in phages that infect Gram-negative hosts [lysA, lysB and holin-like genes are observed not only in genetically diverse phages, but also in phages that are grouped to the same cluster based on gene content. While all phages have the same lysB gene and several groups share the same lysA, only 12 of the 46 distinct holin-like genes are shared between any of the reference phages. This results in 16 different lysis cassette genome organizations within the population of 77 Gordonia phages evaluated. The level of diversity in the linear gene organization and changes to one or more genes within the ~3,000bp lysis cassettes suggest that the cassette itself is unlikely to be acquired by a phage as a single multi-gene module. Instead, it appears that phages acquire and lose individual holin-like genes within the lysis cassettes.A wide variety of genes that encode holin-like proteins containing one-four TM helices were identified immediately downstream of the ve hosts . There ilysA that did not have associated holin-like genes. The DM cluster phage EpicDab does not have a defined holin-like gene in the entire genome. However, EpicDab expresses an exceptionally large major capsid protein with an N-terminal domain of 116 amino acids that has two well defined TM helices and may be cleaved from the native protein. Since EpicDab is a microphage with only 26 genes and a genome of 16,658bp, this novel gene organization may have evolved due to the compactness of the EpicDab genome. The DR phages typified by CaiB also do not have a lysis cassette that contains both holin-like genes and lysA. However, these phages appear to have acquired a forward lysis cassette that contains two lysA and one lysB and a second cassette that contains four consecutive holin-like genes in a distinct operon-like region. The validity that these distal genes are in fact holin-like genes is supported by the finding that homologs of the expressed proteins are found in defined lysis cassettes with lysA in phages that infect other strains of Gram-positive bacteria. This finding is significant as it highlights that there are alternate organizations to the canonical lysis cassette and the linear arrangement of a lysA and holin-like genes may not always be present. A non-linear organization of the lysis genes is also found in the T4 phages . It is also noteworthy that the small 1,600bp holin cassette in the DR phages is not found as an intact module in any of the other phages. Yet the third gene of the holin-like cassette in CaiB (gp18), is present within a lysis cassette that contains a lysA in three of the Gordonia reference phages .The striking diversity observed within the lysis cassette is mostly due to the number and topology of the different membrane or holin-like proteins identified. These holin-proteins have between one-four TM helices and although they have some of the characteristics of the Type I, Type II and Type III holins from phages that infect Gram-negative hosts , 7, the Gordonia phage lysis cassette, each of the holin-like proteins that are predicted to be expressed have a distinct topology and are unique, even if they have the same number of TM helices. Thus, the multiple genes are not simply “duplicated”, and the distinct sizes and topologies of the protein products suggest that each may impart a unique functionality. As described previously, holins function as timer to the lysis pathway and the Type I and Type III holins also function in the generation of large holes that allow the release of the endolysins to metabolize the cell wall components. The type II pinholins only serve as a timer and require the endolysins to be exported from the cell via a SAR. Since SAR regions were not identified in any of the LysA or LysB enzymes, and the Gordonia phages in this report do not appear to express a homolog to the gp1 secretory protein from phage Ms6 [only one aspect of the lysis pathway: either hole formation or timing, not both. In this context, it’s also possible a protein may function as either a holin or antiholin. Thus, the interaction of all of the holin-like proteins would be important in the timing and regulation of the lysis pathway. Unfortunately, the best characterized antiholins are derived from the same gene as the holin using a dual start motif [Currently, all of the well-studied lysis models propose an interaction of no more than two holin-like proteins and in the case of λ and P2 the two proteins are actually generated from a single gene due to a dual start motif , 7. In ahage Ms6 –32, it iGordonia phages genomes contain a cassette with three or more distinct holin-like genes suggest the intriguing possibility that phages acquire multiple holin-like genes to enhance phage fitness and the ability of the phage to diversify the lysis pathway possibly in response to the evolution of the bacteria. The recent report showing that bacteria are evolving host defense that can target the lysis pathway [E. coli and M. smegmatis when the protein is induced from a plasmid construct [Gordonia phages that express multiple holin-like genes, a LysA with several activities and also a LysB may a selective advantage regarding their fitness. In summary, this study provides a framework for the bioinformatic analysis of the putative lysis cassette and characterization of holin-like proteins in phages that infect Gram-positive bacteria. Further wet lab evaluation of these novel lysis cassettes may reveal new lysis pathway models and provide insights into phage fitness, evasion of host defense systems and insights into the genetic engineering of holins with enhanced functionalities.The finding that the majority of  pathway  imply thonstruct . While tS1 TableThe 46 TM proteins as listed in order of consensus prediction of total TM helices. The number of predicted TM helices for each TM prediction program are listed. The final column presents notes related to the consensus prediction.(XLSX)Click here for additional data file.S2 TableThe refence phage and LysB gene number is presented. The first 5 HHpred PDB hits for all LysB proteins are presented by PDB number. The description of each PDB is presented on the bottom of the table.(XLSX)Click here for additional data file.


Autonomy is recognized as important for individual well-being and constitutes one dimension in the KIDSCREEN-instrument measuring health related quality of life (HRQoL) in children and adolescents. However, the autonomy questions in KIDSCREEN are restricted to opportunities to influence leisure time activities, which is a form of autonomy as volition. Yet, there are other aspects of autonomy that might be related to adolescent’s HRQoL. The aims of the present study were first to investigate the psychometric properties of a scale measuring autonomy in adolescence from a control perspective (AAC) including its relation to the autonomy dimension in KIDSCREEN, and second; to investigate AACs ability to predict each of the 10 dimensions constituting KIDSCREEN.n = 154) aged 15–16 years who were recruited from schools located in both low (two schools) and high (two schools) socioeconomic status (SES) areas in Sweden participated in a cross-sectional study. The adolescents answered a questionnaire including a new 6-item scale measuring perceived autonomy and HRQoL assessed by the KIDSCREEN-52 instrument. A factor analyses was computed to investigate the relation between the items in the AAC scale and the autonomy items in the KIDSCREEN instrument. Hierarchical regression analyses were computed to investigate if the AAC scale predicted HRQoL in any of the 10 dimensions in KIDSCREEN after controlling for gender, SES and the original autonomy scale included in KIDSCREEN.Students (The factor analysis showed that all the items from the autonomy scale loaded in one factor and that all the items from the AAC scale loaded in another dimension. The hierarchical regression models showed that the AAC scale uniquely predicted HRQoL in all dimensions of the KIDSCREEN instrument after controlling for gender, SES and the original autonomy scale included in KIDSCREEN-52. A high level of perceived autonomy was associated with a high level of HRQoL for both scales.A new scale for measuring autonomy from a control perspective has been presented and shown to differ from autonomy as volition. Both forms of autonomy are positively related to HRQoL in adolescence 15–16 years old.The online version contains supplementary material available at 10.1186/s12887-022-03607-5. Health and well-being are essential in all human life. Health-related quality of life (HRQoL) is a multi-dimensional measure that relates to individuals’ perception and subjective evaluation of their health and well-being within their specific culture, following the intentions from WHO , 2. ManyKIDSCREEN is a generic instrument measuring HRQoL in children and adolescents 8–18 years old. It was developed as an EU project including 13 European countries and was tested on over 3000 children. The instrument comes in 52, 27 and 10-item versions. All versions cover 10 dimensions: physical well-being, psychological well-being, moods and emotions, self-perception, parent relation and home life, financial resources, peers and social support, bullying, and autonomy . Hence, A close relation between autonomy and different measures of well-being is a common finding. Perceived autonomy in adolescence has been found to be negatively related to depression  and posiHowever, autonomy can be defined in many ways, for example as emotional independence, as volition versus pressure, functional independence and detachment, and its effect on health and well-being is therefore dependent on how it is defined . Van PetDo you have enough free time to be with your mates?) that they concern autonomy as volition, and this is also supported by their positive relationship to the other dimensions in KIDSCREEN.The autonomy dimension in the KIDSCREEN instrument concern children’s opportunities to create social and leisure time  without This conception of autonomy is compared to autonomy from a control perspective in the present study. Autonomy from this perspective emphasizes the importance of power and control for children’s development, in particular their experience of being seen and listened to and their opportunities to take part in decisions that concern themselves . Yet, thThe first aim of the present study was therefore to investigate the psychometric properties of a new scale measuring autonomy in adolescence from the control perspective (AAC)  includinA paper and pencil questionnaire was distributed to 154 students at four Swedish high schools during regular class time in a cross-sectional design. The questionnaire was filled out individually and was completed in approximately 20 min. The students attended grade 9 and were 15–16 years old. Two schools with 93 participating students (47 girls and 46 boys) were in low SES areas (mean income below the median) and two schools with 61 participating students (35 girls and 26 boys) were in high SES areas (mean income above the median). Principals and teachers at all four schools were contacted and all approved the study.HRQoL was measured using the KIDSCREEN-52 questionnaire , a crossDo you find that adults don’t listen to you? (reversed) (2) Do you take part in important decisions concerning your life? (3) Do you find yourself treated unjustly because of your age? (reversed) (4) Do you feel that adults take you seriously? (5) Do you wish you could have a greater influence on your daily life? (6) Do you think your life would be better if more decisions were up to you? Responses were given on a five-point Likert-type scale to each question with alternatives from 0 (‘never’) to 4 . A high value indicated high autonomy.AAC was measured by a 6-item scale developed in the project to measure perceived possibilities for adolescents to influence their life. The scale contained the following questions (translated from Swedish): (1) A principal component analysis (PCA) containing the 6 items from AAC and KSA was first computed determining the optimal number of dimensions and how much of the variance they explained. Next, a promax rotation was employed to find a distinct factor structure.Nine hierarchical regression analyses were computed entering gender and SES in a first block, autonomy as measured by KIDSCREEN-52 in a second block, and autonomy measured by AAC in a third block. HRQoL in all nine dimensions except autonomy served as dependent variables. A two-block analysis with gender and SES in a first block and ACC in the second was employed for a tenth regression in which the autonomy dimension in KIDSCREEN-52 served as dependent variable. Level of significance was set to < 0.05.r(153) = 0.205, p = 0.011. All five items from KSA loaded highest in the first dimension and all six items from AAC loaded highest in the second dimension (Table p < 0.01) although there were also some significant correlations between items across the scales (Table Means (SD) for girls and boys in schools located in high and low SES areas for the AAC scale and all 10 dimensions of HRQoL from KIDSCREEN are shown in Table r15 = 0.205,Next, a series of hierarchical regression analyses were computed in which gender and SES were inserted in a first block, KSA in a second block and ACC in a third block. Each dimension of HRQoL as measured by KIDSCREEN-52 served as dependent variable. An exception was the autonomy dimension of KIDSCREN for which the second block (KSA) was omitted. Collinearity statistics revealed that tolerance for all analyses were above 0.9 and VIF were below 1.1. Hence, multicollinearity was no problem.Physical well-being (8%), Moods and emotions (16%), Self-perception (9%), Autonomy (5%), and Social acceptance and bullying (6%)  accounted together for between 1–16% of the variance in each dimension of HRQoL. They contributed significantly to %) Table .Table 4HKSA (block 2) accounted for between 0–20% of the variance in addition to gender and SES (Δ R2). KSA contributed significantly to all HRQoL dimensions of the KIDSCREEN-52 instrument except to Social acceptance and bullying , Psychological well-being (19%), Moods and emotions (11%), Self-perception (10%), Parent relations and home life (16%), and School environment (19%).ng Table , and wasAutonomy (for which only two blocks were computed). Although the unique contribution of AAC was significant for all dimensions, the contribution was small in half of the cases (5% or less). However, the contributions to the explained variance for Moods and emotions (14%), Self-perception (16%), Parent relations and home life (15%), Social acceptance and bullying (9%), and Financial resources (14%) were substantial. See Table r(8) = 0.038, p = 0.92). Hence, their relation is neither antagonistic nor concurrent.AAC (block 3) accounted for between 3–16% of the variance in addition to gender, SES and KSA. High AAC contributed significantly to all HRQoL dimensions of the KIDSCREEN-52 instrument including motions 1%, Self-pnd for Autonomy) in the regressions revealed that gender predicted HRQoL relative to Physical well-being, Moods and emotions, Self-perception, and Autonomy. Boys reported significantly higher HRQoL than girls in all four of the above dimensions. SES predicted HRQoL relative to Self-perception, Parent relations and home life, School environment, and Social acceptance and bullying. Adolescents from low SES district schools reported significantly higher HRQoL than adolescents from high SES schools for Self-perception, Parent relations and home life, and School environment, whereas the pattern was reversed for HRQoL related to and Social acceptance and bullying  in the present study was in this direction. For the other three dimensions with significant contributions from SES , low SES adolescents reported the highest HRQoL. One reason for this difference might be that SES in the present study was measured indirectly by school location and not on the individual level. This might have weakened the predictive power of SES. Yet, effects of gender and SES were included in the present study for the purpose of control and the sample size was too small to investigate their effect on HRQoL more thoroughly.In agreement with previous studies, we found that HRQoL was higher in 15–16-year-old boys as compared with same aged girls –16, and The small sample size was a major limitation to the main results of this study. However, the relation between AAC and a multidimensional measure of well-being such as KIDSCREEN-52 was a major strength of the present study, demonstrating its associations to many areas in life. It is plausible that the control aspect of autonomy is more important for adolescents than for younger children. Inclusion of younger age groups and ideally longitudinal studies are therefore needed in future studies.To give a young person power and control over her or his life should not be confused with parental neglect. A multitude of sources has shown that parental neglect has aversive effects on children’s development . HoweverThe present research also raises the question of whether HRQoL measures such as KIDSCREEN would be improved if more aspects than volition was measured in the autonomy dimension. This is a challenging thought but must stay as such until some of the limitations of the present study on AAC has been overcome. The KIDSCREEN group might also have some ideas about the relative contribution to HRQoL from each of the included dimensions.Autonomy is a multifaceted concept and has been measured in many ways. A scale for measuring adolescent’s sense of control and influence over things that happen in their lives has been presented and shown to differ from autonomy as volition in adolescence 15–16 years old. Both forms of autonomy are positively related to HRQoL in adolescence.Additional file 1.