Administration of liquid surfactant through an endotracheal tube for the treatment of respiratory distress syndrome has been the standard of care for decades. A skilled health care provider is needed to perform this procedure. In lower-income and middle-income countries (LMICs), healthcare resources are often limited, leading to increased mortality of premature infants, many of whom would benefit from surfactant administration. Therefore, having a simplified procedure for delivery of surfactant without the need for advanced skills could be life-saving, potentially diminish gaps in care, and help ensure more equitable global neonatal survival rates. Modifications to the standard approach of surfactant administration have been put into practice and these include: INtubation-SURfactant-Extubation (INSURE), thin catheter surfactant administration (TCA), aerosolized surfactant, and surfactant administration through laryngeal or supraglottic airways (SALSA). Although there is a need for larger studies to evaluate the comparative effectiveness of these newer methods, these methods are being embraced by the global community and being implemented in various settings throughout the world. Because the SALSA technique does not require laryngoscopy, a provider skilled in laryngoscopy is not required for the procedure. Therefore, because of the ease of use and safety profile, the SALSA technique should be strongly considered as a viable method of delivering surfactant in LMICs. Respiratory distress syndrome (RDS) is one of the most frequently diagnosed diseases in preterm infants in the neonatal intensive care unit (NICU) . ExogenoWhile INSURE requires endotracheal intubation, the TCA techniques are considered less traumatic to the airway than the traditional invasive methods of surfactant administration. These techniques still require operator expertise as laryngoscope insertion is needed . AerosolThe SALSA technique involves placement of a laryngeal mask or supraglottic airway into the posterior pharynx without the need for laryngoscopy. Liquid surfactant is then administered through the device . SurfactIn this manuscript we will review the pathophysiology of RDS and the impact that treatments such as surfactant and continuous positive airway pressure (CPAP) have had on the disease. The evidence, safety, efficacy, and outcomes for each of the newer methods of surfactant administration will also be reviewed and we will draw conclusions as to the applicability of these methods in the LMIC setting. We will conclude by providing instructions on how to perform the SALSA technique as it has unique and specific characteristics that make it ideal for the LMICs setting.RDS is the leading cause of respiratory disease in preterm infants , 26. ThePreterm infants produce limited amounts of surfactant and commonly require exogenous instillation of surfactant to decrease their oxygen requirement and ameliorate the symptoms of RDS. This is why surfactant therapy can be life-saving . Followip = 0.005)  . Similar= 0.005) , 39.While advances in therapies and available approaches in treatment of RDS have led to improved morbidity and mortality in premature infants, there is a lag to disseminate such technology from HICs to LMICs , 41. TheIn an effort to continue improving care and reducing infant mortality in LMICs, the WHO has recommended Sustainable Development Goals (SDG). This collection of 17 goals is designed to achieve a more sustainable future for all by 2030. One aim is for every country to reduce NMR to at least as low as 12 per 1,000 live births and end preventable deaths of newborns and children under 5 years of age . Thanks Continuous positive airway pressure (CPAP) is a simple, cost efficient, non-invasive respiratory support considered an optimal first line therapy for RDS. It aims to support functional residual capacity and prevent the need for mechanical ventilation (MV) , 46. CPADespite these studies coming from HICs, CPAP should be implemented in every LMIC seeking to decrease their NMR as it is an effective treatment for RDS. This can be supported by a systematic review which evaluated the feasibility and efficacy of CPAP in LMICs. Thukral et al. showed that implementing a CPAP program could reduce in-hospital mortality by up to 66% along with a decreased need for MV in preterm infants . BecauseWhen there is a need to administer surfactant, the traditional approach has been to deliver exogenous surfactant through an endotracheal tube (ETT). However, less invasive methods, which may be considered “off label” use based on the country, are now widely being used around the world .Neonatal endotracheal intubation is a life-saving and critical skill, but there are risks associated with it and this needs to be done by a neonatologist or health care provider with expertise in the procedure . This skIn areas without advanced neonatal care, a skilled health care provider capable of successful intubation may not be present in an urgent and timely manner when surfactant is needed in a critically ill infant. In these settings, less invasive methods could be an alternative.INSURE is the most studied mode of surfactant instillation. The prompt removal of an ETT after instillation of exogenous surfactant was both feasible, safe, and beneficial to the premature infant . INSURE via the ETT  of infants failed and required MV after using INSURE . This teSeveral terms are used to describe the variations of the TCA method. The most common are: Less Invasive Surfactant Administration (LISA), Minimally Invasive Surfactant Therapy (MIST), Take Care, and SurE . TCA invvia thin catheters was associated with lower likelihood of mortality, need for mechanical ventilation and bronchopulmonary dysplasia compared with InSurE , while death was not significantly different (10 vs. 7.8%), BPD at 36 weeks' postmenstrual age was lower in the MIST group (37.3 vs. 45.3%)  .Jena et al. of India, have shown that TCA administration can be applicable to an LMIC setting. They found that, compared with the INSURE group, TCA infants had less need for subsequent MV (19 vs. 40%), shorter duration of supplemental oxygen, shorter hospital stay, and a lower rate of supplemental oxygen at 36 weeks' corrected gestational age .The TCA method may be feasible in an LMIC setting, but requires a skilled provider to use a laryngoscope to pass a catheter through the vocal cords. Thus, its applicability is primarily within a NICU environment.p < 0.0001; RR: 0.51 90% CI: 0.41-0.63)  . In a sy41-0.63) .An advantage of aerosolized/nebulized surfactant is that it is the least invasive method to deliver surfactant and requires minimal skills to administer. However, as of the writing of this review, there is currently not a device that is commercially available, although a device is currently under review by the United States Food and Drug Administration. If a device were available, this method will require specific equipment that may be difficult or cost-prohibitive to obtain in LMICs, thereby limiting its application in this setting.SALSA is a less invasive method that has unique characteristics that makes it an ideal candidate for the LMIC setting. The SALSA technique consists of inserting a supraglottic airway device (SAD), commonly known by the trade name, Laryngeal Mask Airway (LMA), into the mouth and advancing it until the device is unable to be advanced further. Proper placement is indicated by listening for bilateral breath sounds and, if available, color change on a carbon dioxide detector. A piece of tubing connected to the surfactant syringe is then placed into the lumen of the device to deliver the surfactant in aliquots just above the vocal cords –23. FiguThe SALSA technique differs from traditional intubation, INSURE, and TCA in that there is no insertion of the device through the vocal cords, therefore, use of a laryngoscope is not required for proper placement of the device. In addition, insertion of the LMA is substantially easier and faster than the insertion of an ETT , and decIn the largest randomized controlled trial of SALSA, placement of the device took an average of 88 s range 12-500 s) and 67% of attempts were successful in <35 s . The dev-500 s anWith decreased need for MV, the SALSA technique may be beneficial for survival of infants in LMICs where access to MV is limited or not available. Because of this, SALSA was introduced in 2018 and has been widely used by physicians in the Republic of Azerbaijan. This has been one contributing factor to their recognition by WHO as one of the fastest progressors globally in meeting the SDG 2030 goals in reduction of the country's NMR , 73.n = 612) in Ethiopia found that only 39% of attendants at deliveries were medical doctors or general practitioners and only half (53%) of the births occurred at a referral hospital (According to the WHO, nearly 15 million babies are born prematurely each year. Preterm birth complications are considered the leading cause of death among children under 5 years of age. This accounted for 1 million deaths in 2015 . United hospital .Studies in adult and pediatric patients show that intubation at referring or community settings, where there is less access to a skilled airway management, increases the risk of poor outcomes, including death , 78, 79.INSURE and TCA methods for administration of surfactant do show a decrease in the need for MV. Because of this, these methods have been implemented as standard practice in many parts of the world. The need for a high degree of skill which is similar to intubation does make this method less desirable for areas where advanced neonatal care and the equipment required to provide this care is not available. Inhaled surfactant, although promising, still requires an approved device to determine whether it will be a widely accepted method, but the cost and additional resources required will likely not allow this method to be widely used outside of high resource settings.SALSA requires minimal skill and resources and is feasible in areas where advanced neonatal care is not widely available . This isAlthough we recognize the limitations of small, unblinded, clinical trials, and that larger trials are necessary to establish a complete safety and efficacy profile, SALSA should become a preferred alternative method to administer surfactant in infants that require this life-saving medication in the LMICs setting. Furthermore, it is ideal for use in situations where there is no provider skilled at intubation, CPAP is available, and/or a minimally invasive method of surfactant administration is desired. If health care providers are trained to administer surfactant using the SALSA technique, it may show benefits in reduction of oxygen needs, intubation and MV, but would also be expected to improve overall morbidity and mortality in newborns with RDS.Unlike intubation which requires significant training to acquire skills, placement of a SAD can be learned quickly with a higher successful rate at first attempt placement . To assihttps://www.youtube.com/watch?v=Iig9l4BgIy4 and https://www.youtube.com/watch?v=ioXGyfVLdyE.Detailed instructions on how to perform the SALSA technique are available in the following links: The benefits of surfactant are clear. The methods to deliver surfactant will, however, continue to evolve. Each of the techniques surveyed in this review have demonstrated safety and efficacy. It is the opinion of the authors that the SALSA technique is uniquely applicable to the LMICs setting. Establishing programs to implement the SALSA technique in LMICs may be advantageous to improve NMR in countries that lack skilled providers but have access to surfactant. One such initiative took place in Azerbaijan and was backed by the Ministry of Health . Other cSimilarly, the SALSA technique could be applied to community hospitals in HICs to help improve outcomes. This may include the prevention of transfers to tertiary care facilities, thus decreasing costs and health care utilization. Quality-improvement programs with stepped-wedge design could give more insight into the challenges of implementation across different hospital systems and how this could be improved. Leveraging social media's power to spread medical science and literature may also be of great benefit to shorten the time it takes to disseminate and implement evidence-based therapies from HICs to LMICs.Finally, it is recognized that at this time SADs are not widely available in most LMICs and that providers may not have experience with the device. As surfactant use becomes more widespread, an investment in education and a dissemination of the tools needed to deliver this medication is equally important. Another limitation is that currently available SADs are too large for infants weighing < ~1,200 g. Development of a cost-effective SAD designed to administer surfactant over a broad range of infant weights would benefit LMICs wishing to use surfactant in combination with CPAP to improve their infant mortality rates. When this occurs, it should be evaluated not only in a HIC setting, but in LMICs where we expect the greatest benefit will be found.SG, PF, and HZ: conception and design. HZ, PF, KR, DK, and SG: draft manuscript preparation. All authors reviewed and approved the final version of the manuscript.PF, KR, and SG have worked for and received payments from ONY Biotech in the past. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AP declared past co-authorships with one of the authors KR and the absence of any ongoing collaboration with any of the authors to the handling editor.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Conclusions: Pharmaco-resistance developed in approximately 20% of patients with PSE and was associated with younger age at stroke onset, stroke type and severity, status epilepticus occurrence, and seizure types.Objectives: The study aimed to explore the clinical predictors of pharmaco-resistance in patients with post-stroke epilepsy (PSE). Methods: Patients with epilepsy secondary to cerebral infarct or spontaneous intracerebral hemorrhage were included. The study outcome was the occurrence of pharmaco-resistance defined as the failure of adequate trials of two tolerated and appropriately chosen and used antiseizure medication schedules, whether as monotherapies or in combination, to achieve sustained seizure freedom. Results: One-hundred and fifty-nine patients with PSE and a median follow-up of 5 (3–9) years were included. The mean age of the patients at stroke onset was 56.7 (14.9) years, and 104 (65.4%) were males. In the study cohort, 29 participants were pharmaco-resistant. Age at stroke onset [odds ratio (OR) 0.97, 95% confidence interval (CI) 0.93–0.99;  Stroke is a leading disease worldwide, with an estimated incidence of 15 million cases annually ,3,4. PosThe aim of this study was to identify the clinical predictors of pharmaco-resistance in patients with PSE treated with antiseizure medications (ASMs).We retrospectively identified patients ≥16 years old who were referred to the Epilepsy Center of the Marche Polytechnic University, Ancona, Italy with a diagnosis of epilepsy secondary to cerebral infarct or spontaneous intracerebral hemorrhage and no history of seizures before stroke. According to the International League Against Epilepsy definition, seizures occurring within 7 days of the onset of stroke were classified as acute symptomatic seizures, seizures occurring beyond 7 days of the onset of stroke were classified as unprovoked seizures, and PSE was diagnosed as the occurrence of one or more unprovoked seizures . Patientt test, Mann–Whitney test or χ2 test, as appropriate. Simple and multivariable logistic regression analyses were performed to identify the predictors of pharmaco-resistance. For multivariate analysis, sex, age at stroke onset, stroke type, occurrence of acute symptomatic post-stroke seizures, seizure type, and any variables with p values < 0.02 from comparisons of baseline characteristics were identified for statistical adjustment. Results were reported as odds ratio (OR) with associated 95% confidence interval (CI). Results were considered significant for p values < 0.05 (two sided). Data analysis was performed using STATA/IC 13.1 statistical package .Values were presented as mean (standard deviation) or median (interquartile range) for continuous variables and as the number (percent) of patients for categorical variables. Comparisons were made through the Student’s Of 172 patients with PSE initially identified, 13 patients were excluded due to follow-up <12 months. Accordingly, 159 patients with PSE and a median follow-up of 5 (3–9) years were included in the analysis. The mean age of the patients at stroke onset was 56.7 (14.9) years, and 104 (65.4%) were males. Cerebral infarct occurred in 102 (64.2%) and cerebral hemorrhage in 57 (35.8%) patients. Blood pressure lowering agents were taken by 121 (76.1%), antidiabetic drugs by 30 (18.9%), and statins by 49 (30.8%) patients.p = 0.016) and severe stroke (p = 0.001) in comparison to responsive patients. Status epilepticus was the first epileptic symptom in 5 (3.9%) responsive and 7 (24.1%) refractory patients (p < 0.001), and focal to bilateral tonic seizures were more commonly observed among patients with drug-resistant epilepsy (p = 0.013).In the study cohort, 29 (18.2%) participants were pharmaco-resistant. Drug-resistance occurred in 13 out of 102 (12.8%) patients with ischemic and 16 out of 57 (28.1%) patients with hemorrhagic stroke. Baseline characteristics of the patients according to treatment responsiveness are shown in p = 0.044), history of intracerebral hemorrhage , severe stroke , status epilepticus as initial presentation of PSE , and focal to bilateral tonic-clonic seizures  had a higher likelihood of being refractory to ASM treatment  can favor refractoriness by decreasing drug sensitivity and cerebral drug uptake . Remarkal models ,17. The l models . Inductil models ,20,21, al models , has beel models . Sproutil models . The mall models . Overalll models .Common neurobiological factors can contribute to both epilepsy severity and pharmaco-resistance, and refractoriness can be inherent to disease severity . In thisThe predominant seizure type in patients with PSE was focal. Focal to bilateral tonic-clonic seizures were found in approximately one third of the cases, and associated with a higher risk of drug-refractoriness. In elderly patients with newly diagnosed epilepsy, seizure freedom was less commonly achieved by patients presenting with seizures evolving to bilateral convulsive than in patients with focal or generalized seizures ,34. A meAge at stroke onset was marginally associated with the risk of pharmaco-resistant PSE, with older age being a protective factor. Decreasing neuronal plasticity with age could smooth epileptogenicity and result in a reduced risk of pharmacoresistant epilepsy following stroke. This explanation has been previously advanced to explain the greater response to treatment observed among older adults . ConsistA not significant higher prevalence of acute symptomatic post-stroke seizures was found among drug-resistant patients. Seizures occurring early after stroke are thought to be a reaction of neuronal cells to acute cerebrovascular injury, and reflect transient cellular biochemical dysfunctions, including variations in calcium and sodium concentrations, glutamate release, and the breakdown of membrane phospholipids. Acute symptomatic seizures have also, however, been associated with an increased risk of late unprovoked seizures, indicating that they might be a hallmark of epileptogenesis . Our finWe did not find any relationship of sex with drug refractoriness. To date, conflicting evidence exists about the predictive role of sex. Sex did not emerge as a significant predictor of refractory epilepsy in two studies recruiting participants with epilepsy of any cause of all ages  and oldeThe current study has merit to provide evidence on the risk of drug-refractory PSE from the clinical perspective. Major strengths include the long-term follow-up of the patients and the real-world design, which allowed the generalizability of the findings to everyday clinical practice. Nonetheless, many shortcomings need to be considered. The retrospective analysis of data is prone to selection bias and misdiagnosis can occur even when medical reports are collected extensively. The inclusion of patients referred to an epilepsy clinic, which may represent a group preselected for age and severity, could limit the representativeness and generalizability of the findings to the general PSE population. Further, no information was available about the comorbidities and medications taken prior to stroke and during the acute phase, as well as whether vascular risk factors were adequately controlled after stroke. Therefore, we could not explore whether agents that have been suggested to reduce the risk of PSE may also influence the development of pharmaco-resistance . The unaThe available data on risks of acquiring PSE are not matched by knowledge on the condition itself and its prognosis, and there remains a large gap in the understanding of the causes and mechanisms of drug-refractoriness. The expected disease course is a reasonable question posed by patients and physicians. The identification of factors that may predict treatment response holds important practical implications to guide clinicians once a diagnosis has been made, and inform the health care system on the resources required when recovery from stroke is complicated by recurrent seizures.


The reporting of Coronavirus Disease 19 (COVID-19) mortality among healthcare workers highlights their vulnerability in managing the COVID-19 pandemic. Some low- and middle-income countries have highlighted the challenges with COVID-19 testing, such as inadequate capacity, untrained laboratory personnel, and inadequate funding. This article describes the components and implementation of a healthcare worker surveillance programme in a designated COVID-19 teaching hospital in Malaysia. In addition, the distribution and characteristics of healthcare workers placed under surveillance are described.th of March 2020 to the 20th of August 2020.A COVID-19 healthcare worker surveillance programme was implemented in University Malaya Medical Centre. The programme involved four teams: contact tracing, risk assessment, surveillance and outbreak investigation. Daily symptom surveillance was conducted over fourteen days for healthcare workers who were assessed to have low-, moderate- and high-risk of contracting COVID-19. A cross-sectional analysis was conducted for data collected over 24 weeks, from the 6A total of 1,174 healthcare workers were placed under surveillance. The majority were females (71.6%), aged between 25 and 34 years old (64.7%), were nursing staff (46.9%) and had no comorbidities (88.8%). A total of 70.9% were categorised as low-risk, 25.7% were moderate-risk, and 3.4% were at high risk of contracting COVID-19. One-third (35.2%) were symptomatic, with the sore throat (23.6%), cough (19.8%) and fever (5.0%) being the most commonly reported symptoms. A total of 17 healthcare workers tested positive for COVID-19, with a prevalence of 0.3% among all the healthcare workers. Risk category and presence of symptoms were associated with a positive COVID-19 test (p<0.001). Fever (p<0.001), cough (p = 0.003), shortness of breath (p = 0.015) and sore throat (p = 0.002) were associated with case positivity.COVID-19 symptom surveillance and risk-based assessment have merits to be included in a healthcare worker surveillance programme to safeguard the health of the workforce. Durinay 2020) . These aay 2020) . In low-ay 2020) , 13.th of January 2020 [Malaysia, an upper-middle-income country, reported its first COVID-19 patient on the 24ary 2020 . Initialary 2020 . As of mary 2020 .Following its designation as a COVID-19 hospital in Malaysia, the University Malaya Medical Centre (UMMC) COVID-19 Task Force conceived a comprehensive plan in ensuring the safety of its healthcare workforce while ensuring continuity of service. The strategies include a) cohorting COVID-19 patients to designated wards, b) development of clinical pathways and guidelines for the management of patients, c) ensuring adequate supply and adherence to PPE usage, d) extensive training of the healthcare workforce, and v) the development of a HCW surveillance programme.The HCW surveillance programme, in particular, leveraged a risk-based testing and isolating strategy that allowed for surveillance while balancing the use of resources. We aim to describe the components and implementation of the HCW surveillance programme in UMMC. In addition, we describe the distribution and characteristics of the HCWs placed under surveillance.th of March 2020 to the 20th of August 2020. Adherence to the surveillance programme by all HCWs is sanctioned as an administrative policy by UMMC to ensure the safety of its workforce. UMMC is a teaching hospital located in Kuala Lumpur and was one of the sixty hospitals designated to manage COVID-19 cases in Malaysia. UMMC has a total of 1,600 beds that caters for paediatric and adult patients, with a comprehensive range of speciality areas. Following its designation as a COVID-19 hospital, 153 beds across eight wards were allocated for COVID-19 patients, with 22 being Intensive Care Unit (ICU) beds. The hospital employed 5,826 HCWs, of which 1,987 staff were assigned to care exclusively for COVID-19 cases.This was a cross-sectional analysis of a HCW surveillance programme in UMMC over 24 weeks from the 6Ethical approval and waiver of written informed consent were obtained from the UMMC Medical Research Ethics Committee (MECID. No 202073–8862). The permission to collate data was obtained from the UMMC COVID-19 Taskforce. Dataset retrieved for this analysis did not contain any personal information, and there was no interaction with any HCWs. HCWs were not intentionally put at risk of exposure for the purpose of this study; they were existing professionals contracted to their routine work obligations at UMMC. There is standard practice for infection control for which HCWs have been trained, and appropriate PPE was provided.Fig 1 [The COVID-19 Operations Room, also known as a hospital emergency operation centre, was set up to prevent and manage COVID-19 outbreaks among UMMC HCWs through activities that focused on early detection and disease containment. The operations room workforce was categorised into four work teams, namely contact tracing, risk assessment, surveillance, and outbreak investigation. The flow of events following the identification of a COVID-19 positive case is illustrated in Fig 1 .The hospital’s Infectious Disease team notified the Contact Tracing team whenever a new COVID-19 case was identified. A new COVID-19 case was either an existing inpatient who was newly diagnosed with COVID-19, a new admission diagnosed with COVID-19 referred to UMMC, or a HCW from UMMC infected with COVID-19. The activation of the contact tracing team would initiate a cascade of activities to gather and collate information pertaining to the movement of the infected person 48 hours before the diagnosis or the onset of symptoms. HCWs who had been in contact with the infected person would be listed as new contacts, while the non-HCW contacts were managed by personnel from respective District Health Offices (DHO) under the Ministry of Health (MOH).. The contacts were then classified into four different risk categories, namely ’no identifiable risk’, ’low risk’, ’medium risk’ or ’high risk’, based on the UMMC guidelines [S2 Appendix. For example, face shield, N95 mask, gloves, coverall suit, shoe cover and proper hand hygiene are recommended for aerosol-generating procedures. Detailed management algorithm of HCWs in high-, medium-, and low-risk groups are shown in . Nasopharyngeal swabs for RT-PCR were taken based on the level of risk and presence of symptoms. Those categorised as ’no identifiable risk’ were not subjected to work restrictions nor placed under active surveillance.The list of new contacts was then relayed to the Risk Assessment team. The level of risk of the new contacts was assessed and stratified according to their risk level based on the “assessment checklist for healthcare workers” idelines . The UMMidelines . A ’low idelines . A ’mediidelines . The ’hiidelines . The recIn the next stage, HCWs classified as low, moderate, and high risk were placed under active surveillance and designated as ’Person Under Surveillance’ (PUS). Their symptoms were monitored for 14 days from the date of their last exposure to a COVID-19 case by the surveillance team. A PUS who developed symptom(s) during the surveillance period was immediately referred to the Risk Assessment team for further management.All epidemiological data were collected from the COVID-19 operation room’s centralised electronic database, which served as a digital repository for HCWs contact tracing, risk assessment and surveillance records. Additional retrievable data included sociodemographic information such as age, sex, comorbidities, and designation of the HCWs. All captured data were stored in a password-protected database which was only accessible to a few authorised personnel .th of April 2020. The outcome variable was HCWs with COVID-19, defined as a person with a laboratory confirmation for SARS-CoV-2 by reverse transcriptase-polymerase chain reaction (RT- PCR) [The exposure variables in this analysis were age, sex, job category, presence of comorbidities, risk category, and symptoms during surveillance. The symptoms, namely fever, cough, dyspnoea, sore throat, arthralgia, myalgia, gastrointestinal (GI) symptoms, and anosmia were self-reported. The symptom surveillance for anosmia was only started on the 4RT- PCR) .th of April 2020 onwards). The PUS would indicate if any symptoms were present and the type of symptoms (if any). Those who did not reply by afternoon each day were contacted by the surveillance team via text messaging or a phone call.During the initial phase, the surveillance team used an instant messaging system (WhatsApp application) to disseminate a standardised daily symptoms surveillance message to all PUS. This message enquired on any new symptom(s) for the day, any worsening of symptoms and the date of last exposure to COVID-19 patients. The selection of symptoms listed were fever, cough, breathing difficulty, myalgia, arthralgia, and any gastrointestinal symptom . The surveillance text message contained a link directing them to an online feedback form in the UMMC staff portal, and their responses were recorded in the database. Surveillance messages were sent at 6 am daily to elicit feedback on their general wellbeing. In the event that a PUS failed to reply, a second message was then sent at 10 am and a final reminder at 12 pm. Any PUS who failed to respond was contacted by the surveillance team via text messages or phone call.P <0.05. The prevalence of COVID-19 was calculated as the proportion of HCWs diagnosed as per case definition over the total number of staff in UMMC.Descriptive analysis was performed for the baseline characteristics. Pearson chi-square tests were used to compare the proportions of HCWs with and without COVID-19; Fischer’s exact tests were conducted when more than 20% of the cells had expected frequencies below five [A post hoc analysis was conducted to calculate the sensitivity and specificity of the risk-based assessment in identifying cases and non-cases. All analyses were conducted using the IBM SPSS version 23 .Fig 2. From a total of 5,826 HCWs in UMMC during the study period, almost a quarter of them  were exposed to COVID-19 infection and had their risk assessed. After excluding HCWs who had ‘no identifiable risk’ and ‘community exposure with casual contact’, a total of 1,193 HCWs were placed under surveillance, of which 1,174 HCWs had work-related exposure. All 1,174 HCWs who had work-related exposure were included for analysis.The surveillance flowchart, along with the total number of HCWs included in the study, is detailed in Tables 1 and 2, respectively. Throughout the study period, a total of 17 HCWs (12 HCWs had work-related exposure and 5 HCWs had community exposure–close contact) tested positive for COVID-19. This translates into a prevalence of 0.3%  COVID-19 case positivity among the HCW population in UMMC. As soon as each of these HCWs were tested positive, isolation measures were initiated, which constituted a 14-day admission to a COVID-19 designated ward regardless of the presence or absence of symptoms. Following these actions, no onward transmissions were recorded from infected HCWs. All HCWs who were placed under the surveillance program completed the stipulated surveillance duration. The mean daily response rate  was 96.3% (95% CI: 95.6–97.0) with a range from 88% to 100%.Baseline characteristics and surveillance outputs for all HCWs who had work-related exposure  are presented in Table 3 shows the univariable analysis for a potential association between variables of interest and case positivity of HCWs for COVID-19. There was no significant association between HCWs baseline characteristics and the positive COVID-19 test. The risk of exposure, however, was shown to be associated with positive COVID-19 tests (p<0.001). All of the 12 HCWs with work-related exposure who tested positive had at least moderate risk of exposure. Similarly, HCWs having symptoms were significantly associated with a positive COVID-19 test (p<0.001). When specific symptoms were investigated further, fever (p<0.001) and respiratory symptoms–cough (p = 0.003), shortness of breath (p = 0.015) and sore throat (p = 0.002) were found to be significantly associated with case positivity while other symptoms such as arthralgia, myalgia, gastrointestinal symptoms, and anosmia had no significant association.. The ability to correctly identify HCWs who did not have COVID-19 or the specificity was around 72%. It should be noted, however, that not all HCWs in the low-risk category were tested. From the 832 HCWs in the low-risk category, only 282 HCWs (33.9%) were tested, with all of them yielding negative results. After excluding HCWs who were not tested, the specificity of this risk-based assessment was 46.1%. (= 282/612). Thus, the result needs to be interpreted cautiously.In the post hoc analysis, the risk-based assessment employed in this surveillance was found to have a sensitivity approaching 100% in identifying positive COVID-19 cases In this article, we describe how UMMC, a teaching hospital in an upper-middle-income country, adapted a surveillance system to safeguard the welfare of its HCWs against the COVID-19 pandemic. The HCW surveillance programme leveraged a risk-based testing and isolating strategy which do not require high resources for its operation.The establishment of the Operations Room represents the cornerstone of UMMCs COVID-19 prevention, containment and mitigation strategy. Throughout the 24 weeks, teams running the operations room tried to optimise the HCW surveillance system. The technological upgrade from the WhatsApp application (Phase 1) to the online portal-based surveillance system (Phase 2) was necessary to respond to the increasing number of PUS and in anticipation of the additional workload that ensued. This was because Phase 1 of the surveillance system required manual data entry that was time-consuming and laborious. Therefore, a partially automated mechanism, i.e. Phase 2 of the surveillance system, was developed by digitalising and integrating the surveillance processes into the existing UMMC portal for HCWs. This system allowed responses from the PUS to be gathered and analysed, producing a more streamlined workflow and organised database. Furthermore, the evolution from Phase 1 to Phase 2 not only improved the efficiency and sustainability of the surveillance system but was also resource-effective.The shift from a manual to an online reporting mechanism was met with some resistance from a small number of HCWs in the early stages. We received feedback that the portal-based surveillance monitoring was tedious as it required the PUS to log onto the UMMC portal and fill up the surveillance reporting form on their own. In order to promote acceptability and compliance, continuous education and training were done to familiarise users with the portal, keeping non-respondent rates at a minimal number. We also ensured that all PUS were given an adequate explanation on how and why the surveillance was done . These aEvery PUS who failed to respond to the daily surveillance messages was contacted. For PUS who did not respond for three consecutive days, their names were submitted to the hospital management for further action. The PUS compliance was further enhanced by email reminders from the Hospital Director. The effort and resources put into developing this surveillance system bridged a service gap in a time of need and serves as an investment for the future.In this study, the characteristics of the HCWs under surveillance was comparable to a study done in Wuhan whereby the younger aged population, females and also nurses predominated . The relIt is important to note the significant heterogeneity of symptoms experienced by the COVID-19 positive HCWs in different countries , 26, 27.The unique characteristic of our risk-based assessment is that the assignment of risk categories during the risk assessment is not dependent on the symptoms. This is important as there is growing evidence on asymptomatic and pre-symptomatic infection of COVID-19 . Our risGiven its feasibility and practicality, this risk-based assessment may be suitable to be implemented in healthcare settings with high numbers of COVID-19 cases and limited resources. Other low- and middle-income countries may benefit from our risk-based assessment as challenges with COVID-19 testing such as inadequate capacity, untrained laboratory personnel, and inadequate funding have been highlighted , 13. WhiThe decision to prescribe home surveillance or no work restriction is dependent on HCWs’ risks. For example, HCWs with moderate and high-risk were put on home surveillance for 7 and 14 days, respectively, with specific protocol on testing for COVID-19. On the other hand, low-risk asymptomatic HCWs had no work restriction. The increased demand for workforce during this pandemic has posed challenges in the United Kingdom, with retired doctors being asked to consider returning to work and clinical staffs redeployed to areas of greatest need . During We have progressively developed a functional HCW surveillance system since the first COVID-19 case was admitted and made further modifications as the number of cases and new evidence surfaced. Besides that, we described the implementation of a HCW surveillance program and the surrounding issues and outputs that can be used to strengthen the program. Our study findings may potentially benefit other healthcare facilities where the resource for expanded screening is limited.As in any surveillance system, cases can be missed. Due to the risk-based assessment practice in our surveillance system, asymptomatic cases may have been missed, particularly for HCWs in the low-risk category. Airborne transmission of SARS-CoV-2 can occur under special circumstances, further raising the possibility of undiagnosed cases in our study . If testThe value of a well-designed surveillance programme and the importance to innovate methods in tandem with evolving situations is described in our article. The combination of contact tracing, risk assessment and symptom surveillance activities proved to be effective in containing the transmission of COVID-19 among HCWs while optimising the use of resources. Findings from our surveillance program suggest that risk-based assessment and symptom surveillance were associated with COVID-19 positivity, highlighting their roles and importance in a COVID-19 surveillance program to safeguard the health of the workforce in an upper-middle-income country.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S4 Appendix(DOCX)Click here for additional data file.S5 Appendix(DOCX)Click here for additional data file.S6 Appendix(PDF)Click here for additional data file.


Gray literature from religious leaders about the Great Religions were also considered. Results from these sources contributing to the knowledge base of cultural, spiritual and religious dimensions of perinatal palliative care are considered in this paper.For perinatal palliative care (PPC) to be truly holistic, it is imperative that clinicians are conversant in the cultural, spiritual and religious needs of parents. That cultural, spiritual and religious needs for parents should be sensitively attended to are widely touted in the PPC literature and extant protocols, however there is little guidance available to the clinician as to how to meet these needs. The objective of this review article is to report what is known about the cultural, spiritual and religious practices of parents and how this might impact neonates who are born with a life-limiting fetal diagnosis (LLFD). The following religions will be considered—Islam, Buddhism, Hinduism, Judaism, and Christianity—in terms of what may be helpful for clinicians to consider regarding rituals and doctrine related to PPC. Data Sources include PubMed, Ovid, PsycInfo, CINAHL, and Medline from Jan 2000–June 2020 using the terms “perinatal palliative care,” “perinatal hospice,” “cultur Perinatal palliative care (PPC) is a specialized branch of pediatric palliative care that considers an interdisciplinary strategy for the care of newborns with life-limiting or life-threatening conditions. The World Health Organization defines palliative care as the “primary goal for the provision of a good quality of life for those with life-threatening diseases.” This expands to palliative care for children and infants as a specialized field that extends to the family of children and infants. The WHO has set up principles of palliative care for children, and it includes the following points: “(a) Complete care of the infant must be taken including mind, soul, and body; (b) Moral support to the family should be provided; (c) Palliative care should start when the decision for not providing any more intensive care has been made; and (d) Care should be implemented even when resources are limited” .For PPC to be truly holistic, it is imperative that clinicians must be conversant in the cultural, spiritual, and religious needs of parents. The need for cultural, spiritual, and religious considerations to be sensitively attended to is widely touted in the literature on PPC and extant protocols; however, there is little guidance available to the clinician on meeting these needs.In recent times, most life-limiting fetal diagnoses (LLFD) are made during the prenatal period at a time that should represent hope and joy for parents and families. When faced with an LLFD, however, these hopes and dreams of a healthy child fade. It is at the margins of life and death that people consider the existential questions of life and their spirituality, and, at times, they may even question their religious beliefs. The death of their child will lead to maladaptive grief, long-term diminished quality of life, and symptoms linked to psychological morbidity if parents are unable to reconcile these very personal needs . ReligioWhile it is not possible for the clinician to be knowledgeable about all religious tenets and cultural norms encountered in practice, having the knowledge of the most common religions and how these religious tenets apply to infants with an LLFD or that are born at the margins of viability can assist parents greatly. Sadeghi et al.  identifiThe large variations in religious practice are known from the outset, and it is beyond the reach of this review to identify and accept all of these variations exhaustively. The objective of this review is to report on what is known about the cultural, spiritual, and religious practices of parents and how this might impact the neonates who are born with an LLFD. The following religions will be considered in terms of what may be helpful for clinicians to consider regarding rituals and doctrine related to PPC: Islam, Buddhism, Hinduism, Judaism, and Christianity.In addition, cultural and spiritual needs will be addressed.*,” “spiritual*,” and “religiou*.” The inclusion criteria included all empirical and research studies published in English that focused on the cultural, spiritual, and religious needs of parents who opted to continue with their pregnancy, where the fetus had a life-limiting condition or had received PPC. Results from these studies contributing to the knowledge base of cultural and religious dimensions of PPC are considered in this review. Data sources for this review included PubMed, Ovid, PsycInfo, CINAHL, and Medline. The data from January 2000 to August 2020 were collected using the terms “perinatal palliative care,” “perinatal hospice,” “culturIn addition to these published papers, sources discussing the principles of the Great Religions were also included to provide a background about religious doctrine, beliefs, and current global demographics.We live in a multicultural, multi-faith world, where religion is seemingly more important than it has ever been, with four out of five people identifying as belonging to an organized faith . ChristiReligion is somewhat simpler to define than spirituality. It considers beliefs, practices, and rituals and may also include beliefs about spirits, both good (angels) and bad (demons). Religion can be observed either publicly, as part of a community, or privately, and often as both . It is eIn a discussion paper, McGuirl and Campbell  claimed In the Southeast and East Asian community, religious and spiritual values may also directly affect the pregnancy decisions linked to genetics. Leung et al.  stated tIt is critical that clinicians are aware of the importance of inquiring about the social and religious affiliations of a patient, as these do not always correlate with the specified religious affiliation of the patient and these are important guiding principles in perinatal decision-making.Allah, translated literally as “God”). Clinicians should be aware of the Five Pillars of Islam and work with families to understand how this belief may influence their decision-making when having a baby with an LLFD. For Muslims, the Five Pillars are five broad principles that allow them to live a good and responsible life. These include the following: The Declaration of Faith (Shahada); praying five times a day ; giving charity (Zakah); fasting during the month of Ramadan (Sawm); and a pilgrimage to Mecca at least once (Hajj). It is important that healthcare providers are cognizant and do not confuse Islamic traditions with cultural traditions.Islam impacts all aspects of daily living: from eating to clothing to health practices. Islam has been pursued for more than 1,400 years, originating from Mohammad in Mecca, Arabia, who is believed to be the last prophet sent by God , the pervasive belief is that curative medical interventions come from Allah, and healthcare professionals are the medium for delivering the will of Allah. The Quran provides guidance and is considered the source of healing for psychological and spiritual distress. If family members, including infants and children, require surgical or medical measures, the Quran can be used as an addendum to resolve theological, social, and cultural needs .To facilitate decision-making, rigorous ethical codes are required that adhere to the Islamic social and legal standards . When anin utero is confirmed. The infant can receive PPC if born alive , or the sum of one's actions (and inactions) in their current and previous states of existence. The sum of these karmic actions decides their fate in future existences, influencing how they will be reincarnated and what they will be reincarnated as. Karma, however, is not isolated to Hinduism and Buddhism: in America, 27% of the population were reported to have belief in reincarnation and “some form of karma” . Comprised of 6.9% of the global population, Buddhists recognize suffering as central to human existence. Suffering is unavoidable and life-limiting illness serves to prompt reflection on the ultimate meaning of life. Death is associated with rebirth (reincarnation), and having serene surroundings at the time of death and during the dying process is important to the dignity of the person and for their rebirth . Althougf karma” . In 2014f karma” .Essentially, when the death of their infant becomes imminent, Buddhist parents need to be able to focus on caring for the spiritual state of the infant rather than unnaturally prolonging the life of their child, as this will encourage a good rebirth. It is important for medication to not interfere with consciousness. When caring for Buddhist families, the clinician will benefit from demonstrating the “right understanding”  and an understanding of mindfulness (the whole-body-and-mind awareness of the present moment), which in turn creates good karma for the child and encourages the well-being of the child. In both its present state and as a living being, this will benefit the child in the current life and for the next life to come .Christianity is a monotheistic Abrahamic faith. Its ideology is based on the life and teachings of Jesus Christ of Nazareth, practiced by over 30% of the global population. Its followers, known as Christians, include many denominations: in the Western world, Roman Catholic; Protestant ; in the East, Eastern Catholic; Eastern Orthodox; Oriental Orthodox Church of the East (Nestorian); and in Nontrinitarian, Jehovah's Witness; Latter Day Saint; and Oneness Pentecostal.In Christianity, Jesus is considered the savior, and although beliefs vary between sub-denominations, most Christians view illness as a natural process of the body and even as a testing of their faith. While the death of an infant represents profound loss, most Christians view death as being part of the will of God . Table 4In a qualitative multicenter study, Boss et al.  describePrayer is an important component for Christians, regardless of the sub-denomination, and may be directed to one or all the holy trinity: God, the Holy Spirit, and/or Jesus Christ. Baptism, which is a Christian rite of entry into Christianity, almost always through the use of water, is another important ritual, usually performed near the time of birth. According to Birgit , the onuCaulfield et al.  have recAccess to emergency baptism services is important, because, in the early stages of grief of parents whose infant had died, 37% of the parents  said that their religious beliefs were challenged and that they questioned their faith . ParentsBrahman), but other gods and goddesses are still acknowledged by their followers. In the scriptures and wisdom heritages of the Vedic tradition, however, all of them have their individual origins  is the primary purpose. Similar to Buddhism, disease and illness are considered the result of karmic actions, and death is simply a passage because the atma has no beginning or end  affords personhood to the fetus at 180 days of gestation as the embryo begins to develop awareness and beings to feel pain (Dharmashastras (the treatises of Hinduism) view the termination of pregnancy (the “killing” of a human fetus is referred to as “bhrunahatya”) as a sin; however, in less traditional Hindu families, termination may not be seen this way.In terms of infants born with an LLFD, the Clinicians should be aware that more importance is placed on the male child in Hinduism, given that they continue the lineage of the family, including the name. Therefore, it is more likely that the death of a male infant will be more acutely felt in traditional Hindu families than the death of a female infant . In the Rabbi. Psalms, as the last prayer of confession (vidui), are conducted at the bedside of the person , reconstructionist (an evolving civilization of the Jewish people), conservative , and orthodox . Jewish clergy are known as e person .Nefel”: the same word is often applied to an abortus or an infant born with major life-incompatible defects. The word Nefel, in Jewish culture, refers to an infant who lacks sufficient hair and nail growth; is born in the eighth month of pregnancy; and survives for <30 days. Relevant social rules are different for a Nefel in Judaism compared to a well term infant ; however, nowadays this is mostly untrue. . It has been reported that Jewish parents still feel a keen sense of loss and grief when an infant dies before the thirty-day benchmark .Though used interchangeably, faith and spirituality are different from each other. Spirituality is more difficult to define because there are no common characteristics that were agreed upon. It is a broader concept that each individual defines for themselves . SpirituIn an explorative study about relevant sense-making activities, whether through prayer and/or meditation, first-time mothers established two key themes to help them deal with the life-threatening illness of their infants: (a) looking for meaning circles and (b) using intuitive/spiritual experiences to find meaning . SimilarTo help parents make meaning and meet their spiritual needs when faced with perinatal death, the parents should be encouraged to consider how their infant has touched other lives besides their own; this includes other family members, particularly grandparents, who would have had their own hopes and dreams for the child. Raingruber and Milstein  propose In 2017, Hawthorne et al. explored the disparities between gender, race/ethnicity, and religion in the use of spiritual and religious coping strategies by parents after the death of a child. The study found that non-Hispanic black mothers were more likely to use religious coping strategies than non-Hispanic white mothers. Christian parents used more religious coping practices than the other groups, and mothers reported considerably more use of religious and spiritual coping practices than fathers . This stCulture refers to the collective deposit of information, experience, opinions, principles, behaviors, meanings, hierarchies, faith, notions of time, responsibilities, spatial relationships, concepts of the world, and material objects and belongings acquired through person and community by a group of people over different periods of time .In 2008, Davies et al. conducted a survey demonstrating that almost 40% of healthcare professionals cited “cultural differences” as a barrier to providing palliative care. This was particularly so among ethnic minorities whose cultures were poorly understood, including Latino, Indian, and Native and African Americans. Cultural gaps extended to language barriers, religious differences, and mistrust of healthcare providers and care of health practitioners who did not have the same racial or cultural context and who did not recognize these differences . In a stNo matter how committed, highly trained, and dedicated the healthcare team is, when caring for parents and their infant with an LLFD, an expert understanding of how families function, spiritually, cross-culturally, and existentially, presents a considerable challenge in PPC.It is not surprising that families rely on their faith and spirituality in times of crisis to help them make sense of the path to the end of life. This analysis revealed why healthcare practitioners need to become familiar with the definitions of the moral and religious ideas that arise in their clinical field or specialization for parents and families. Regardless of the religious, spiritual, or cultural beliefs, all parents facing the death of their infant due to an LLFD require a compassionate, supportive, and knowledgeable healthcare service. Life and death are momentous occasions in the life cycle, and when the two occur in close proximity, beliefs and religious ceremonies become even more profound. All members of the healthcare team providing care to parents from the moment of the diagnosis through the palliation of to death of the infant require a unique skill set, including the ability to pay attention to, and the knowledge of, religious, spiritual, and cultural needs of the parents.VK conceptualized and designed the review, drafted the manuscript, and is solely responsible for data collection and interpretation of the literature data.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lower limb alignment is crucial in total knee arthroplasty (TKA). Previous studies have shown that robotics and personalized three-dimensional (3D) preoperative planning could improve postoperative alignment accuracy compared with conventional TKA, but comparison between the above two techniques has never been reported. The authors hypothesized that robotics may be superior to personalized 3D preoperative planning in terms of postoperative alignment in primary TKA, with similar patient-reported outcome measures (PROMs) but higher cost and longer operative time.A consecutive series of patients who received TKA in our center from September 2020 to January 2021 were enrolled retrospectively. After 1:2 matching, 52 and 104 patients were included and divided into study group for robotics and control group for personalized 3D preoperative planning, respectively. Multiple postoperative alignment angles were measured, and clinical features such as operation and tourniquet time, length of hospital stay and hemoglobin (Hb) were recorded. Knee Society Score (KSS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) were used to evaluate clinical results.P < 0.05). It also had less outliers in hip–knee–ankle angle (HKA), FTC, lateral femoral component angle (LFC) and lateral tibial component angle (LTC) (P < 0.05). Hb loss of robotics group was significantly lower than control group (P < 0.001), while the operation and tourniquet time were longer (P < 0.001). There was no significant difference in KSS and WOMAC scores between two groups.Compared with control group, robotics group had significantly lower frontal femoral component angle (FFC) and frontal tibial component angle (FTC) absolute deviation (Compared with control group, patients in robotics group had significantly less malalignment, malposition, Hb loss, but similar PROMs. The operations in robotics group spent longer operation time and cost more compared with control group.Trial registration: The Chinese Clinical Trial Registry, ChiCTR2000036235. Registered 22 August 2020, http://www.chictr.org.cn/showproj.aspx?proj=59300.III.The online version contains supplementary material available at 10.1186/s13018-022-03115-3. Substantial studies have demonstrated that accurate alignment and prosthesis positioning in total knee arthroplasty (TKA) are closely related to satisfactory postoperative outcomes and prosthesis longevity –5. AlthoThe alignment and prosthesis positioning accuracy among navigation, PSI, robotics and conventional TKA were compared in the previous large sample meta-analysis , 14. A BTo achieve accurate alignment and prosthesis positioning with a cost-effective method, we reported previously a verified technique which used personalized 3D preoperative planning . It coulThe previous study confirmed that personalized 3D preoperative planning could improve resection accuracy compared with conventional TKA , while tMedical records and imaging data were retrospectively collected from a consecutive series of TKA performed in our center from September 2020 to January 2021. The robotics group included patients with knee osteoarthritis, who underwent TKA with the Skywalker™ robotics system ; the 3D preoperative planning group included patients with knee osteoarthritis who underwent TKA with personalized 3D preoperative planning. Patients failed to achieve pre- and postoperative full-length weight-bearing radiographs (FLX) or did not meet Paley’s criteria  were excβ = 0.95, α = 0.05). The power analysis by PASS 15.0 revealed that 48 in robotics group and 96 in 3D preoperative planning group would be an appropriate sample size. To reduce the influence of selection bias and potential confounding factors in this retrospective study, the gender, left or right, age, body mass index (BMI) and preoperative hip–knee–ankle angle (HKA) were selected to perform a 1:2 matching with the “nearest” method by R software . Finally, 52 robot-assisted TKAs and 104 personalized 3D preoperative planning TKAs were compared in this study.Previous literatures indicated that malalignment rates of robotics and 3D preoperative planning after TKA was about 2% and 18%, respectively , 19, 20. or the previous article [The personalized 3D preoperative planning  and Legi1 article  for moreThe Skywalker™ robotics system and Advance® medial-pivot knee prosthesis  were used in robotics group. A patient-specific 3D model was formulated automatically after importing the patients' lower limb CT data into the Skywalker™ robotics system. Multiple feature points were marked in the 3D model, such as the center point of femoral head, knee joint and ankle joint, the most prominent point of lateral femoral epicondyle, and the most concave point of medial femoral epicondyle. Then, the appropriate prosthesis positioning and alignment parameters were selected in preview to form a preoperative planning. During the surgery, the navigation markers made by radix lens  were installed and the patient's anatomical characteristics were registered to fit the preoperative plan. Under the help of optical measurement technology, the robotic arm with a cutting guide at the distal end automatically moved to the appropriate position and assisted the surgeons to complete accurate resection with saw.In both groups, nerve block anesthesia and medial parapatellar approach were conducted. A tourniquet was applied before skin incision and released after the closure of joint capsule. In order to reduce total blood loss, tranexamic acid was routinely used. No patella replacement was performed, and all cases were conducted following mechanical alignment. Discharge criteria included that there were no obvious swelling, no extension lag, active bending ≥ 90°, walking distance with assistance ≥ 200 m and VAS pain score ≤ 4. Patients who met all the above criteria could be discharged. The hospital stay included postoperative rehabilitation programs education.2), operation time (min), tourniquet time (min), hospital stay (day) and hemoglobin (Hb) loss at 1 and 3 days after operation (g/L) were collected via the electronic medical record management system. Meanwhile, KSS and WOMAC scores were obtained preoperatively and 3, 6, 12 months after operation during outpatient follow-ups. Based on the guidance [Preoperative FLX (within 1 month before surgery) and FLX of the latest follow-up were collected. Preoperative HKA and postoperative frontal femoral component (FFC) angle, frontal tibial component (FTC) angle, lateral femoral component (LFC) angle, lateral tibial component (LTC) angle and HKA  was used to evaluate intra-rater and inter-rater consistency in FLX measurement. ICC values less than 0.5, between 0.5 and 0.75, between 0.75 and 0.9, and greater than 0.90 were indicative of poor, moderate, good and excellent reproducibility, respectively [P < 0.05 was considered statistically significant.The ectively . StatistAll baseline characteristics were similar between two groups after 1:2 matching (Table P < 0.05). The postoperative HKA and FFC were significantly different between two groups (P < 0.05), while the two groups had similar postoperative FTC, LFC and LTC. The two groups had similar absolute deviations from the target value in HKA, LFC and LTC, but the robotics group was significantly better in FFC and FTC absolute deviation (P < 0.05). Besides, the robotics group had significantly less outliers compared with the 3D preoperative planning group in HKA, FTC, LFC and LTC (P < 0.05)  Table .Table 2CP < 0.001), while the robotics group had lower Hb loss at 1 and 3 days after operation (P < 0.001)  and less outliers in HKA, FTC, LFC and LTC (P < 0.05), compared with the personalized 3D preoperative planning group  (Table The Hb loss at 1 and 3 days after operation was significantly lower in robotic group (P < 0.001) , which is the only prostheses that the robotics system could recognize. Although prostheses types differences had no effect on the comparison of alignment accuracy between robotics and the personalized 3D preoperative planning, this made the comparison of postoperative outcomes a little confused because various prosthesis designs might influence PROMs –34. SecoRobotics could significantly improve alignment accuracy, but the expensive start-up costs  discourCompared with 3D preoperative planning group, patients in robotics group had significantly less malalignment, malposition, Hb loss, but similar PROMs. The operations in robotics group spent longer operation time and cost more compared with 3D preoperative planning group.Additional file 1: Detailed procedure of the personalized 3D preoperative planning.


Molecular markers play an important role in predicting clinical outcomes in pancreatic adenocarcinoma (PAAD) patients. Analysis of the ferroptosis-related genes may provide novel potential targets for the prognosis and treatment of PAAD.RNA-sequence and clinical data of PAAD was downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) public databases. The PAAD samples were clustered by a non-negative matrix factorization (NMF) algorithm. The differentially expressed genes (DEGs) between different subtypes were used by “limma_3.42.2” package. The R software package clusterProfiler was used for functional enrichment analysis. Then, a multivariate Cox proportional and LASSO regression were used to develop a ferroptosis-related gene signature for pancreatic adenocarcinoma. A nomogram and corrected curves were constructed. Finally, the expression and function of these signature genes were explored by qRT-PCR, immunohistochemistry (IHC) and proliferation, migration and invasion assays.The 173 samples were divided into 3 categories  and a 3-gene signature model  was constructed. The prognostic model showed good independent prognostic ability in PAAD. In the GSE62452 external validation set, the molecular model also showed good risk prediction. KM-curve analysis showed that there were significant differences between the high and low-risk groups, samples with a high-risk score had a worse prognosis. The predictive efficiency of the 3-gene signature-based nomogram was significantly better than that of traditional clinical features. For comparison with other models, that our model, with a reasonable number of genes, yields a more effective result. The results obtained with qPCR and IHC assays showed that ALOX5 was highly expressed, whether ALOX12 and CISD1 were expressed at low levels in tissue samples. Finally, function assays results suggested that ALOX5 may be an oncogene and ALOX12 and CISD1 may be tumor suppressor genes.We present a novel prognostic molecular model for PAAD based on ferroptosis-related genes, which serves as a potentially effective tool for prognostic differentiation in pancreatic cancer patients.The online version contains supplementary material available at 10.1186/s12935-021-02431-8. Pancreatic cancer has a low 5-year survival rate and is one of the cancers that have high mortality . There wFerroptosis, a type of cell death, plays a vital role in inhibiting tumorigenesis by removing cells that either has a deficiency or overabundance of key nutrients or cells damaged by environmental pressure . Unlike Recently, research has focused on the immune infiltration and its role in cancer. Previous studies have demonstrated that infiltrating immune cells can be isolated from tumors suggesting that tumor immune infiltration is a crucial biological process of cancers . FurtherWith the development of next-generation sequencing technology, gene transcription profiles in PAAD can be better understood. Based on The Cancer Genome Atlas (TCGA) and the comprehensive Gene Expression Omnibus (GEO) databases, many genes be found contribute to the development of PAAD. Recent studies have shown that CA9, CXCL9, and GIMAP7 genes specifically regulate the expression of FoxO1, thereby regulating immune infiltration in PAAD . FurtherIn this study, we identified ferroptosis-associated genes and constructed molecular subtypes of PAAD based on TCGA and GEO databases. Finally, we established a 3-gene signature prognostic model and verified its ability to predict the prognostic risk and immune infiltrates of PAAD. This serves as a potentially effective tool for prognostic risk prediction in patients with pancreatic cancer.Expression data and corresponding clinical follow-up information from the TCGA-PAAD data set were downloaded using the UCSC genome browser database. GSE62452 chip data sets with survival time were selected from Gene Expression Omnibus (GEO) database. The GSE62452 data set is Microarray gene-expression profiles of 69 pancreatic tumors and 61 adjacent non-tumor tissue from patients with pancreatic ductal adenocarcinoma. ([HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array [transcript (gene) version]). Henceforth, samples that lacked clinical follow-up information were removed and the expression of multiple gene symbols was considered the median value.Firstly, 60 ferroptosis-related genes were retrieved from the literature –18  method of the GSVA  package was used to identify the immune score-based relationships among the molecular subtypes in the TCGA-PAAD data set. The scores of 28 immune cells were assessed  and thenA total of 173 samples in the TCGA-PAAD data set were divided into a training set and a test set. To prevent the random allocation bias from affecting the stability of subsequent modeling, all samples were put back into random grouping, two hundred times in advance. Using the training data set, a univariate Cox proportional risk regression model was constructed for ferroptosis-related genes (n = 60) and survival data was constructed using the coxph function with survival R package. Since 58 ferroptosis-related genes had expression profile data in our data set, only these were selected for the univariate Cox regression analysis, and P < 0.05 was selected as the threshold for filtering. The R software package “glmnet_4.10–1”  was usedThe risk scores of each sample were calculated separately based on the expression level of the sample. KM-curve analysis showed significant differences between the high and low expression groups. Furthermore, we used the R software package timeROC_0.4  to conduTo identify the independence of the 3-gene signature model in clinical applications, we performed Cox regression analysis on the TCGA-PAAD training dataset. Based on the results of univariate and multivariate analyses, we used the TCGA-PAAD training dataset to construct a histogram. In addition, corrected curves were used to analyze the prediction accuracy of nomogram at 1, 3, and 5 years.PAAD tissues were derived from surgically resected specimens and snap-frozen in liquid nitrogen until RNA extraction. None of the patients received chemotherapy or radiation therapy before surgery. All patients signed informed consent forms provided by the Eastern Hepatobiliary Surgery Hospital. This study was approved by the Ethics Committee of the Eastern Hepatobiliary Surgery Hospital.RNA was extracted from tissues using the TRIzol reagent  and was reverse-transcribed into cDNA using the QuantiTect Reverse Transcription Kit . Quantitative PCR (qPCR) uses real-time fluorescence to measure the quantity of DNA present at each cycle during a PCR. Real-time qPCR analyses were quantified with SYBR-Green , and expression levels were normalized to GAPDH levels.Immunohistochemistry was performed by two-step method according to the instructions . Pancreatic cancer samples were fixed in 10% formalin, embedded in paraffin, and processed into 5-µm sequential sections. The samples were de-waxed with ethanol and blocked to inhibit the endogenous peroxidase activity. After this, samples were heated in a microwave for antigen retrieval, cooled to room temperature, and blocked using goat serum for 30 min at 37 °C. The samples were incubated overnight at 4 °C with rabbit anti-ALOX5 (ab169755), anti-ALOX12 (ab211506), and anti-CISD1 (ab203096)  (1:200), followed by incubation with horseradish peroxidase-coupled goat anti-rabbit secondary antibody  at 37 °C for 30 min. The samples were then stained with 3,3′-Diaminobenzidine (DAB). Cell nuclei were stained blue with hematoxylin. The sections were then dehydrated, cleared with xylene, and mounted. ALOX5, ALOX12, and CISD1 expressions were determined by immunohistochemistry (IHC) using the streptavidin peroxidase method, with adjacent tissues serving as the controls. The experimental procedure was performed as per the manufacturer’s instructions. Image-Pro Plus 6.0 Software  was used to analyze protein expression and perform statistical analysis of the results obtained by IHC.2 in a humidified incubator. The Panc 02.03 cell lines are derived from human primary pancreatic cancer and are epithelial-like cells. The Panc 02.03 cell lines were cultured in RPMI-1640  with 15% fetal bovine serum at 37 °C under 5% CO2 in a humidified incubator. Si-ALOX5 (No: CAT#: SR319325) was purchased from Origene . Transfection was performed using Lipofectamine 3000 reagent  according to the instructions and cell transfection efficiency was 82%.The human ALOX12 and CISD coding sequences were cloned into the pEZ-M03 Vector.The human PAAD cell line T3M4 and Panc 02.03 were provided by the National Collection Authenticated Cell Cultures . The T3M4 cell lines are derived from the metastatic lymph node tissue of human pancreatic cancer and are epithelial-like cells. The T3M4 cell lines were cultured in DMEM (Dulbecco’ modified eagle medium) . Supplemented with 10% fetal bovine serum  at 37 °C under 5% COThe si-ALOX5 transfected in T3M4 cell lines, and ALOX12 or CISD transfected in Panc 02.03 cell lines. Forty-eight hours post infection, the cells were collected and seeded into 96-well plates at a concentration of 2000 cells per well. Cell viability was detected by Cell Counting Kit-8 assay  according to the manufacturer’s protocol after 48 h. Te absorbance at 450 nm was measured using an automatic microplate reader . All Cell Counting Kit-8 assay were performed in five times.5 cells were added in the upper chamber. The cells were harvested, resuspended in serum-free media and placed into the upper chamber of a Transwell membrane filter  for the migration assays or in the upper chamber of a transwell membrane filter coated with Matrigel (Corning) for the invasion assays.The T3M4 and Panc 02.03 cells were added in the upper chamber. After incubation for 24 h at 37 °C, the Transwell chamber was removed. The cells were stained with methanol and 0.1% crystal violet, imaged, and the relative cell density was measured by ImageJ . ImageJ software was used to analyze and calculate the migration and invasion area of the cells. The migration and invasion index (%) depicts a proportion of an area where cells have invaded in percentage and is calculated as epithelium area divided by the total area of invaded cells area. The area describes the overall area (μm2) of invaded cells. Evaluation of invasive capacity was performed by counting invading cells under a microscope (40 × 10). Five random fields of view were analyzed for each chamber. All cell migration and invasion assays were performed in five times.The si-ALOX5 transfected in T3M4 cell lines, and ALOX12 or CISD transfected in Panc 02.03 cell lines. Forty-eight hours post infection, the cells were collected. For the migration assay, 800 μl DMEM with 20% serum was added to the lower chamber of a Transwell plate , and 1.5 × 10All data were analyzed using the SPSS 21.0 statistical software program . Graphs were generated with GraphPad Prism 8.0 software . Student’s t-tests were performed. For a two-tailed t-test, P < 0.05 was essential for considering the results to be statistically significant.After data preprocessing, there were 173 samples in TCGA-PAAD, with 88 samples in the training set and 85 samples in the test set. GSE62452 included 66 samples. The clinical statistical information of the samples is shown in Table The 173 samples were divided into 3 categories: C1, C2, and C3. The expression of prognostic ferroptosis-related genes in the three categories is shown in Fig. With the help of the limma_3.42.2 package, 4,903 DEGs were identified between Cluster 1 and Cluster 2, among which 2,717 genes were up-regulated and 2,186 genes were down-regulated  (cell markers are from the reference ) and theThe R package survival coxph function is used to perform a univariate Cox proportional hazard regression model for ferroptosis-related genes (n = 60) and survival data in training set. Because only 58 ferroptosis-related genes are expressed in our expression profile data and we selected these genes for single-factor cox regression analysis, and selected P < 0.05 as the threshold for filtering. Finally, there were 10 genes related to prognosis. Next, LASSO regression was used to further compress the 10 genes to reduce the number of genes in the risk model. The changing trajectory of each independent variable is shown in Fig. RiskScore = 0.289* ALOX5 + (− 1.359)*ALOX12 + (− 1.053)* CISD1.Further, we calculated the risk score of each sample based on the expression level of the sample. In TCGA-training set, KM-curve analysis showed that there were significant differences between the high and low-risk groups Fig. C. The riAnalysis of the GSE62452 showed that the risk score distribution was consistent with that of the training set and that the high-risk score samples had a worse prognosis . It is the link that connects metabolism, redox biology, and human health. Accumulating evidence indicates that ferroptosis can be triggered to treat cancer to eradicate aggressive malignancies that are resistant to conventional therapies . It is cDue to the insidious and aggressive nature of PAAD, it is difficult to detect and prevent PAAD at an early stage. At the time of consultation, approximately 80% of patients have locally advanced or metastatic cancer . AlthougThe signature we constructed contained three genes, namely, ALOX5, ALOX12, and CISD1, all of which are closely related to tumors genesis and development. 5-Lipoxygenase (ALOX5) is a non-heme iron-containing dioxygenase that catalyzes the peroxidation of polyunsaturated fatty acids, such as arachidonic acid . ALOX5 iIn this study, we present evidence that ALOX5, ALOX12, and CISD1 may have prognostic value in PAAD. Specifically, our in vitro results suggest that ALOX5 may be an oncogene, while ALOX12 and CISD1 may be tumor suppressor genes in PAAD. The results of our study indicate that the established 3-gene signature model could be an effective prognostic tool for patients with PAAD. However, the limitations associated with this study emphasize the need for additional analysis before the clinical application of this signature. The samples used in our study were obtained retrospectively and the study solely focused on the prognostic value and clinical significance of ferroptosis. To validate our findings for clinical applications, we need to include prospective samples and evaluate the prognostic values of other biological processes characteristic to the development of cancer.In conclusion, we propose a 3-gene signature  predictive model based on ferroptosis-related genes in PAAD. Despite the many drawbacks of the current analysis, this model may serve as an interesting molecular diagnostic tool to assess the prognosis and possible risk factors of PAAD.Additional file 1.  Different expressed genes (DEGs) between three clusters.Additional file 2.  KEGG pathway analysis and GO functional enrichment analysis.


Arabidopsis thaliana, RELATIVE OF EARLY FLOWERING 6 (REF6)/JUMONJI DOMAIN-CONTAINING PROTEIN 12 demethylates H3K27me3 by recognizing a specific DNA motif. However, little is known about how REF6 activates target gene expression after recognition, especially in environmental responses. In response to warm ambient temperature, plants undergo thermomorphogenesis, which involves accelerated growth, early flowering and changes in morphology. Here we show that REF6 regulates thermomorphogenesis and cooperates with the transcription factor PHYTOCHROME INTERACTING FACTOR 4 to synergistically activate thermoresponsive genes under warm ambient temperature. The ref6 loss-of-function mutants exhibited attenuated hypocotyl elongation at warm temperature, partially due to downregulation of GIBBERELLIN 20-OXIDASE 2 and BASIC HELIX-LOOP-HELIX 87. REF6 enzymatic activity is necessary for warm ambient temperature responses. Together, our results provide direct evidence of an epigenetic modifier and a transcription factor working together to respond to the environment.Dynamic trimethylation of histone H3 at Lys27 (H3K27me3) affects gene expression and controls plant development and environmental responses. In  Arabidopsis, the main H3K27me3 demethylases are EARLY FLOWERING 6 (ELF6/JMJ 11) [H3K27me3, a conserved and facultative repressive histone mark found in euchromatin, is crucial for tissue-specific gene expression and developmental regulation in multicellular eukaryotes ,2. The d/JMJ 11) , JMJ13 [/JMJ 11)  and JMJ3/JMJ 11) ,11. ThesFLOWERING LOCUS C by the transcription factor BRASSINAZOLE-RESISTANT1 [Despite their functional redundancy, these H3K27me3 demethylases have distinct targeting mechanisms and biological functions . For insSISTANT1 , where iSISTANT1  and prevSISTANT1 ,14; DNA SISTANT1 ,16. REF6SISTANT1 , organ bSISTANT1 , leaf seSISTANT1  and seedSISTANT1 . InteresSISTANT1 , abscisiSISTANT1 , ethylenSISTANT1  and brasSISTANT1 . HoweverPlants have evolved diverse strategies to respond to natural environmental fluctuations ,24. AmbiYUCCA 8, TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 and CYTOCHROME P450 FAMILY 79B) [DWARF4 and BRASSINOSTEROID-6-OXIDASE 2) [FT promoter to mediate flowering responses [SPEECHLESS to restrict stomatal development under warm conditions [The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) acts as a central regulatory hub in thermomorphogenetic responses . PIF4 meILY 79B) ,30 and BIDASE 2)  to mediaesponses , and binnditions . In addinditions ,27.Arabidopsis [FLOWERING LOCUS C promoter [Multiple regulatory mechanisms are involved in the extensive network of plant thermoresponsive growth, including transcriptional regulation, RNA metabolism and protein stability . Among tbidopsis ,38. The bidopsis . JMJ13 abidopsis . JMJ30 ppromoter , and funpromoter .These reports revealed that histone methylation controls the balance between vegetative and reproductive growth at warm ambient temperature . Howeverref6 mutants is insensitive to warm ambient temperature. We then used hypocotyl elongation as a system to investigate the function of REF6 in targeting, H3K27me3 demethylation and activation of target genes. Three-day-old seedlings grown at 22°C were transferred to 22°C or 28°C for 3 days in long-day conditions  and REF6 complementation lines , were recorded after 3 days of treatment at 22°C or 28°C  at 22°C, the mutants exhibited markedly attenuated responses at 28°C compared with the wild-type seedlings . REF6-HA transgenic lines rescued the attenuated hypocotyl elongation of the ref6 mutants at 28°C -specific demethylases, namely JMJ11/ELF6 ,13, JMJ1REF6 expression and REF6 targeting activity at warm ambient temperature. The mRNA levels of REF6, as measured by reverse transcription-quantitative Polymerase Chain Reaction (PCR) (RT-qPCR), did not change at 22°C and 28°C , suggesting that REF6 expression is not induced by warm ambient temperature. To test whether elevated temperature affects the recruitment of REF6 to its target genes, we profiled the genome-wide localization of REF6 in wild-type Col using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) with anti-REF6 antibody [ref6-5 mutant was included as a negative control. Two biological replicates of REF6 ChIP-seq showed a high correlation  with each other . A total of 1954 and 2136 peaks covering 1834 and 1981 genes were bound by REF6 at 22°C and 28°C, respectively. These were highly correlated (r = 0.98) and overlapped  with each other . These results demonstrated that REF6 transcript levels, REF6 targeting and REF6 enzymatic activity are temperature insensitive.We then checked the H3K27me3 levels in wild-type Col and ref6 mutant influence target gene expression at warm temperatures. We therefore carried out an RNA sequencing (RNA-seq) analysis, using RNA from seedlings of wild-type Col and ref6-5 mutant treated at 22°C and 28°C (Supplementary Table 3). A total of 74 million (79%), 76 million (80%), 77 million (82%) and 76 million (82%) clean reads of highly correlated RNA-seq replicates were uniquely aligned to the Arabidopsis reference genome, and were mapped to 17 092, 16 981, 17 189 and 17 127 genes in the wild-type Col and ref6-5 mutants at 22°C and 28°C, respectively Reads Per Kilobase Million (RPKM > 1) (Supplementary Table 3). RNA-seq data from three replicate samples showed high correlation .Because REF6 is an H3K27me3 demethylase and H3K27me3 is associated with gene repression, we next investigated the relationship between REF6 targeting and gene activation, and whether the increased H3K27me3 levels in the ref6-5 mutants at 22°C and 28°C . For this analysis, we first calculated the fold change of transcript levels comparing 28°C to 22°C in wild-type Col and ref6 mutants  in wild-type Col at 28°C compared with 22°C, and inefficiently induced in ref6 mutants  encodes an enzyme that is involved in GA biosynthesis and temperature responses [BASIC HELIX-LOOP-HELIX 87 (bHLH87) was reported as a PIF4 downstream target in Arabidopsis [GA20ox2 and bHLH87 were upregulated by elevated temperature in wild-type Col, but were inefficiently induced in ref6-5 mutants with high levels of H3K27me3  with an independent batch of ChIP samples yielded consistent results . Evaluation of hypocotyl elongation at warm temperature in the wild type, double mutants and the corresponding parental lines showed that both ga20ox2 and bhlh87 are thermo-insensitive, similar to the ref6-5 mutant  that abolishes its enzymatic activity  [REF6 transcript levels comparable to those of wild-type Col were chosen for further analysis . Phenotypic analysis showed that REF6 H246A-HA ref6-1 transgenic lines did not rescue the attenuated hypocotyl elongation of the ref6 mutants at 28°C  . Transge8°C Fig. A and B, bHLH87 and GA20ox2 . These results indicate that REF6 and PIF4 cooperate with each other to regulate some thermoresponsive genes.The bHLH transcription factor PIF4 accelerates hypocotyl growth at warm temperatures and acts as a central regulatory hub in ambient-temperature signaling, in which various signaling pathways modulate PIF4 activity, and PIF4 integrates hormone signaling in thermoresponsive regulation of growth ,26,28. BbHLH87, we performed electrophoretic mobility shift assays (EMSAs) using DNA fragments from bHLH87 that contain the CATATG and CTCTGTTT motifs . The DNA probes containing the CATATG motif and CTCTGTTT motif were incubated with maltose-binding-protein (MBP)-tagged PIF4 (PIF4-MBP) and Glutathione-S-transferase (GST)-tagged C-terminal REF6 (GST-REF6C). Consistent with the ChIP-seq data, PIF4 and REF6 can directly bind to bHLH87 DNA probes . Genetic analysis showed that pif4-101 ref6-5 plants displayed similar phenotypes to pif4-101 plants . Thermoresponsive hormone signaling in different plant organs should be coordinated to facilitate synchronized growth and acclimation to elevated temperatures [Plants actively sense temperature and, under warm ambient temperatures, they promote the biosynthesis and localization of signaling molecules, such as auxin, BR and GA, to optimize plant morphology in a coordinated manner ,47. Our bHLH87 locus under elevated temperature suggest that bHLH87 activation is regulated by both H3K27me3 demethylation and the thermoresponsive transcription factor PIF4, but neither REF6 nor PIF4 alone can activate bHLH87 expression  and DNA methylation ,16. Hereion Fig. . The tar2/sec. Seedlings from each sample were scanned using ZEISS SteREO Discovery V20, and the hypocotyl length was measured using ImageJ software .Surface-sterilized seeds were sown on half-strength Murashige and Skoog (MS) medium supplemented with 0.8% (w/v) agar and 1% (w/v) sucrose. The plates were stratified at 4°C in darkness for 3 days, then transferred into a growth chamber  and germinated at 22°C for 3 days, then transferred to 28°C (treatment) or maintained at 22°C (control) at ZT0 (Zeitgeber Time) for another 3 days under a 16-h light/8-h dark cycle. The plants were grown under white fluorescent light with a fluence rate of ∼75 μmol/mAbout 2.5 g of seedlings was used for ChIP assay, as previously described ,16, usinArabidopsis thaliana TAIR10 reference genome using Bowtie2 (version 2.2.1) [https://deeptools.readthedocs.io/en/develop/) with the ‘–normalizeUsing RPKM –extendReads 200 –binSize 50’ options. Then the output BigWig files were visualized using the Integrative Genomics Viewer [All sequencing reads of ChIP-seq were aligned to the n 2.2.1)  with defn 2.2.1) . After fn 2.2.1) , only uns Viewer . MACS2 vs Viewer  was useds Viewer . Then ths Viewer  when a sq-value < 0.05, log2 (fold change) > 2.For RNA-seq data, all reads were mapped to the TAIR10 reference genome using HISAT2 (version 2.0.5)  with defChIP-seq and RNA-seq data from this article can be found in the Gene Expression Omnibus data library under accession number GSE181292.nwab213_Supplemental_FilesClick here for additional data file.