Can a behavioral nudge delivered through text messages with a reserved date for vaccination over a 2-week period accelerate employee adherence with a health system COVID-19 vaccination policy?In this randomized clinical trial of 2000 participants, the behavioral nudge delivered through text messages significantly increased adherence to the health system COVID-19 vaccination policy by 4.9 percentage points compared with the control group during the 2-week intervention period. At the 4-week time point near the vaccination policy deadline, there was no longer a significant difference in the overall adherence rate between groups.This randomized clinical trial found that a behavioral nudge delivered by text message with a reserved date for vaccination accelerated adherence to a health system COVID-19 vaccination policy; however, other approaches may be needed to change overall adherence rates by the time of the policy deadline. This randomized clinical trial evaluates use of a text message behavioral nudge to accelerate adherence to a health system’s COVID-19 vaccination policy. Many organizations implemented COVID-19 vaccination requirements during the pandemic, but the best way to increase adherence to these policies is unknown.To evaluate if behavioral nudges delivered through text messages could accelerate adherence to a health system’s COVID-19 vaccination policy.This randomized clinical trial was conducted within Ascension health system from October 11 to November 8, 2021. Participants included health system employees in the Midwest or South US who were not adherent with the vaccination policy 1 month before its deadline. Data were analyzed from November 17, 2021, to February 25, 2022.Participants were randomly assigned to control or to receive a text message intervention that stated a vaccine had been reserved for the participant, with a scheduled date for vaccination within a 2-week period. Participants could reschedule to a different date within the period or upload a copy of their vaccination card. Follow-up text message reminders were sent the day before and the day of the appointment.The primary outcome was adherence to the health system’s vaccination policy during the 2-week intervention. Secondary outcomes included time to vaccination during a 4-week follow-up period.P = .02). Among participants who became adherent by the end of the 4-week follow-up period, the text message nudge significantly reduced time to adherence by a mean of 2.4  days (P < .001) and a median of 5.0  days (P < .001) compared with the control group. At 4 weeks, overall vaccination adherence was no longer different between groups .The sample included 2000 participants , with 1000 participants randomized to the control group and 1000 participants randomized to the intervention group. Overall, there were 164 Hispanic participants (8.2%), 46 non-Hispanic Asian participants (2.3%), 202 non-Hispanic Black participants (10.1%), and 1418 non-Hispanic White participants (70.9%). By the end of the 2-week intervention, 363 participants in the text message nudge group (36.3%) and 318 participants in the control group (31.8%) were adherent with the vaccination policy, representing a significant increase of 4.9  percentage points in adjusted analyses comparing the nudge group with the control group (This randomized clinical trial found that a behavioral nudge delivered through text messages accelerated adherence to a health system’s COVID-19 vaccination policy but did change overall adherence by the time of the policy deadline.NCT05037201ClinicalTrials.gov Identifier:  VHA employees who did not obtain vaccination by the deadline were suspended.9 In November 2021, the Department of Labor’s Occupational Safety and Health Administration (OSHA) expanded this policy to organizations with 100 or more employees, and the Centers for Medicare & Medicaid Services (CMS) announced a new requirement that all health care workers at CMS-covered facilities be vaccinated.10 By the Fall of 2021, more than 150 health systems across the United States had announced new policy requirements for their employees to receive COVID-19 vaccination. While there is precedent for vaccine mandates,11 acceptance among the general public has varied.7More recently, policy makers and large employers have implemented mandates for employees to receive COVID-19 vaccination. On July 29, 2021, President Biden signed an Executive Order that federal employees had to become vaccinated or participate in weekly COVID-19 testing.14 While these efforts have been a tremendous success, several challenges remain. First, increasing vaccination rates may still take time as many mandates provide 4 or more months between their announcement and their deadlines. Second, organizations need to plan to replace staff who chose to leave instead of getting vaccinated, which can be difficult to predict until very close to the mandate deadline. From an employer perspective, vaccine adherence includes the employee both being vaccinated and submitting documentation, or otherwise having an approved vaccination exemption. These steps are often delayed. Therefore, there is a significant need for new and innovative approaches to accelerate adherence to a COVID-19 vaccine mandate.Early reports indicated that these vaccine mandates increased employee vaccination rates to greater than 90% at a number of organizations.15 Nudges have been demonstrated to increase other types of vaccination, such as influenza.16 A randomized clinical trial conducted at a university found that emailing participants a default time and location for vaccination (opt-out condition) led to a 36% relative increase in vaccination compared with individuals who were sent an email to schedule an appointment (opt-in condition).17 In another study, patients from 2 large health systems were sent text messages in the days preceding a visit with their primary care clinician.18 The trial randomly assigned patients to receive 1 of 19 different text messaging approaches or to be in a control group. On average, nudges sent by text message increased vaccination by 5% compared with the control group, and several of the top-performing messages used the phrase “the vaccine has been reserved for you,” with up to an 11% increase in vaccination. This phrase leverages the principle of loss aversion, which has found that individuals are more motivated to avoid losing something they feel is already theirs than to do an equivalent amount of work to gain a similar benefit. Nudges have also been used for COVID-19 vaccination efforts. In a randomized clinical trial conducted at UCLA Health, researchers found that telling patients a vaccine was available to them and they should claim it increased COVID-19 vaccination by 6% compared with a control group.19 However, this trial was implemented earlier in the pandemic, when demand for vaccination exceeded supply. Therefore, we lack good evidence on how to use nudges among individuals who may be more vaccine hesitant.Nudges are subtle changes to the way information is framed or how choices are offered that can have an outsized impact on behavior.In this study, our objective was to conduct a randomized clinical trial to evaluate whether a behavioral nudge sent by text message could accelerate adherence to a health system’s policy requiring COVID-19 vaccination. The text message provided the participant with an appointment date for vaccination, with options to change the date or upload vaccination documentation if they had already been vaccinated. The intervention was conducted in the month before the vaccination policy deadline and targeted a more vaccine-hesitant group of employees.CONSORT) reporting guideline.The protocol for this randomized clinical trial was approved by the Ascension St. Vincent Institutional Review Board and is available in This was a 2-arm randomized clinical trial conducted among 2000 health system employees from the Midwest and South regions of the United States who were not adherent with a policy requiring COVID-19 vaccination announced by Ascension health system on July 27, 2021, with a deadline of November 12, 2021. Ascension is 1 of the largest nonprofit health systems in the United States and operates more than 140 hospitals. The trial was conducted in the final month before the deadline and included a 2-week prespecified intervention period , during which participants in the intervention were notified by text message of a default appointment date for COVID-19 vaccination. The prespecified secondary outcome followed adherence rates for an additional 2-week follow-up period until November 8, 2021. Prior to the intervention, all participants received communications about the vaccination requirement through multiple channels, including email, but not through text messages. Employees not adherent by the deadline would be placed on suspension.Eligible participants included health system employees who were identified as having not submitted documentation of vaccination by October 5, 2021, the time data were obtained for randomization. Participants were classified as physicians, physician support roles, nursing, nursing support roles, or other type of employee. Data on participant characteristics were obtained from human resources (HR) records, and data on vaccination adherence were obtained from Associate and Occupational Health records at Ascension’s health systems. Race and ethnicity were self-reported in HR records and categorized as Hispanic, non-Hispanic Asian, non-Hispanic Black, non-Hispanic White, and other  or unknown.From the available list of employees who were not adherent and with a documented phone number and email address on record, we randomly selected 2000 for participation in the trial. From this group, we randomly assigned half to the control group and the other half to the intervention group using an electronic randomization process. All investigators and statisticians were blinded to group assignment until the trial and analysis plan were completed.Prior to the initiation of the trial, all health system employees were informed of the vaccination requirement and the potential for job suspension or termination for nonadherence by November 12, 2021. These communications began when the mandate was implemented on July 27, 2021, and were delivered mostly by email and the intranet, as well as verbal communication during meetings with their hiring manager or supervisor. These types of communications continued during the trial for both groups. The control group received no other interventions.The intervention group received an initial text message on October 11, 2021, stating that as an employee of the health system, vaccination was required; that a vaccine has been reserved for them with a specific date during the upcoming 2 weeks; and indicating they could come in at any time during hours of operation. This was followed by a confirmation text message. Examples of the text messages are available in the eFigure in The primary outcome measure was adherence to the vaccine policy by the end of 2 weeks after the start of the intervention. This measure required that an employee received at least 1 dose of a vaccine and submitted documentation of vaccination. We selected 2 weeks because the default appointment was set for within 2 weeks of the initial text message. Secondary outcome measures were time to vaccine documentation submission (mean and median days) within 4 weeks of the start of the intervention.A priori power calculations estimated that a sample of 1450 participants would provide at least 90% power to detect a 5–percentage point difference in the adherence rate between the intervention and control groups. This assumed a baseline adherence rate of 20% and used a 2-sided α = .05.20 The model was adjusted for employee job role , which was the only participant characteristic that differed significantly between randomized study arms. A quantile regression model was used to estimate adjusted differences in median days to adherence and bootstrapping with 200 cycles was used to estimate 95% CIs.22All randomly assigned participants were included in the intention-to-treat analysis. Unadjusted analyses tabulated the percentage of participants in each group who became adherent over time. To obtain the adjusted differences in adherence in percentage points along with 95% CIs, we fit a linear probability model using the participant as the unit of analysis.t tests or χ2 tests, as appropriate. We also conducted a per-protocol analysis excluding participants who were adherent prior to intervention implementation or those granted a vaccine exemption by the policy deadline. We used 2-sided hypothesis tests with a significance level of P = .05. All analyses were performed in R statistical software version 4.0.5 . Data were analyzed from November 17, 2021, to February 25, 2022.We conducted exploratory subgroup analyses by participant characteristics with In this randomized clinical trial, 2000 health system employees were randomized, with 1000 participants in the intervention group and 1000 participants in the control group . ParticiP = .003). In the adjusted intention-to-treat analysis during the 2-week intervention period, 363 intervention participants (36.3%) were adherent by the end of the period, compared with 318 control participants (31.8%), a significant absolute increase of 4.9  percentage points in vaccination adherence in the text message nudge group compared with the control group (P = .02), representing a 15.4% relative increase. No adverse events were reported during the trial.When the intervention began, the overall vaccination adherence rate at the health system was 83.7%. P = .007)  who either became adherent before the intervention was implemented or had a vaccine exemption approved by the policy deadline, with a 6.0  percentage point increase in the text message nudge group compared with the control group (P < .001) and a median of 5.0  days (P < .001) compared with the control group  days (ol group . The perIn this randomized clinical trial, a nudge delivered by text message stating the COVID-19 vaccine had been reserved for them on a specific appointment date significantly increased adherence to the health system vaccination policy during the 2-week intervention. The nudge also significantly accelerated the time to adherence within 4 weeks. These findings demonstrate the potential impact of using nudges to enhance timely adherence to a vaccine requirement. However, overall adherence did not differ by the time of the vaccination policy deadline, indicating that other approaches may be needed.18 However, it differs from a UCLA Health study that used text message nudges for COVID-19 much earlier in the pandemic among a larger group of individuals who had not been vaccinated and who were likely not as vaccine hesitant.19 Importantly, the 15.4% relative increase in vaccination adherence within this trial was larger in magnitude than the 5% relative increase in influenza vaccination found in the study by Milkman et al18 and the 6% relative increase in COVID-19 vaccination found in the study by Dai et al,19 each of which were likely implemented in less vaccine-hesitant populations than in our study.Our findings have several important implications for policy makers and large employers who have implemented or are considering a mandate for COVID-19 vaccination. First, our work demonstrates that behavioral nudges can be effective for accelerating vaccination adherence among a more vaccine-hesitant population. The COVID-19 vaccine was first made available to health care workers in late 2020; therefore, nearly a year had passed and the participants of this randomized clinical trial had not been vaccinated. Our work builds on previous nudge interventions that framed influenza vaccination as opt-out and told participants the vaccine was reserved for them.Second, the behavioral nudges implemented in this trial were particularly important for accelerating the time to adherence. For large employers, faster adherence can offer both a safer environment for the workforce and better management of future staffing needs. An employee can face delays in both obtaining vaccination and submitting documentation. The intervention prompted action by providing a prespecified date for vaccination but also enabled employees to reschedule if another date worked better for them. Once vaccinated, participants could use the link in the text message to upload a picture of their vaccination card. Since most previous communications were by email or verbally, this text message approach may have offered enhanced salience. These vaccination reservations may have also simply made it easier for participants to follow through with vaccination, which indicates that health systems should consider looking for ways to improve structural changes to the delivery of health care services.14 this demonstrates the strong association of these types of policies with increasing vaccination. However, it also demonstrates that many employees will put off vaccination unless prompted earlier, highlighting the importance of using behavioral nudges to improve the speed of adherence. These findings also indicate that other approaches may be needed to change overall adherence rates when interventions are implemented close to the policy deadline. Future research could evaluate these types of approaches when implemented closer to the mandate announcement than its deadline.Third, in the final week before the vaccination policy deadline, approximately 15% of the sample became adherent, and rates between groups became similar. Similar to prior reports,23 Adherence to booster vaccination has been low.24 Behavioral nudges could also provide an important approach to address this issue.Fourth, the ongoing COVID-19 pandemic and new strains of the virus indicate that regular vaccination may be needed for some period. In November 2021, the Centers for Disease Control and Prevention recommended that all adults receive a booster vaccination at least 3 months after completing the initial series.Our study has limitations. First, it was conducted among employees from within the same health system, which could limit the generalizability of the results to other populations. Second, we had a shorter follow-up period, given that the trial was conducted in the last month before a COVID-19 vaccination requirement deadline. Third, we focused on adherence to the health system vaccination policy, which includes both obtaining vaccination and submitting documentation. Participants obtaining vaccination outside of Ascension facilities could have delayed submitting documentation. However, from an employer perspective, adherence relied on documentation submission. We did not have data on participants who received vaccination but did not submit documentation of it. Fourth, adherence was prespecified as obtaining the first COVID-19 vaccination dose, and some vaccines require a second dose to complete the series. This was designed to be aligned with health system adherence policies. Fifth, we were unable to quantify how many participants had phone numbers that were outdated or a landline, thereby preventing them from receiving the text message nudge. However, this represents a pragmatic implementation of the approach and would bias our results toward the null.The findings of this randomized clinical trial suggest that behavioral nudges delivered through text messages offered a scalable approach to accelerate adherence and could be adopted more broadly to improve COVID-19 vaccination efforts. However, other approaches may be needed for interventions implemented close to the policy deadline to impact overall adherence rates.


The relationship between nurse staffing levels and patient safety is well recognised. Inadequate provision of nursing staff is associated with increased medical error, as well as higher morbidity and mortality. Defining what constitutes safe nurse staffing levels is complex. A range of guidance and planning tools are available to inform staffing decisions. The Society for Acute Medicine (SAM) recommend a ‘nurse-to-bed‘ratio of greater than 1:6. Whether this standard accurately reflects the pattern and intensity of work on the Acute Medical Unit (AMU) is unclear.Nurse staffing levels in AMUs were explored using the Society for Acute Medicine Benchmarking Audit 2019 (SAMBA19). Data from 122 acute hospitals were analysed. Nurse-to-bed ratios were calculated and compared. Estimates of the total nursing time available within the acute care system were compared to estimates of the time required to perform nursing activities.The total number of AMU beds across all 122 units was 4997. The mean daytime nurse-to-bed ratio was 1:4.3 and the mean night time nurse-to-bed ratio was 1:5.2. The SAM standard of a nurse to bed ratio of greater than 1:6 was achieved in 99 units (81.9%) during daytime hours and achieved by 74 units (60.6%) at night.The estimated time required to deliver direct clinical care was 35,698 h. A deficit of 4128 h (11.5% of time required) was estimated, representing the time difference between the total number of nursing hours available with current staffing and the estimated time needed for direct clinical care across all participating units.This UK-wide study suggests a significant proportion of AMUs do not meet the recommenced SAM staffing levels, particularly at night. A difference was observed between the total number of nursing hours within the acute care system and the estimated time required to perform direct nursing activities. This suggests a workforce shortage of nurses within acute care at the system level. Acute Medical Units (AMUs) are a core component of the acute care pathway in the UK . The AMUNursing on the AMU provides a unique set of challenges. High rates of patient turnover, in combination with high levels of patient acuity, create a demanding environment enriched for risk. Year-on-year increases in the number of emergency medical admissions and seasonal surges in demand compound this problem , 12. LowAcknowledgement of the importance of staffing levels has not translated into a clear understanding of what constitutes safe and adequate staffing. The Society for Acute Medicine (SAM) has published relevant guidelines, recommending nurse-to-bed ratios of 1:4 for patients on trolleys and 1:6 for patients in beds . While tThe Society for Acute Medicine Benchmarking Audit (SAMBA) has run annually since 2012, and by convention takes place on the penultimate Thursday in June . SAMBA rRecruitment to SAMBA19 was open to all hospitals in the UK receiving acutely unwell  medical patients. Non-acute and community hospitals were excluded. SAMBA19 was undertaken on Thursday 27th June 2019, between 00:00 and 23:59. SAMBA19 was the first SAMBA survey to collect data regarding nurse staffing levels and skill-mix.SAMBA is a cross-sectional audit with an observational design. Patients aged 16 years or above who were seen for either admission or assessment as part of the internal medicine admission process (‘take’) or ambulatory emergency care (AEC) were eligible for inclusion. Patient level data collection was performed voluntarily by local clinicians, overseen by a named responsible consultant physician. The SAMBA protocol is publicly available .Prior to the collection of patient level data, a lead acute medicine clinician at each participating AMU completed an online questionnaire to provide organisational and unit level data, relating to staffing levels on the AMU. Data on staffing levels could only be submitted once from each AMU. The questions regarding nursing numbers, level of nursing experience and the specific roles performed by staff were developed by a group of nurses affiliated with SAM. Total number of nurses present on the AMU during the 24-h data collection window was measured, with nurse-to-bed ratios during daytime (08:00–19:59) and nighttime 20:00–07:59) hours calculated using the reported number of available beds within each AMU (occupied and unoccupied). The number of non-registered nurses, band 5 nurses, band 6 nurses and band 7 or above nurses were recorded separately [:00–07:59The proportion of units which achieved the SAM standard was calculated. In order to determine whether the use of non-registered nurses was higher in units with fewer registered nurses, the ratios of registered to non-registered nurses were compared between units with nursing levels above and below the recommended SAM standard.The unit and patient level data collected were used to generate an estimate of the total number of nursing hours available within all contributing AMUs. This estimate was compared with an estimate of the anticipated time required to complete nursing tasks informed by the relevant NICE guidance on safe staffing levels . The totTo estimate the amount of nursing time required to complete tasks at the system level. This was achieved by multiplying the anticipated number of occupied beds, or the number of AMU admissions, by the proportion of patients deemed likely to require each nursing intervention and the time the time taken to perform each nursing intervention as stated by the NICE guidelines.These estimates were made under multiple assumptions. A bed-occupancy rate of 90% was used to inform the estimate, consistent with national averages during the study period . It was All admitted patients were assumed to require administration of medications (including assessment of whether ‘as required’ medications were needed), 80% were assumed to require administration of intravenous medication . IntraveThe administration of oral and parenteral medications and clinical tasks associated with care coordination were deemed appropriate for registered nursing staff in accordance with NMC guidance . Tasks sSAMBA19 collected paired unit and patient data from 130 AMUs. Complete data relating to nurse staffing and workforce planning was provided by 122 AMUs (93.8%), with individual patient level data collected for 5849 patients seen through these units, 8 AMUs did not provide data on nurse staffing levels and were not included in the analysis.The most common working pattern, employed by 70 units (57.4%) was exclusive 12-h shifts. A combination of 12-h and 8-h shifts were implemented in 51 units (41.8%). Only one unit used solely 8-h shifts. During the daytime, 1159 nurses were on duty; this included 913 staff nurses and 246 senior nurses (ratio 3.7:1). At nighttime, 970 nurses were on duty; this included 801 staff nurses and 169 senior nurses (ratio 4.7:1).Nurse-to-bed ratios were calculated. The total number of AMU inpatient beds across all 122 units was 5028. The mean daytime nurse-to-bed ratio was 1:4.7 and the mean nighttime nurse-to-bed ratio was 1:5.7). The SAM standard of a nurse-to-bed ratio of greater than 1:6 was achieved in 104 units (85.2%) during daytime hours and achieved by 80 units (65.6%) at night. The variation in nurse to bed ratios at day and night is provided in Fig. The number of non-registered nurses on duty during the evaluation period was recorded. During the day, 562 non-registered nurses were on duty, a ratio of registered to non-registered nursing nurses of 2.1:1. At night, 466 non-registered staff were on duty, a ratio of registered to non-registered nurses of 2.1:1. In AMUs with staffing levels above the SAM standard these ratios were 2.1:1 and 2.1:1 during the day and night respectively. The corresponding ratios in AMUs with staffing levels below the SAM standard were 1.8:1 and 1.5:1. The ratio of non-registered to registered nurses at daytime and night time is provide in Fig. The total amount of registered nursing time available during the 24-h evaluation period was 22,344 h, assuming 75% of the total workforce were working a standard 12-h shift and the remainder 8-h shifts (assuming mandated break periods were observed). The total amount of non-registered nursing time was 10,280 h, assuming the same shift pattern.The total amount of time required to complete direct nursing tasks was estimated. The calculations and assumptions used to derive this estimate are provided in Table There was a total deficit of 2738 h between the estimated time required to deliver all care and the actual time available. An additional 238 non-registered nursing staff working 12 h shifts would be required to meet the predicted deficit.SAMBA19 provides insight into nurse staffing levels in acute medical care at the system-level. The results suggest a short-fall in staffing levels within the 24-h study period. SAM recommend a nurse to bed ratio of 1:6, but this was not achieved in approximately 20% of AMUs during daytime hours. Performance against the standard was considerably poorer at night. Nighttime staffing was also characterised by a lower ratio of band 6 or higher nurses to more junior colleagues.Estimates of the time required to perform direct clinical tasks, based on current NICE guidelines, suggest a significant gap between the time required to deliver care and the total number of nursing hours available. These estimates only calculate the nursing time required for direct clinical care. The provision of direct clinical care does not include time between tasks or documentation, which represent a significant proportion, if not the majority, of time used in delivering nursing care. A previous observational study using a validated tool to measure working patterns and the time distribution of nursing tasks suggested only a fifth of available time was devoted to activities involving direct patient contact . The stuNurse to bed ratios in isolation are recognised as a poor measure for staffing requirements as they do not adequately account for the multitude of factors that determine whether demand and supply are adequately matched , 29. NICThe deficit in the number of hours to provide care was driven by tasks that could be appropriate for non-registered nurses. Classification of tasks on this basis is a simplification of reality. It would be expected that a proportion of a registered nurses time would involve delivering basic care in addition to more complex tasks which would be inappropriate for non-registered colleagues. Although our estimates suggest a surplus of hours to perform tasks only appropriate for registered nurses, this did not account for the indirect time required to perform these tasks, such as documentation, collecting of equipment and communicating with other members of the medical team. Not accounting for this indirect time is likely to have led to a significant under-estimate of the time required to conduct these tasks. Increasing the number of non-registered nurses may help reduce some of the gap between anticipated workload and available staff but is unlikely to provide a solution to inadequate staffing levels in isolation. High ratios of non-registered to registered nurses have been linked to higher mortality during in-patient care . The devThe NHS staff survey highlighted the relationship between overstretched staff with excessive workloads and poor morale . These wWe provide a system-wide estimate of the mis-match between nurse staffing levels and the time required to deliver basic nursing care. We used broad generalisations which are likely to under-appreciate variation between AMUs. The assumptions used to estimate the total number of nursing hours required to complete direct clinical care are likely to lack precision. The proportion of patients predicted to require each intervention was based on a subjective assessment informed by staff nurses and clinicians with experience working in acute work. The same is true of the estimates of time required to complete tasks, although these values were taken directly from the relevant NICE guidelines. The estimates within the NICE guidelines may not accurately represent reality. While these limitations are likely to affect the accuracy of our findings, the large disparity in the estimate of available versus required nursing time to deliver acute medical care supports the conclusion that current nursing levels are insufficient. We used conservative estimates in relation to bed occupancy which may have underestimated demand. The single day-of-care methodology employed by SAMBA does not account for seasonal and diurnal variation or the weekend affect which are known to affect demand on the AMU .Our findings assume all nursing time is devoted to direct clinical care, this ignores the time required to document case-records, find equipment or undertake unexpected tasks. Therefore, we contend that any inaccuracies in our study findings underestimate the extent of the problem. Our findings provide a system level description of nurse staffing levels at the system level but our study design precludes meaningful analysis of how nurse staffing levels impact on clinical care, patient experience and outcomes.There are approximately 225 AMUs within the UK; our evaluation therefore represents 58% of AMU’s within the NHS . There iThis UK-wide study suggests that despite reasonable compliance with current SAM staffing levels, when based on a nurse per bed ratio, in reality acute medical care faces a staffing crisis among nurses. There is not enough nursing time within the system to complete direct clinical tasks, as predicted by our study findings. Ultimately our findings highlight the need for an increase in the number of nurses working in AMUs. A concerted effort to recruit and retain nursing staff is urgently required. A valid fear would be declining quality in patient care, especially if the current situation deteriorates further.


The COVID-19 pandemic has a unprecedented impact on the way individuals make sense of the interconnected nature of themselves in relation to the world. This study investigated the mediating role of transcendental awareness and compassion in the association of interconnectedness with a sense of civic duty and collective action participation during COVID-19 using a longitudinal design. A total of 336 young adult participants were recruited at baseline and were asked to complete measures of interconnectedness, transcendental awareness, compassion, civic duty, and collective action participation at three time points over a 6-month period. Path analysis was used to test the hypothesized mediation model. The results showed that compassion fully mediated the positive association between interconnectedness and collective action participation and partially mediated the positive association between interconnectedness and civic duty. Transcendental awareness also partially mediated the positive association between interconnectedness and civic duty but not collective action participation. This study highlighted the potential of interconnectedness in promoting civic duty and engagement in collective action through transcendental awareness and compassion during the COVID-19 pandemic. The coronavirus COVID-19) pandemic that began in 2019 is said to cause more “mass trauma” than World War II [9 pandemiDisasters, whether natural or human-caused, or other incidents of mass trauma such as a worldwide pandemic can cause a myriad of direct  and indirect  consequences on affected populations . COVID-1In fact, emerging evidence suggested that the pandemic brought about a sense of community among fellow citizens ,11. To dCOVID-19 has shed light on the interconnected nature of all phenomena, raising individual and societal awareness on how our behaviors can affect each other. This is consistent with ideas espoused by existential positive psychology, whereby individuals tend to re-negotiate their assumptions about the world when confronted by adversities that threaten our understanding of the self and our place in the world ,17. IndeAdopted from Buddhist philosophy, interconnectedness has been operationalized as “an awareness that the existence of all phenomena in the world is the result of the fulfilment of different causes and conditions, in which no entity can substantiate independently without relying on other factors” . Cross-sTranscendental awareness is having the realization and appreciation of the intricate nature of oneself in relation to the world at multiple perspectives  during the COVID-19 pandemic. It is a perception and understanding of the world that is beyond one’s self-interest, with a sense of connectedness and relationships with other individuals and ecology in the world . Having Compassion is an approach-oriented emotion that entails a feeling of care toward those who are suffering and a motivation to relieve the suffering ,34. AccoBased on the literature reviewed, the present study aimed to investigate the relationship between interconnectedness, transcendental awareness, compassion, civic duty, and collective action participation in the context of the COVID-19 pandemic using a 6-month longitudinal design across three time points. We hypothesized that interconnectedness at baseline is associated with greater transcendental awareness at Time 2 (3rd month) and greater compassion at Time 2 (3rd month), which in turn is associated with greater civic duty and collective action participation at Time 3 (6th month).The present study is part of a larger research study named Mindful Flourishing that aimed to promote mindfulness and flourishing among young adults in Hong Kong through a mindfulness ambassador program and a mindfulness-related mobile application . Upon obA total of 336 participants gave their informed consent after they were given written instructions and explanation of the study online. The response rates of these 336 participants were 90% (Time 2), 82% (Time 3), and 80% (Time 4). Since 31 participants only completed the baseline questionnaires but not any of the subsequent time points, they were excluded from analyses, resulting in a final sample size of 305. The present study used the first three waves of data to test the mediation model. The age range of the participants was from 18 to 38 years old ; 258 (85.7%) of them were female; 237 (78.0%) of them indicated their education level as undergraduate and 22 (7.4%) of them as postgraduate; 212 (70.4%) of them had no religion while 9 (3.0%) of them were Buddhist and 77(25.7%) were Christian or Catholic; 165 (54.8%) of them had never tried any mindfulness exercises before they participated in the study.Participants were asked to complete the following questionnaires at all time points except that the measure of transcendental awareness was administered only from Time 2–4.The 12-item Interconnectedness Scale  that wasEight items in Chinese were constructed by the authors to examine respondents’ transcendental awareness over the past three months during COVID-19. The items were assessed on a 6-point scale on degree of agreement . Composite score was obtained by averaging the scores of all eight items. Higher scores indicated greater agreement on having thoughts about the interconnected nature between people and society across the globe during COVID-19. The items are developed based on the interconnected conditions raised in previous commentaries about the pandemic ,13,14,15The 5-item Santa Clara Brief Compassion Scale (SCBCS)  was usedThe 12-item civic duty subscale from the Active and Engaged Citizenship Scale  was usedThe Collective Action Scale was adapted from the scale used among collective action for sexual minorities in Hong Kong . The 14-Analysis on Pearson correlation among the study variables was conducted using SPSS (Version 25). Path analysis was used to test the hypothesized mediation model using Mplus Version 8.7 . Intercop < 0.05 and their BC 95% CI did not include zero, the hypothesized mediating effects were supported [Bootstrapping technique was used to test the hypothesized indirect effects of the mediation model . About 5upported . To evalupported . If bothupported ,55. MissThe bivariate associations between the mindfulness practice experience, interconnectedness, transcendental awareness, compassion, civic duty, and collective action participation were investigated by Pearson correlation. The results showed that mindfulness practice experience was significantly associated with the four studied variables, while most of the studied variables were significantly correlated with one another. Given the significant association of mindfulness practice experience with other variables, past studies have shown that mindfulness is associated with interconnectedness, compassion, and self-transcendence ; mindful2 (1) = 2.60, p = 0.11; CFI = 0.99; TLI = 0.91; RMSEA = 0.07; SRMR = 0.02  and with compassion at Time 2  were both significant  and collective action participation at Time 3  remained significant. After controlling for interconnectedness at Time 1 and compassion at Time 2, the association of transcendental awareness at Time 2 remained significantly associated only with civic duty at Time 3  but not collective action participation at Time 3 . The direct effect of interconnectedness at Time 1 on civic duty at Time 3 , but not on collective action participation at Time 3 , was significant. Result suggested satisfactory goodness-of-fit for the mediation model: χR = 0.02 . The asscant see . After cβ = 0.14, p < 0.001, 95% CI 0.09 to 0.21) and collective action participation at Time 3  through compassion at Time 2 were all significant. Through transcendental awareness, the indirect effect of interconnectedness at Time 1 on civic duty at Time 3 , but not on collective action participation at Time 3  was significant. Altogether, the results implied that compassion and transcendental awareness partially mediated the association between interconnectedness and civic duty. Furthermore, compassion fully mediated the association between interconnectedness and collective action participation.The specific indirect effects of interconnectedness at Time 1 on both civic duty (The present study investigated the mediating roles of transcendental awareness during the pandemic and compassion in the relationships of interconnectedness with civic duty and collective action participation on a sample of young adults longitudinally over a 6-month period. Our findings supported the potential of transcendental awareness and compassion as mechanisms underlying the effect of interconnectedness on a sense of civic duty and compassion on the effect of interconnectedness on collective action participation for young adults in Hong Kong. The mediation model was generally supported and several major findings are worth highlighting. First, this study supported the positive relationship between interconnectedness and civic duty. This is consistent with the findings from Yu et al.  that fouThe results suggested that these mechanisms warrant further investigation to examine how we can more effectively motivate one to contribute to the common good in the society. This might be achieved by examining the distinct contributions between interconnectedness, transcendental awareness, and compassion on prosociality and collective action using experimental induction or specific interventions . In addiThe present study carries several practical implications. Past research has shown contemplative practices such as mindfulness, loving-kindness, or compassion meditation to increase positive emotions, social connectedness, decrease mental distress and physical stress as indicated by biomarkers ,61,62. TThe results should be interpreted with its limitations in mind. First, transcendental awareness during COVID-19 is measured using self-constructed items that were not previously validated. Second, this study was conducted with a group of young adults who are predominantly female, thus, generalizability of the results should be made with caution. The study was also correlational in nature and could at best illuminate on temporal relationships; causality cannot be drawn. Notwithstanding these limitations, our study provided valuable insights into understanding how interconnectedness may be related to being compassionate and having transcendental awareness that may arise during the pandemic and how they may be harnessed to improve a sense of civic duty and encourage collective action participation.At the time of writing, the world is still adapting to the COVID-19 pandemic. With a new virus variants spawning, the pandemic continues to pose uncertainties and expose vulnerabilities in our communities ,65. Our


In their comment, Anenburg and Guzmics (hereafter A&G) describe one experiment performed to assess whether they could replicate the observed immiscible carbonatite liquid from the Laacher See volcano1. Their experimental run product shows that the starting material did not melt, that the run was quite obviously subsolidus and, consequently, contained no carbonatitic liquids. However, the absence of melt can be explained by the experimental techniques A&G used. We were surprised to see that the experiment was run in a piston cylinder apparatus with uncalibrated assemblies, very similar to those used in a previous experimental study2. Piston-Cylinder assemblies suffer from large friction, especially at low run temperatures, and this is the reason why most modern experimental laboratories use these assemblies only at run temperatures of well above 1000 °C, and only after careful calibration . There is a plethora of information about different piston cylinder assemblies and the associated problems with friction in the literature . The use of uncalibrated piston-cylinder assemblies without friction correction led A&G to substantially underestimate the actual pressure of their run. As the solidus and the liquidus of a volatile element-rich system critically depend on pressure, we are not surprised that the single experiment of A&G did not melt at all. However, we agree with A&G that experiments are needed to map out the new immiscibility gap between phonolitic melts and SiO2-rich Ca-carbonatites1. However, these are difficult experiments that need to be well documented and they need to be carried in a well-calibrated high-pressure apparatus.In a recent paper, we present evidence for immiscible Ca- and Si-rich carbonatite and phonolite melts that were found in hauyne-hosted melt inclusions from the Laacher See volcano in the Eifel, Germany2. However, several experimental studies clearly show that crustal carbonatite melts can easily contain between ≥12  and 30 wt.% of SiO2 . It is quite obvious that these experimental results, and our observations at the Laacher See volcano1, do not agree with a simplistic model  that aims to explain the origin of Ca-rich carbonatites, in essence, by loss of Na (and gain of Si amongst other elements) from a parental Na-rich carbonatite melt to the surrounding wall rocks. Whilst we do not claim that this process may not occur in nature at all, our findings simply imply that the model may not be applicable to all carbonatites and our observations clearly show that SiO2-rich carbonatite melts are stable at crustal conditions. However, more research on these melts is needed and future studies may show that the validity of previous genetic models of crustal carbonatites need to be reconsidered.A&G claim in their comment that carbonatite melts at crustal pressures and temperatures cannot contain more than 5% SiOFurthermore, A&G speculate that our reported carbonatite analyses suffered from analytical artefacts, namely alkali-loss, mixed analyses of phonolite-carbonatite melts and hauyne, and droplets of silicate in the carbonatite melt.some of the melt inclusions reported by ref. 1 show the incomplete physical separation of silicate and carbonate melts as they represent early stages of liquid immiscibility. However, A&G falsely conclude that all carbonatite melts contain such silicate droplets and that all analyses could be contaminated. This is clearly not the case. The high z-contrast between silicate and carbonatite melt allows accurate identification of carbonatite (Lc) droplets in silicate glass (Ls) and vice versa. Consequently, we strictly avoided analysing “contaminated” carbonatite blebs and hence there was no need for corrections of any kind. The existence of pure, silicate droplet-free carbonatite melt blebs as identified by high-resolution backscattered electron imaging is exemplarily demonstrated by high-resolution FE-microprobe maps confirming the absence of Ls droplets (melt inclusions (i) and (ii) in Fig. not analysed.As to the droplets, Given the small size, most of the carbonatite blebs were analysed using EPMA spot sizes of 2–5 µm, short counting times (5 s) and a beam current of 10 nA. As migration of alkalis during analysis can indeed be a problem, we recorded the peak signals of Na and K, as well as Si and Ca, on multiple carbonatite blebs as a function of time and spot size. Figure 3 and a hauyne contribution of 10% to the overall carbonatite signal would result in SO3-contents of >1.6 wt.%, clearly above the Lc average SO3 concentration of 0.38 wt.%1. A significant contribution of hauyne to the carbonatite analyses can thus be ruled out.Although A&G do not report quantitative analyses of the phases identified in their single subsolidus experiment, they claim to have found hauyne and raise the possibility that hauyne might have also contributed to our carbonatite analyses. We want to stress that we never found any hauyne inclusion in neither the carbonatite nor the silicate part of the melt inclusions. Nevertheless, as hauyne is the host mineral of the melt inclusions care must be taken to avoid mixed analyses. The hauyne contains around 12 wt.% SOFinally, A&G criticise the fact that the immiscible carbonatite and phonolite melts are very different from previously reported immiscible melt pairs, but this is exactly what makes our new findings so exciting and we believe the immiscible melts from the Laacher See volcano will spur further research into the origin of carbonatites.


During the COVID-19 first wave in France, the capacity of intensive care unit (ICU) beds almost doubled, mainly because of the opening of temporary ICUs with staff and equipment from anaesthesia.We aim to investigate if the initial management in temporary ICU is associated with a change in ICU mortality and short-term prognosis.Retrospective single-centre cohort study.Surgical ICU of the Bichat Claude Bernard University Hospital during the COVID-19 “first wave” (from 18 March to 10 April 2020).All consecutive patients older than 18 years of age with laboratory-confirmed SARS-CoV-2 infection and/or typical radiological patterns were included during their first stay in the ICU for COVID-19.Patients were admitted to a temporary ICU if no room was available in the classical ICU and if they needed invasive mechanical ventilation but no renal replacement therapy or Extracorporeal Membrane Oxygenation (ECMO) in the short term. The temporary ICUs were managed by mixed teams (from the ICU and anaesthesiology departments) following a common protocol and staff meetings.ICU mortalityAmong the 59 patients admitted, 37 (62.7%) patients had initial management in the temporary ICU. They had the same characteristics on admission and the same medical management as patients admitted to the classical ICU. ICU mortality was similar in the 2 groups . SAPS-II and ECMO use were associated with mortality in multivariate analysis but not admission to the temporary ICU.In an overload context of the ICU of a geographical area, our temporary ICU model allowed access to intensive care for all patients requiring it without endangering them. During the first coronavirus disease 2019 (COVID-19) wave (March-May 2020), intensive care units (ICU) in many areas of Europe were overwhelmed by patients developing severe hypoxemic pneumonia and acute respiratory distress syndrome (ARDS) . To manaFrench authorities activated the national crisis plan (Plan Blanc)  to postpThis exceptional situation and numerous organizational changes required to create temporary ICUs have led to questions concerning the quality of the care provided. This issue has been minimally addressed in the literature to date, but some studies suggest that ICU overflow or a high rate of occupancy of beds compatible with invasive mechanical ventilation are associated with ICU mortality , 11.This study aimed to evaluate whether the initial management of patients admitted for COVID-19 in the temporary ICU was associated with a modification of the short-term prognosis.th to April 10th, 2020).This retrospective monocentric study was conducted on a cohort of patients admitted to the surgical ICU for COVID-19 during the “first wave” at the Bichat-Claude Bernard University Hospital in Paris  was provided by the French Institutional Review Board  on 21 December 2020. This committee waived the need for signed informed consent. The French law  mentions that no specific permission or license is required for observational retrospective studies. This was the case for the current investigation which did not modify the physician’s management of the patients.All consecutive patients older than 18 years of age with laboratory-confirmed SARS-CoV-2 infection and/or typical radiological patterns were included during their first stay in the intensive care unit for COVID-19. Lung transplant patients and postoperative patients for whom a therapeutic limitation had previously been decided were excluded from this analysis.Two groups of patients were considered depending where they were admitted in: classical ICU or temporary ICUPatients admitted to the temporary ICU were selected according to our predefined criteria Fig. . The rulTemporary ICUs were opened in the recovery room (12 beds) and in 2 operating theatres (4 beds in each room). Therefore, all the temporary ICU beds were opened in common rooms. The beds were not ICU beds but standard beds. The respirators were ICU ventilators (Dräger Evita 4®) and anaesthesia ventilators (Dräger Primus® and Perseus®). Cushions for ventral decubitus installation were ordered in emergency. Renal replacement therapy (RRT) could not be performed in the temporary ICU because no specific water circuit was available and no nurse was trained on this technique. ECMO could be started in temporary ICUs with a subsequent transfer to the classical ICU for care continuation because of a lack of space between beds in temporary ICUs and because nurses were not trained in this technique.The classical ICU comprised 8 ICU beds and 6 continuous care beds upgraded to ICU beds dedicated to COVID-19-infected patients (while maintaining 4 ICU beds for non-COVID-19 patients). This upgrading did not imply any structural modification but only modified the patient/nurse ratio. The ventilators used were Dräger Evita V500®, Dräger Evita XL®, Dräger Evita 4®, Dräger Evita 2 dura®, Dräger Savina®, and General Electric Carescape R860®.At the peak of the crisis, 34 ICU beds for COVID-19 patients were opened in our service. We kept 4 ICU beds for non-COVID-19 patients during the entire period.National health authorities organized ICU patients by interregional transfer to increase available beds in overcrowded areas . Our serA senior intensive care physician supervised each sector (comprising 6 to 12 beds) supported by a team of physicians comprising senior intensive care physicians from other sectors  and anaesthesia and intensive care residents from all listed sectors. The senior supervisor remained in the same area throughout the crisis. In summary, on a weekday, a senior intensive care physician took care of 6 to 12 patients, assisted by senior anaesthesiologists and / or residents who each took care of 3 patients.Duty lists for night shifts had to be reinforced: as usual, the senior and resident physicians of the ICU managed the classical ICU (18 beds), emergency calls and admissions regulation. The senior anaesthesiologist duty list was kept for surgical emergencies. The on-call senior anaesthesiologist duty list was transformed into a duty list for temporary ICUs in the recovery room (12 beds). A resident duty list was created for the temporary ICU in the recovery room. A senior duty list was created for the temporary ICU in the operating theatre (maximum 8 beds).On weekends, patients were managed by the duty teams, assisted by an additional senior intensive care physician during the day.Initial management was structured using a written protocol.Written transmissions were available on a secured cloud.A daily staff meeting was organized, comprising all the classical ICU senior physicians and where the teams from each sector came once at a time to shorten their presence and avoid crowded meetings. This staff ensured homogeneous care throughout the entire department and permitted senior ICU physicians to have a global vision of the patients and organize transfers from one sector to another if needed or if a room was available in the classical ICU.The nurse-patient ratio is regulated in ICU in France and cannot exceed 2.5 patients to 1 nurse. This ratio has been respected during the COVID-19 crisis.The organization of the service during the crisis was as follows:2, PaO2/FiO2 ratio, PaCO2), and standard laboratory data. The PaO2/FiO2 ratio was calculated only for patients with IMV. We recorded if the patient had chest computed tomography imaging (CT-scan), the time between admission in ICU and CT-scan and the degree of lung involvement on chest CT-scan  days. The other 27 patients were transferred to the classical ICU within four [2–8] days, with an overall ICU LOS of 26.5 [16-33] days.No differences were found in medical management or patient outcomes Table .2/FiO2 ratio on admission, prone positioning, ECMO, the absence of chest-CT-scan and the absence of interregional transfer. Initial management in the temporary ICU was not associated with mortality (Table In univariate analysis, factors associated with mortality were the BMI, SAPS 2 on admission, PaOty Table .Table 2CNo multiresistant bacterial acquisition was observed in either ICU.To our best knowledge, this study is the first to describe and investigate the prognosis of patients managed in a temporary ICU. Beyond the medical value, this study demonstrates a specific episode of our recent sanitary history. It describes the adaptation of a French ICU team to a massive pandemic to increase ICU beds by threefold, without affecting patient care.Patients initially admitted to the temporary ICU did not present an increased mortality rate compared with those initially admitted to the classical ICU.Nevertheless, our results can roughly be generalized to all Temporary ICUs: there has been many different ways to manage Temporary ICUs. We have described here our way to make a temporary ICU work.Ventilation strategies might have differed between ICU categories and, consequently, IMV duration, without sufficient power to relate this effect.First, as the crisis evolved and the number of infected patients increased, the number of ICU beds decreased, and the demand for resuscitation beds increased. A shift in skills occurred with the implementation of noninvasive ventilation techniques and high-flow oxygen therapy in the classical ward. Although 27% of patients admitted to the classical ICU were not ventilated, only 3% of those admitted to the temporary ICU were not under IMV.Second, ventilator availability in both ICUs differed. Ferré et al. used a transport ventilator without identifying consequences regarding the outcome . BecauseAlthough the temporary ICUs were set in common rooms, we did not observe multiresistant bacterial transmission. The COVID-19 patient isolation protocol and strict adherence to hospital hygiene rules helped achieve this outcome.Our cohort is small. To avoid biases, we have chosen to limit our observation to the first pandemic wave, only in our department. Indeed, the understanding of the disease and the practices have evolved over time. Similarly, the operating modes of the temporary ICU varied from one hospital to another.We did not use noninvasive mechanical ventilation during the first wave because of the fear of an increased risk of contamination of healthcare workers without encouraging data in the literature at that time on acute hypoxemic respiratory failure .Admission to temporary ICU was indicated for patients under IMV or with a short term indication of IMV. Nevertheless, as staging severity was difficult to assess with this new disease, we made some staging mistakes and admitted to temporary ICU patients who quickly improved and were not placed under IMV. This explains why a few patients without IMV were admitted to temporary ICU.Chest CT-scans were performed before admission to the ICU. We note that patients who were going to die had less frequently a chest CT-scan, probably because they were considered too severe to tolerate this procedure before admission to the ICU. Also, at the time of the first wave, the degree of lung involvement was not yet assessed and did not help us to evaluate the severity of patients.During the COVID-19 first wave, our team doubled its capacity to receive patients requiring intensive care because of the creation of temporary ICUs. In our cohort, 62.7% of patients were initially managed in temporary ICUs. The temporary ICU allowed access to intensive care for all patients requiring it immediately. This benefit could not be modelled in our study. However, during the first pandemic wave, on a national level, population access to ICUs was correlated with the global mortality observed , 21. SimThe mortality observed in our cohort and associated risk factors are consistent with the main studies published during the related period , 24, 25.Temporary ICUs are an operating model developed in a health crisis context and should not represent a long-term strategy to increase regular ICU capacities. Although no loss of opportunity occurred related to managing patients presenting respiratory distress linked to SARS-CoV-2 infection during the first wave, this model certainly has collateral effects that are more difficult to measure.We did not measure potential side effects that may have crucial consequences on the survivor’s life : rate ofThe potential psychological impacts of the development of temporary ICUs for all the actors in this particularly anxiety-provoking episode are not established: patients suffering from a new disease, patients ‘relatives who could not visit them, healthcare workers who had to work in unfamiliar surroundings–31.The development of temporary ICUs required surgical deprogramming, the consequences of which are difficult to assess at this time. In November 2020, the French hospital federation published the first global and national estimate of the impact of the epidemic on non-COVID-19 activity . CompareThe medicalization of these new units was ensured by the redeployment of 2,500 doctors, 80% of whom were anaesthesiologists, and 715 residents, 80% of whom were taking the anaesthesia and intensive care course. This unprecedented reinforcement clearly illustrates the value of a dual ability in anaesthesiology and intensive care.We did not observe increased mortality associated with initial management in the temporary ICU. This management in the temporary ICU, in the context of saturation of the classical ICU, managed by mixed teams made it possible to save lives without endangering these patients. The consequences in terms of morbidity can only be evaluated in larger groups.


X-ray free-electron laser (XFEL) is the latest generation of the X-ray source that could become an invaluable technique in structural biology. XFEL has ultrashort pulse duration, extreme peak brilliance, and high spatial coherence, which could enable the observation of the biological molecules in near nature state at room temperature without crystallization. However, for biological systems, due to their low diffraction power and complexity of sample delivery, experiments and data analysis are not straightforward, making it extremely challenging to reconstruct three-dimensional (3D) structures from single particle XFEL data. Given the current limitations to the amount and resolution of the data from such XFEL experiments, we propose a new hybrid approach for characterizing biomolecular conformational transitions by using a single 2D low-resolution XFEL diffraction pattern in combination with another known conformation. In our method, we represent the molecular structure with a coarse-grained model, the Gaussian mixture model, to describe large conformational transitions from low-resolution XFEL data. We obtain plausible 3D structural models that are consistent with the XFEL diffraction pattern by deforming an initial structural model to maximize the similarity between the target pattern and the simulated diffraction patterns from the candidate models. We tested the proposed algorithm on two biomolecules of different sizes with different complexities of conformational transitions, adenylate kinase, and elongation factor 2, using synthetic XFEL data. The results show that, with the proposed algorithm, we can successfully describe the conformational transitions by flexibly fitting the coarse-grained model of one conformation to become consistent with an XFEL diffraction pattern simulated from another conformation. In addition, we showed that the incident beam orientation has some effect on the accuracy of the 3D structure modeling and discussed the reasons for the inaccuracies for certain orientations. The proposed method could serve as an alternative approach for retrieving information on 3D conformational transitions from the XFEL diffraction patterns to interpret experimental data. Since the molecules are represented by Gaussian kernels and no atomic structure is needed in principle, such a method could also be used as a tool to seek initial models for 3D reconstruction algorithms. Structural information of the biological molecules is necessary for understanding their functions, and thus determination of their structure is one of the primary interests in biology. To determine the 3D structure of biomolecules, multiple experimental techniques have been developed. X-ray crystallography is the most widely used technique to determine the 3D structure at the atomic level . HoweverX-ray free-electron laser (XFEL) is the latest generation of the X-ray source that could become an invaluable technique in structural biology. XFEL can create significantly strong, coherent X-rays in a femto-second pulse form , enablinab initio. In such a situation, hybrid approaches, which combine the computational simulations with experimental data, could be used to obtain information on 3D structures  sampling. We refine the initial model iteratively during the MC sampling to maximize the similarity between the XFEL diffraction patterns simulated from candidate models and the target XFEL diffraction pattern. The basic assumption is that the similarity between diffraction patterns correlates reasonably well with the similarity of 3D models, while it is expected that multiple 3D models could match equally well to the target diffraction pattern. Such a strategy can avoid the difficulties of 3D reconstruction from limited data sets along with phase retrieval procedures. Dasgupta et al. developed an algorithm following the same strategy for atomic force microscopy (AFM) studies , 2021 toThe proposed algorithm was tested on two biomolecular complexes of different sizes with different complexities of conformational transitions, adenylate kinase, and elongation factor 2 using synthetic XFEL diffraction patterns. Our results show that the proposed algorithm can successfully describe the conformational transitions by refining the overall 3D shapes of biomolecules against a single simulated XFEL diffraction pattern. In addition, we showed that the incident beam orientation has some effect on the accuracy of the 3D structure modeling and discussed why the accuracy is low in some orientations. The proposed method could serve as an alternative approach for retrieving 3D information from the XFEL diffraction, which provides new insight into the interpretation of experimental data. Since the molecules are represented by Gaussian kernels and no atomic structure is needed in principle, this method could also be used as a tool to seek initial models for 3D reconstruction algorithms.To evaluate our proposed algorithm, we performed tests on two proteins . For eacE. coli adenylate kinase (AK), a protein comprising 214 residues. Two X-ray structures with different conformations, PDB 1ake , a protein that consists of 842 residues. Two X-ray structures of EF2, PDB 1n0u  and 1n0vIn the MC conformational sampling process, we need to efficiently generate a large number of simulated diffraction patterns from structure models in order to compare the 3D candidate models to a target XFEL diffraction pattern. In addition, the current target systems in XFEL experiments are large macromolecular complexes, where atomic details are not essential, and our aim is to provide a method to describe large-scale protein conformational transitions. Therefore, we employ Gaussian mixture models (GMMs) as a coarse-grained approach to represent the structures instead of atomically detailed models. GMMs were found able to capture shape details at a specified resolution . Furtherith Gaussian distribution in three-dimensional space, In the Gaussian mixture model (GMM), a macromolecule is represented by the sum of gmconvert  .d), and that the detector is set perpendicular to Z-axis with its center being at , where d is the distance between object and detector (d > 0). In elastic scattering, the intensity of diffraction at , I , is proportional to I  relative to the intensity at the image center, and, in the following, the diffraction patterns are discussed using k values corresponding to pixels as the coordinates, i.e., In our formalism, we assumed that the incident beam comes from the positive side of Z-axis, i.e., the wave vector of the incident beam is 2 near the center. Following the above protocol, we simulated the initial and target diffraction patterns for AK and EF2 , and then corresponding XFEL diffraction patterns are simulated (jth and kth patterns). Then the updated kth representation (or candidate) is accepted or rejected based on the correlation coefficient between kth pattern to the target pattern and jth to the target pattern. The Metropolis scheme depends on the difference between correlation coefficients at an arbitrary temperature T. In this study, T was set to 1.0.In our modeling process, we optimize the initial model based on the target XFEL diffraction pattern simulated from the target conformation . This opIn the above scheme, Gaussian kernels should not extensively overlap each other during conformation transitions. Therefore, repulsive interactions between the kernels during the updates are also considered. The overlap between two three dimensional Gaussian kernels ix Σij . This ovix Σij ξijN=ξijBefore performing the Metropolis method with two-dimensional comparison, candidate models in which any of the normalized overlaps between the pairs of kernels was greater than a threshold Our algorithm uses the MC scheme to refine the initial model based on the assumption that increases in diffraction patterns similarity correlate reasonably well with increases in the 3D model similarity between the candidate and target representations. Therefore, to evaluate the performance of the algorithm, we measured the 3D similarity between the initial model and the target structure  and the 3D similarity between the final candidate models and the target structure . The 3D similarities were calculated using the ‘Fit in Map’ tool in Chimera  to superThe algorithm was tested on a large conformational transition observed between two conformations of AK. Our aim is to obtain a low-resolution model of the open state AK conformation (PDB ID: 4ake) from the corresponding XFEL diffraction pattern. The initial model is the closed state AK conformation (PDB ID: 1ake). Since AK has three distinct domains, we used 3 Gaussian kernels to represent the initial model , and the target is apo conformation (PDB ID: 1n0u). The reference diffraction pattern was simulated from the GMM model of the target conformation with 819 Gaussian kernels, which corresponds to one kernel per residue. Low-resolution models with 7 Gaussian kernels built from the holo and apo conformations  to generate corresponding initial and target GMM representations. In combination, there are 84 modeling cases, and for each orientation, 10 trial runs were performed. The average and highest 3D CC  for 84 orientations are shown in Furthermore, we consider simulated XFEL diffraction patterns from EF2 in different orientations. The initial holo conformation and target apo conformation were rotated by the same rule as the AK test. The average and highest 3D CC  for 84 orientations are shown in We could observe from In the proposed structure optimization approach, the initial model needs to have an orientation similar to the orientation that the XFEL diffraction pattern represents. This is a common requirement for flexible fitting approaches, including the fittings to 3D information such as electron density maps. The orientation of a protein molecule observed in XFEL diffraction patterns can also be estimated by calculating correlation coefficients . HoweverFor the same target diffraction pattern of EF2 as via 3D reconstruction from a large dataset.In this study, we proposed a new hybrid method that can be applied to single particle XFEL diffraction patterns to produce a low-resolution 3D model and study conformational transitions. In the current single particle XFEL experiments on biological systems, it is still difficult to perform 3D reconstruction directly from the diffraction patterns. We explored the strategies to obtain plausible 3D structural models by optimizing a known structure to maximize the similarity between the target XFEL diffraction pattern and simulated diffraction pattern from candidate models using MC sampling. A set of Gaussian kernels are used to represent the candidate models. There were significant increases in the similarity between the candidate models and target conformation in most cases. Thus, our method could successfully refine initial models of AK and EF2 to infer target conformations using just one XFEL diffraction pattern. In addition, as the biomolecules are represented by Gaussian kernels, this approach could be used to study the conformational changes of the molecules where only low-resolution structural information without atomic details is available. Therefore, the proposed algorithm can be a new approach to studying the dynamics of biomolecules from XFEL experiments, as an addition to the structure determination


The spatial and temporal segregation of cargo proteins within the TGN, which is mediated with different sets of regulators including small GTPases and cargo adaptors, is a fundamental process in the sorting machinery. Recent studies with powerful imaging technologies have suggested that the TGN possesses spatially distinct subdomains or zones for different trafficking pathways. In this review, we will summarize the spatially and dynamically characteristic features of the plant TGN and their relation to cargo protein trafficking.Membrane trafficking contributes to distinct protein compositions of organelles and is essential for proper organellar maintenance and functions. The  Membrane trafficking tightly regulates protein localization among organelles and plays a fundamental role in numerous biological processes such as cell growth, development, and stress responses. It is an evolutionarily conserved system among eukaryotes and consists of four fundamental processes: (1) forming transport carriers and sorting cargo proteins on donor organelle membranes; (2) transporting the carriers from the donor to the target organelles; (3) tethering; and (4) fusing them with the target organelle membrane . These pFirst, Arf/Sar GTPases activated by their guanine-nucleotide exchange factor (GEF) promote the recruitment of coat proteins on the donor membrane. Coat proteins play important roles in cargo protein recognition and membrane distortion . RAB GTPcis cisterna of the Golgi apparatus receives cargo proteins from the ER, whereas the trans cisterna sends them to their destinations, including the plasma membrane (PM) and vacuoles, via the trans Golgi network  where approximately one-third of proteins are synthesized. In normal anterograde membrane trafficking, the ork TGN; . Therefoork TGN; . In thistrans-side of the Golgi apparatus  was found to impair the biogenesis of the TGN, while the Golgi apparatus became over-stacked and elongated in Arabidopsis  are derived from a common ancestral complex and are thought to have been present in the last eukaryotic common ancestor . Hence,  the TGN .ap1γ1 ap1γ2 double mutants cannot be obtained (ap1μ2 (major isoform of AP-1 μ subunits) mutants have retarded growth and are unable to produce progenies  polar recycling from the brefeldin A (BFA) compartment to the PM or vacuoles is compromised in ap1m2-1/hap13-1 seedlings. BRASSINOSTEROID INSENSITIVE1 recycling from the BFA compartment to the PM is also compromised in ap1m2-1/hap13-1 seedlings  as components of the GI-TGN, while AP-4 seems to stay with the GA-TGN . Electro the TGN . In supp and TGN . It has  and TGN . These f and TGN . It shou and TGN .Arabidopsis, AP-4 components were reported to be responsible for the vacuolar sorting of GFP-CT24, which is an artificial cargo protein with β-conglycinin-derived vacuolar targeting signals  and KINASE ON THE INSIDE (KOIN) are sorted at the TGN by different machineries and transported to the opposite sides  of the root endodermal PM. These different de novo pathways have also been demonstrated using BFA and Endosidin16 in combination with or without the protein synthesis inhibitor cycloheximide. Trafficking of IRK is compromised with BFA but not Endosidin16, whereas that of KOIN is sensitive to Endosidin16 but not BFA , JST (grant number: JPMJCR20E5 to TU), Grant for Basic Science Research Project from Sumitomo Foundation (to TU), and JSPS Research Fellowship for Young Scientists (to YS).The 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.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.


Preeclampsia (PE) is a serious disease that can be fatal for the mother and fetus. The two-stage theory has been proposed as its cause, with the first stage comprising poor placentation associated with the failure of fertilized egg implantation. Successful implantation and placentation require maternal immunotolerance of the fertilized egg as a semi-allograft and appropriate extravillous trophoblast (EVT) invasion of the decidua and myometrium. The disturbance of EVT invasion during implantation in PE results in impaired spiral artery remodeling. PE is thought to be caused by hypoxia during remodeling failure–derived poor placentation, which results in chronic inflammation. High-mobility group protein A (HMGA) is involved in the growth and invasion of cancer cells and likely in the growth and invasion of trophoblasts. Its mechanism of action is associated with immunotolerance. Thus, HMGA is thought to play a pivotal role in successful pregnancy, and its dysfunction may be related to the pathogenesis of PE. The evaluation of HMGA function and its changes in PE might confirm that it is a reliable biomarker of PE and provide prospects for PE treatment through the induction of EVT proliferation and invasion during the implantation. The high-mobility group (HMG) of chromosomal proteins regulates DNA-dependent biochemical processes . HMG proHMG proteins are involved in autoimmune diseases ,5. HMGB1HMGA has HMGA1 and HMGA2 forms. HMGA1 is a 20-kDa protein with three AT hooks, which are DNA-binding domains. It has been reported to activate transcription of the insulin receptor . It bindPE is a hypertensive disorder of pregnancy that is associated with proteinuria and/or organ failure and is a major cause of maternal and neonatal death. PE is characterized by vascular constriction, which results in maternal organ failure, and increased vascular resistance of the placental and systemic circulation, which leads to reduced uteroplacental blood flow followed by placental dysfunction  and fetaSkjærven et al.  reportedHMGA has three AT hooks that bind AT-rich DNA; it modulates the chromatin conformation of target DNA and has HMGA1 and HMGA2 forms synthesized by alternative splicing . HMGA1 iFedele et al.  reportedFurthermore, the up-regulation of matrix metalloproteinase-2 (MMP-2) by HMGA1 promotes lung cancer transformation, and the blocking of MMP-2 expression inhibits cancer cell migration and invasion . HMGA1 iHMGA2 is a non-histone architectural transcription factor that alters the chromatin structure and regulates gene transcription. It is known to influence processes such as the cell cycle, DNA damage repair, apoptosis, senescence, and EMT . HMGA2 ohmga2 gene results in skeletal muscle damage [Transforming growth factor-β (TGF-β) is important for appropriate placentation, and its dysfunction may be related to the pathogenesis of PE . It can e damage ,47.HMGA2 plays an important role in embryogenesis; it is highly expressed in murine and human fetal tissues, but not in adult tissues, with the exception of the uterine myometrium ,48. HMGAWest et al.  reportedOn the other hand, Bamberger et al.  found thXian et al. reported that HMGA1 could amplify Wnt/β-catenin signaling . It is kAlthough HMGAs are expressed strongly in the placenta and are thought to be involved in placentation and the regulation of trophoblast function, the expressions of HMGA1 and HMGA2 in the placenta differ markedly . HMGA1 iInterferon (IFN)-β is an immune modulator that promotes tolerance of paternal antigens at the maternal–fetal interface , and HMGIn a PE mouse model used to investigate the mechanism of HMGA1′s involvement in the pathogenesis of PE, we demonstrated that HMGA1 played an important role in the regulation of EVT migration and proliferation . In normIn addition to HMGA, there are other HMG-proteins that reported to be associated with the pathogenesis of PE. One of them is HMGN1, which encodes a protein associated with active chromatin and transcription. HMGN1 could stimulate cytokine production and be capable of stimulating immune tolerance by a TLR4-dependent pathway . FurtherHMGB1 is a cytokine mediator of inflammation and known as to be released from macrophages and dendric cells . In partHMGAs are thought to play an important role in normal placentation by creating an optimal microenvironment for spiral artery remodeling and EVTs invasion; disturbance of these processes may play a major role in the pathogenesis of PE through poor placentation resulting in the reduction of uteroplacental perfusion. In previous work, we found that HMGA1 translocation from trophoblast nuclei to the cytoplasm contributed to the impairment of EVT invasion . Thus, H


Sexual quality of life (QoL) is affected during and after breast cancer (BC) treatment. The purpose was to investigate sexual and global QoL and patient-reported measures to address this issue in young women (< 51 years) with BC after the acute treatment phase, during adjuvant endocrine therapy.Three EORTC questionnaires and an additional specific questionnaire, developed for the study, were used to assess sexual and global QoL and patient-reported supportive measures in BC patients who had received their endocrine therapy for at least 24 months. Among the 54 eligible patients, 45 (83%) agreed to participate in the study.We showed a deterioration in sexual QoL and poor communication with healthcare professionals. Most patients (88.9%) declared that it was important that sexuality should be discussed with caregivers and that the partner should also be involved. Most patients (60%) had taken at least one action to overcome their sexual problems. Most of these interventions (63%) originated from the patient herself.Sexual QoL is a major issue in young BC patients and is poorly addressed by healthcare professionals. Most of the supportive methods used by the patients to overcome these side effects were on their own initiative. Communication and counseling on sexuality by healthcare professionals need to be improved during BC treatment. Patients suggested supportive measures they would find useful and appropriate to develop in the clinic. The final goal is to improve the sexual QoL of BC patients with the appropriate intervention and support.The online version contains supplementary material available at 10.1007/s00520-022-06810-3. Advances in BC diagnosis and treatment have led to the improvement of the patients’ prognosis, with a 5-year survival in wealthier countries now reaching 90% . The disWe previously published the results of a longitudinal assessment analyzing the sexual and global QoL of 106 women with hormone receptor-positive (HR +) BC during the first year of endocrine therapy, using the EORTC SHQ-22 and global HRQLQ . This stIn this new study, we focused on young patients less than 51 years old, a population whose sexual QoL is more frequently and severely affected following BC care –13. We aThis unicentric prospective study (CUPIDON 2) was conducted at the Institut du Cancer de Montpellier (ICM). The protocol was approved by the French Ethics Committee and the internal review board of the institution. Patients received an information letter and all provided written consent before enrolment. The primary objective was to evaluate the sexual QoL of women younger than 51 years old with HR + BC who had received their endocrine therapy for at least 24 months using the EORTC SHQ-22 questionnaire. The secondary objectives were: (1) to evaluate the global QoL at the same time-point using the EORTC QLQ-C30  and QLQ-General health and socio-demographic information, including employment status, physical activity, and partner status, were self-reported by participants. Age, weight, size, menopausal status, pathology report, type of treatment  as well as comorbidities were collected from the medical records.Patients were invited to complete the three EORTC HRQLQ only once.The general EORTC QLQ-C30 questionnaire dedicated to all cancer patients includes 30 items assessing the global health status with 5 functional scores  and 9 symptom scores .The specific EORTC QLQ-BR23 module dedicated to breast cancer patients includes 23 items allowing to assess 4 functional scores  and 4 symptom scores .The EORTC SHQ-22 is a multi-dimensional QoL instrument used to measure sexual health in patients with cancer (men or women). This new tool covers both sexual functioning and psychosexual components. It includes 8 items on sexual satisfaction, 3 items on sexual pain, and 11 single items in an integrative approach, leading to 7 functional scales and 4 symptom scales.In these questionnaires, higher scores in the functioning scales indicate a better functional level, whereas higher scores in the symptom scales indicate the severity of problems. A user agreement for the use of the EORTC SHQ-22 was signed.In addition, a homemade questionnaire containing 11 questions was proposed to patients in order to explore (1) the information regarding sexuality received during treatment, (2) the supportive measures or therapeutic interventions used by patients to overcome sexual dysfunction, and (3) the need and acceptability for specific management of sexual dysfunction. This specific Cupidon questionnaire is reproduced in extenso in the In this descriptive study, the sample size was based on the recruitment capacity of our institution, which represented a potential of around 50 patients meeting the inclusion criteria of the study for the planned duration. Considering an 80% acceptance rate , a total of 40 patients were expected to participate. The inclusion of at least 40 patients permits to estimate (for descriptive purposes) the mean scores of the dimensions of the EORTC SHQ-22 (ranging from 0 to 100) and its 95% confidence intervals (95% CI) with a width of 15 and hypothesizing a standard deviation of 24 .Scorings of the three EORTC HRQLQ were calculated according to the EORTC Scoring Manuals, and results were presented as means and standard deviations (SD). Categorical variables were described using frequencies and percentages.Statistical analysis was performed using the Stata v16 software .Among the 65 patients screened during consultations between February and April 2020, to participate in the study, 11 were not eligible , and 9 refused to participate. A total of 45 patients have been included and completed the four questionnaires. Clinical and socio-demographic characteristics are summarized in Table Mean scores for the EORTC SHQ-22 questionnaire are shown in Table Vaginal dryness was the most important symptom (a mean score of 57.66). Fatigue appeared important (mean score of 49.24) as well as pain related to intercourses (a mean score of 31.98). Worrying about incontinence was rare (mean score 9.09).Mean scores for the EORTC QLQ-C30 and the EORTC QLQ-BR23 are shown in Table According to the EORTC QLQ-C30, the global health status score was 69.63. The mean scores of physical functioning, role functioning, and social functioning appeared better than those of emotional and cognitive functioning.Regarding the symptom scales, the highest scores were fatigue and insomnia .According to the EORTC QLQ-BR23, the score for the body image was 66.30, while the score for the sexual functioning scale appeared low (a mean score of 35.19). Sexual enjoyment appeared better (a mean score of 62.75), but only 75% of patients answered this item. The side effects of systemic therapy and local breast and arm symptoms had similar scores. With a minimum of 2 years since the end of the chemotherapy, women were no more concerned by hair loss.The answers to the specific Cupidon questionnaire are shown in Table Most patients (73.3%) had not received or did not remember receiving any information about sexuality from their oncologist or any other caregiver. Among the 12 patients who had received information, 10 (83.3%) were satisfied with the received information. Most patients (86.7%) had never discussed potential sexual problems with their oncologist or caregiver.When asked why they did not discuss about sex with them, most patients declared having no question about sexuality  and feeling it was a minor issue as compared to cancer and the cancer treatment (66.7%). Only a minority of patients felt that the caregivers were too busy to discuss (25.7%) or felt discomfort (30.8%) to talk about the subject.At the same time, most patients (88.9%) declared it important that sexuality and its potential problems should be discussed with caregivers and that the partner should be involved in the discussion (73.4%). Half of the patients (53.3%) wished for a systematic initial consultation with a sexologist, and 75.6% wished that such a consultation would be available when needed.n = 5), with a general practitioner (n = 2), and with a midwife (n = 1).The majority of patients (60%) had tried at least one method to overcome their sexual problems, in order of frequency: local treatment like vaginal moisturizer (48.9%), consultation with a psychologist (17.8%), while a consultation with a sexologist remained rare (2.2%). Most of these interventions (63%) originated from the patient herself. Eight patients declared having used other help: consultation with a gynecologist (Patients suggested as desirable the following interventions in order of frequency: consultation with a caregiver trained in sexology (91.1%), vaginal moisturizer (77.8%), pelvic floor physical therapy (73.4%), combined consultation with her partner and a caregiver (71.1%), consultation with a sexologist (66.7%), online psychotherapy (46.7%), psychiatric consultation (40%), and group consultation (24.4%).This study described the sexual and global QoL of 45 young BC women during adjuvant endocrine treatment for at least 24 months, as well as the actions taken and suggested by these women to overcome the encountered problems.First of all, women displayed a high level of interest in the study, as demonstrated by the 83% participation rate and a high rate of response to the items of the questionnaire.The scores observed for the sexual QoL evaluation with the EORTC SHQ-22 were similar to those previously observed using the same questionnaire in a population without age restriction  during eThe global QoL deterioration analyzed with the QLQ-C30 and BR23 was also similar to what we previously reported , with thWe explored the actions taken and proposed by patients to overcome these sexual dysfunctions using a homemade questionnaire specific to our study. Firstly, most patients had not received any information; either a fortiori help from their oncologist nor any caregiver. A vast majority of them declared that is was important that sexuality should be discussed and that their partner should be involved. However, among the women who had received information from their oncologist or any caregiver, the vast majority were satisfied with the received information, which is altogether encouraging, since these caregivers were not trained before the study.The majority of patients used interventions to overcome sexual problems on their own initiative, underlying the need for specific management of this issue. They stated, mostly not based on their personal experience, that it would be desirable to develop, by order of frequency: consultation with a caregiver trained in sexology, vaginal moisturizer, pelvic floor physical therapy, consultation with their partner, consultation with a sexologist, and less frequently, any kind of psychotherapy.Most patients declared that sexuality remains a minor issue compared to cancer treatment as the main reason to explain why they did not discuss it with their oncologist or caregiver. This seemingly contradictory response might reflect the patients’ ambivalence about the theme and/or the fact that they want to prioritize the time with their oncologist to focus on the medical information regarding the cancer and its treatment. The role of the oncologist is probably mostly to raise the subject with the patient and question the need for such a dedicated consultation.The main limitations of our study are the low number of patients included and the absence of longitudinal evaluation. Moreover, a memory bias might have altered the content of the reported events when patients were asked about their history of sexual problems. The strengths are the homogeneity of this young women’s population as well as the use of standardized EORTC questionnaires integrating physical, psychological, and social aspects to evaluate sexual QoL. Moreover, despite the fact that the specific Cupidon questionnaire is not a validated research instrument, asking women their opinion on the actions that could be useful and appropriate to develop in clinic is crucial. This latter information from the involved women going through this issue is precious and should be used to identify the actions to be taken to support these women. The key proposals from the affected patients should be considered to tailor the patient’s management, with the final goal being to improve the QoL of our patients with adequate intervention and support.Supplementary file1 (DOCX 25 KB)Below is the link to the electronic supplementary material.