A healthy diet throughout the life course improves health and reduces the risk of disease. There is a need for new knowledge of the relation between diet and health, but existing methods to collect information on food and nutrient intake have their limitations. Evaluations of new tools to assess dietary intake are needed, especially in old people, where the introduction of new technology might impose challenges.We aimed to examine the usability of a new web-based dietary assessment tool in older adult women and men.A total of 60 women and men  aged 60-74 years recruited by convenience and snowball sampling completed a 24-hour web-based dietary recall using the newly developed Norwegian version of Measure Your Food On One Day (myfood24). Total energy and nutrient intakes were calculated in myfood24, primarily on the basis of the Norwegian Food Composition Table. No guidance or support was provided to complete the recall. Usability was assessed using the system usability scale (SUS), where an SUS score of ≥68 was considered satisfactory. We examined the responses to single SUS items and the mean (SD) SUS score in groups stratified by sex, age, educational level, and device used to complete the recall .The mean total energy intake was 5815 (SD 3093) kJ. A total of 14% of participants had an energy intake of <2100 kJ  and none had an intake of >16,800 kJ . Mean energy proportions from carbohydrates, fat, protein, alcohol, and fiber was within the national recommendations. The mean SUS score was 55.5 (SD 18.6), and 27% of participants had SUS scores above the satisfactory product cut-off. Higher SUS scores were associated with younger age and lower education, but not with the type of device used.We found the overall usability of a new web-based dietary assessment tool to be less than satisfactory in accordance with standard usability criteria in a sample of 60-74–year-old Norwegians. The observed total energy intakes suggest that several of the participants underreported their intake during the completion of the dietary recall. Implementing web-based dietary assessment tools in older adults is feasible, but guidance and support might be needed to ensure valid completion. A healthy diet throughout the life course improves health and reduces the risk of disease . The WorLife expectancy is increasing, and the world’s population is ageing . TherefoMeasure Your Food On One Day (myfood24) is a self-administered web-based dietary data collection tool and analysis software developed  and valiThe UK version of myfood24 has demonstrated reasonable response proportions for repeated 24-hour dietary recalls in 60-85–year-old individuals in the United Kingdom , and theWe recruited participants by combining targeted convenience and snowball sampling to ensure an even distribution of sex, age (between 60-74 years), and educational level . Only individuals with an email address and access to a smartphone, tablet device, or computer were invited. We approached individuals accessible to the researchers through their personal networks (convenience sampling), and further invited individuals approached by already recruited individuals . A total of 72 (convenience sample size) women and men aged 60-74 years (58% women) were invited to complete a single dietary recall. Invitations were sent in 3 rounds between May 12 and June 8, 2021, of which the first 2 groups received 1 reminder after 2 weeks .myfood24 ,10 dietaThe System Usability Scale (SUS) is a postsession questionnaire  and one The invitees were sent an email with a weblink to myfood24 and an SMS text message to their mobile phone with information that the email had been sent. The email included a brief description of the aim of the study  and a common  weblink to complete the 24-hour dietary recall and a questionnaire about usability and demographics. No detailed guidance about how to complete the myfood24 was provided; that is, no preparation or tutoring.By clicking the weblink in the email, the participant was directed to myfood24 and asked to report all food and drink intake from the previous day. After completion of the 24-hour dietary recall , the participant was automatically redirected to a separate external web-based questionnaire created using Nettskjema , a web-bAccording to the Norwegian Health Research Act , analysit test for differences between age groups . We used Excel  to present the response to each usability item as frequencies. Participants’ free-text comments were grouped into themes.The SUS score was calculated in accordance with Sauro 2011 . The scoA total of 60 women and men participated . Study sample characteristics and energy intakes are shown in The response to each SUS item is presented in P=.02). For each group stratified by sex, education, and device used, there was a tendency toward higher SUS scores in the younger compared to the older age group, but only significantly different among those with higher education.A total of 16 participants (27%) had SUS scores of ≥68 . In total, 18 participants had left comments in the free-text option. These comments (examples in parentheses) could thematically be grouped to (1) lack of categories of meals or foods (“Found nowhere to register supper”), (2) complexity (“Unnecessary complex at the same time as not nuanced enough to catch important differences between foods”), (3) wording (“Why is the word ‘generic’ used?”), or (4) technical problems .In this study of the usability of a new web-based dietary assessment tool among older adult Norwegian women and men who were provided no guidance or support, we found the overall usability to be less than satisfactory and the overall total energy intake to imply error (underreporting) in completion of the dietary recall. In total, 17% of participants had energy intakes above 8000 kJ, and 27% had usability scores within the range indicating a satisfactory product.The E% for all macronutrients were within the recommendations from the Norwegian Health Directorate , but in Our results deviate considerably from the findings from a usability study of the German version of myfood24 , who fouDuring the development of the original UK version of myfood24, a small sample of adults aged above 65 years was included to report expectations from myfood24 . The samNonetheless, digital illiteracy is a concern for self-administered web-based assessment tools, particularly among older adults. In a feasibility study of myfood24 among 60-85–year-old individuals in the United Kingdom , those cEven though the overall SUS score in our study was lower than that considered satisfactory, we do believe that myfood24 is a useful tool to measure dietary intake. The participants in our study were solely provided information about the aim of the study, with no guidance or support in completing the recall. We believe that additional tailored instruction on how to complete the recall could have improved the overall perception of usability considerably and led to less underreporting. Moreover, usability questions highlighting the need for technical support or training revealed that technical challenges did not constitute a major barrier. Thus, we believe that technical difficulty was not the main contributor to the observed SUS score. In telephone surveys by Statistics Norway, 70% of 75-79–year-old individuals reported daily use of the internet in 2021 compared to 40% in 2015 , implyinThe study was set up to be anonymous to protect participant privacy; therefore, the participants received unidentifiable weblinks. Thus, a major study limitation is that we could not link SUS scores and dietary intakes on an individual level, to examine the correlation between intake and SUS scores. Further, we collected no information regarding whether the participants were comfortable with using technology. We invited only women and men with an email address and access to a device to complete the recall, and we did not examine reasons for nonparticipation. People without email addresses or those who did not participate could be less digitally literate. Thus, lower SUS scores could be anticipated among nonparticipants. A strength of the study is testing of the tool in a mixed group of older adults .In this population of older adult Norwegian women and men, the overall usability score for a web-based 24-hour dietary recall assessment tool, myfood24, was lower than what is considered satisfactory according to standard usability criteria. Implementing myfood24 in older adults is feasible, but additional guidance and support might be needed to ensure valid completion, and more work is needed for adapting myfood24 for use among older adult Norwegians.


We explore the routes to cancer diagnosis to further undertanding of the inequality in the reduction in detection of new cancers since the start of the pandemic. We use different data sets to assess stages in the cancer pathway: primary care data for primary care consultations, routine and urgent referrals and published analysis of cancer registry data for appointments and first treatments.Primary and cancer care.In this study we combine multiple data sets to perform a population-based cohort study on different areas of the cancer pathway. For primary care analysis, we use a random sample of 5 00 000 patients from the Clinical Practice Research Datalink. Postreferral we perform a secondary data analysis on the Cancer Wait Times data and the National Cancer Registry Analysis Service COVID-19 data equity pack.Primary care: consultation, urgent cancer referral and routine referral rates, then appointments following an urgent cancer referral, and first treatments for new cancer, for all and by quintile of patient’s local area index of multiple deprivation.Primary care contacts and urgent cancer referrals in England fell by 11.6% (95% CI 11.4% to 11.7%) and 20.2% (95% CI 18.1% to 22.3%) respectively between the start of the first non-pharmaceutical intervention in March 2020 and the end of January 2021, while routine referrals had not recovered to prepandemic levels. Reductions in first treatments for newly diagnosed cancers are down 16.3% (95% CI 15.9% to 16.6%). The reduction in the number of 2-week wait referrals and first treatments for all cancer has been largest for those living in poorer areas, despite having a smaller reduction in primary care contact.Our results further evidence the strain on primary care and the presence of the inverse care law, and the dire need to address the inequalities so sharply brought into focus by the pandemic. We need to address the disconnect between the importance we place on the role of primary care and the resources we devote to it. This study draws from multiple data sets along the complex, multidisciplinary cancer pathway.We use a rich primary care data set containing patient level primary care activity linked to patients’ local area socioeconomic indicator.Our primary care patient sample is relatively small (500 000 active patients from January 2016 to January 2021); however, the data produces results that closely mirror the rates of consultation and urgent cancer referral per patient produced in publicly available national data sets.Primary care is key part of the pathway for early cancer diagnosis through both routine and 2-week wait referrals.Cancer diagnosis rates have experienced a sustained fall since the start of the COVID-19 pandemic and introduction of non-pharmaceutical interventions ‘lockdowns’.The fall in urgent cancer referral is larger than the fall in primary care contacts, implying that the content of consultations has shifted away from potential cancer diagnosis.Despite having a smaller reduction in primary care contact through the pandemic, patients living in poorer areas have had larger reductions in urgent cancer referrals and first treatments for new cancer.Government, patients and primary care staff must work together to catch up on missing diagnosis.Resilience in primary care is key for the cancer diagnosis pathway and must be developed for future disruptions, particularly in poorer areas where care is more complex.The COVID-19 pandemic has had a profound impact on UK’s health system. Each part of the UK’s National Health Service (NHS) has been impacted in different ways, and we are still feeling many of the consequences of both the COVID-19 pandemic and the public health measures put in place to manage it . Cancer is one of the most complicated diseases that the UK health system must manage, being responsible for over one in four UK deaths in 2019. Cancer outcomes are acutely sensitive to changes in social determinants, patient pathways and service provision. Delays in both diagnosis and treatment have significant impacts on patient outcomes.4Primary care sits at the heart of the cancer patient pathway and is the most crucial interface for early diagnosis and referral to hospital-based care, in addition to their wider support of patient with undergoing and after treatment. As models of cancer care have evolved in light of both technical advances and an ageing comorbid population, primary care has become an increasingly important aspect of integrated cancer care and an expansion of general practitioner (GP) roles in cancer care.6It is important to reflect that prior to the start of the COVID-19 pandemic, primary care had seen significant declines in overall resourcing relative to the funding of the rest of the NHS and compared with growing levels of disease burden that is managed in primary care. In addition, there is growing evidence that primary care has been under greater pressure in more deprived areas, with higher levels of staff turnover,11Thus, to understand the COVID-19’s impact on primary care and the downstream impact on cancer outcomes we need to see that the pandemic arrived when the system that was already struggling to cope. Prior to COVID-19, the central role of primary care as agents of change in reducing inequalities had been the subject of much debate yet could do little in the face of political avoidance of health equity.Box 1COVID-19 was officially declared a pandemic by the WHO on 11 March 2020, and the Government announced its first full lockdown in England and the wider United Kingdom on 23rd March. In the following months England’s NPI were eased, schools reopened in phases, non-essential shops reopened and in August the population were encouraged to eat out. Some restrictions were re-imposed in September and October, on the 5th of November 2020 a second brief national locked lasted until 2nd December. On the 6th of January 2021, a third national lockdown was introduced.53It is now clear that the UK experience of the pandemic was one of the worst in the world, both in terms of excess mortality (both COVID-19 and non-COVID-19) and the impact of NPI (lockdowns) on both the ability of health services to continue provide care and the impact of messaging (stay at home) on patients’ timely presentation for care.We perform a population-based cohort study using the following three separate sources.Primary care electronic health records were obtained from the Clinical Practice Research Datalink Aurum database (henceforth CPRD). We included patient records from 1 January 2016 to 31 January 2021. Prepandemic data were included to establish long-term trends and patterns of seasonality in primary care use and referrals to secondary care. Similar to recent analysis of the COVID-19 pandemic,CPRD contains anonymised patient primary care data from approximately 7% of the UK population and is broadly representative in terms of age, sex and ethnicity.https://www.gov.uk/government/statistics/english-indices-of-deprivation-2015)https://www.datadictionary.nhs.uk/nhs_business_definitions/lower_layer_super_output_area.html%23:%7E:text=Lower Layer Super Output Areas,statistics in England and Wales). About 5 00 000 patients were randomly sampled from the CPRD population in England who were eligible for linkage within the defined study period.The period of eligibility for study inclusion starts on the latest of the study start date (1 January 2016) or the patient’s registration to their practice. A patient’s period of eligibility ends on the earliest of leaving their practice, the end of data collection from their practice or their death. Primary care records from CPRD were linked to the deciled index of multiple deprivation (IMD) from 2015 .Cancer waiting time (CWT) measure performance against the NHS Constitution Standards, recording the number of patients screened, referred to oncology specialists, diagnosed and treated for cancer. These measures are used by local and national organisations to monitor the timely delivery of services to patients, and they are published quarterly by NHS Digital  and National Cancer Registry Analysis Service (NCRAS) that have two published data sets,http://www.ncin.org.uk/view?rid=4346 (accessed on 24 January 2022)).The number of urgent suspected 2-week wait referrals (http://www.ncin.org.uk/view?rid=4347 (accessed on 24 January 2022)).First definitive treatments for cancer  (‘EMIS consultation source identifier’ and ‘Consultation source code identifier’) (These variables contain strings that categorise the patient record input and are selected by the staff member completing the record). In line with the approach taken by Carey et al 2012 for CPRD Gold data, we use a combination of the consultation code and the category of the record to identify consultations . To help with the comparability of consultations in the two periods, we removed primary care appointments that included a influenza vaccine.Using the observation file in CPRD Aurum, we were also able to identify where patients had influenza vaccinations. We look to exclude influenza vaccines from our analysis on the basis that the programme was expanded in 2020/2021 to achieve maximum uptake .CWT data are reported monthly, our pre-NPI data are therefore from 1 October 2009 to 31 March 2020 and our post-NPI onset period is from 1 April 2020 to 31 January 2021.et al.https://www.causesofdeath.org/docs/standard.pdf).We perform a linear regression of consultations, urgent and routine referrals from CPRD data and appointments following an urgent cancer referral and first treatments from CWT data over time to estimate expected values for the post-NPI onset period, based on predicted values from the data pre-NPI. To account for seasonality and time trends, we include months as a categorical variable and time as a continuous variable, the approach taken by Carr 93-95.The analysis presented in the equity data pack compares new instances of first treatments in months during the pandemic (1 April 2020–31 January 2021) compared with the same months in 2019/2020. The analysis includes a 95% CI for the changes, based on rate ratios under an assumption that the population is the same in the pre-COVID-19 baseline and COVID-19 months. This is calculated using the exact method described in Breslow & Day 1987, pp https://digital.nhs.uk/data-and-information/publications/statistical/appointments-in-general-practice/march-2021).In the calendar year of 2019, before the COVID-19 pandemic and the associated NPI, there was an average of 39 127 primary care consultations per 100 000 patient-months. This equates to 4.70 attended appointments per registered patient or an estimated 266 million appointments in primary care nationally in 2019  referral was 314 per 100 000 patient-months, equating to an estimated 2.12 million for the NHS in England. Following the first NPI, urgent cancer referrals from primary care  fell to Routine referrals however have shown a different trajectory in that their rates did not recover to prepandemic levels . As a shPatient demographics and patient-time and total numbers of observed consultations and routine and urgent referrals in our CPRD sample are presented in The knock-on effect of the reductions in patients’ primary care appointments and referrals can be observed in the national CWT data. The number of first appointments with a cancer specialist following an urgent cancer referral has fallen by approximately the same amount as estimated for the referrals themselves: 19.2% (95% CI 19.1% to 19.3%). The number of cancer first treatments (following a diagnosis and decision to treat) was 16.3% (95% CI 15.9 to 16.6) lower than expected or 43 600 (95% CI 42 500 to 44 700) missing first treatments from 1 April 2020 to 31 January 2021 .  and gynaecological cancers  and greater reductions for lung  and urological  cancers . To showThere are inequalities in primary care use in England, with the people who live in the poorest areas have higher rates of consultation than those in richer areas once we adjust for age. The most deprived quintile was expected to have 43 184 consultations per 100 000 patient-months , 15% morThe reduction of consultations over the period 22 March 2020 to 30 January 2021 was smallest for those in most deprived areas. Their reduction in consultations for the non-age-standardised figures was 9.6% (9.2%–9.9%), while for the least deprived the reduction was 12.4% (95% CI 13.2% to 13.9%) . Weekly Despite a smaller reduction in primary care contacts, we observe the largest reduction in both urgent cancer referrals and first treatments for cancer for patients living in the most deprived areas. The NCRAS data equity pack presents the number of urgent cancer referrals and first cancer treatments by IMD quintile (They do not age-standardise their results.). There was a greater percentage reduction in urgent cancer referrals for those living in the most deprived areas in England, who experienced a 17.6% (95% CI 17.2% to 18.0%) reduction between 1 April 2020 and 31 January 2021 compared with the same period 12 months before, while referrals for the least deprived quintile fell by proportionately less: 15.3% (95% CI 14.9% to 15.6%). This equates to a reduction of 61 500 referrals for the most deprived and 62 600 or the least: without adjusting for age, the most deprived quintile had a smaller proportion of the prepandemic urgent cancer referrals, with 350 000 referrals compared with 410 000 for the least deprived quintile from April 2019 to January 2020.At the same time, rates of new treatment for cancer for the people living in the most deprived 20% of England experienced a 15.8% (95% CI 14.6% to 17.0%) reduction between 1 April 2020 and 31 January 2021 compared with the same period 12 months before (6 610 missing first treatments). The reduction for the least deprived was 12.6% (95% CI 11.5% to 13.7%) which equates to 6880 missing first treatments.Despite having more access to primary care for patients in more deprived areas (9.7% reduction for most deprived compared with 12.5% for the least deprived), urgent cancer referrals and newly diagnosed cancers have been disrupted by the pandemic more for people living in poorer areas.The coronavirus SARS-CoV-2 (COVID-19) pandemic has had a profound impact on the management of patients with cancer.1 21There are also new potential barriers to the pathway that have resulted and may exacerbate these findings. For example, decreases in health-seeking behaviour due to the fear of acquiring COVID-19 infection through interactions with healthcare settings, increasing the use of remote consultations,24Similar issues have also been identified within the health systems of other high-income countries. Primary care providers in eight European countries experienced similar issues in how to rapidly transform services in the wake to COVID-19.31–34While it was already known that there had been a substantial reduction in the number of overall cancer-related referrals,This reduction in cancer pathways through primary care needs to be put in the context of wider disruptions. The suspension of national cancer screening programmes meant that around 2 million people were not screened for cancer through national programmes.36In our analysis of urgent cancer referrals by site in relation to reductions seen in first treatments, significant differences were seen, which is also reflected in the international evidence. Urological cancers  have been particularly impacted with greater than 25% decrease both in urgent referrals and first treatments. This suggests that outcomes will be particularly impacted in this group. Lung, skin and lower gastrointestinal  cancer also experienced significant declines in urgent referrals; in the Netherlands, there was a 60% reduction in skin cancer diagnosis during the first wave.28Breast cancer was the least impacted of all in terms of urgent referrals but experienced a 25% reduction in first treatments. This highlights how much breast cancer diagnosis relies on screening programmes which have suffered badly as a result of the pandemic in England44Differences in systems, populations and NPI from the pandemic present high levels of complexity in tackling the recovery at both national and local levels. Although it is possible that, in many countries, some patients with cancer have already been ‘lost’ to the system, that is, died of COVID-19 or other non-COVID-19 comorbidities, a significant number will now present with later stage disease, creating further pressure on acute cancer care.Our findings also reflect socioeconomic inequalities, with more profound decrease in urgent cancer referrals and first treatments for the most deprived populations despite relatively better preservation of consultation rates. This is unexpected and extremely worrying, indicating greater disruption to the diagnostic pathway for patients living in more deprived areas, whose cancer outcomes were typically worse than their less deprived counterparts prepandemic.This study uses multiple data sets to analyse a complex and disjointed pathway. We include a primary care data set that uses a relatively small 500 000) patient sample. However, the CPRD data produce results that closely mirror the rates of consultation per patient (and their reduction) produced in NHS Digital’s appointments data.0 000 patOur data reflect a disruption to a complex interaction of several systemic issues that place a great deal of impetus on the role of primary care in ensuring early diagnosis of cancer. Primary care was already under strain prepandemic, with low levels of investment and workforce deficits.7 8 10Early cancer diagnosis requires concordance of each participant and mechanism—including patients’ awareness and ability to present with cancer symptoms, the ability of GPs to detect and urgently refer possible cancer cases and sufficient diagnostic capacity (in terms of both workforce and equipment) to enable swift referrals and minimise delays to diagnosis and treatment. Every one of these nodes on the pathway to early diagnosis has been affected by the pandemic and the national policy response. However, further work is required as there is currently little understanding and even less evidence about how much each disruption is ultimately impacting cancer pathways.The impact of the pandemic on cancer diagnosis and time to treatment shown here is very serious. However, what is more concerning is the unequal and inequitable impact on those worst off. Cancer as a disease area ‘magnifies what we know to be true about the totality of the health care system. It exposes all its strengths and weaknesses’.


The interaction between two proteins may involve local movements, such as small side-chains re-positioning or more global allosteric movements, such as domain rearrangement. We studied how one can build a precise and detailed protein-protein interface using existing protein-protein docking methods, and how it can be possible to enhance the initial structures using molecular dynamics simulations and data-driven human inspection. We present how this strategy was applied to the modeling of RHOA-ARHGEF1 interaction using similar complexes of RHOA bound to other members of the Rho guanine nucleotide exchange factor family for comparative assessment. In parallel, a more crude approach based on structural superimposition and molecular replacement was also assessed. Both models were then successfully refined using molecular dynamics simulations leading to protein structures where the major data from scientific literature could be recovered. We expect that the detailed strategy used in this work will prove useful for other protein-protein interface design. The RHOA-ARHGEF1 interface modeled here will be extremely useful for the design of inhibitors targeting this protein-protein interaction (PPI). This virtual step is even critical for drug discovery, as examplified with the successful targeting of 50 PPIs by small molecules  described Ottmann, , but onlThe binding mode prediction of two proteins is very challenging since: (i) minor to major local structural rearrangements may be triggered upon protein recognition, (ii) one protein may recognize multiple proteins, and (iii) cofactors/nucleic acids may be involved to enhance or stabilize the interaction. The analysis of the various existing protein-protein interfaces available in the Protein Data Bank indicates also that (i) the interface area varies greatly between protein families, (ii) the composition in amino acids in this interface may be biased, and (iii) the binding lifetime is transient. Due to the complexity of modeling these diverse PPIs, there are many methods developed, and a global evaluation called CAPRI is performed periodically. We use the most robust and successful methods validated in this competition for protein-protein docking evaluation of the optimal binding mode of our example , the second protein partner is Rho Guanine nucleotide Exchange Factor 1 (gene ARHGEF1). RHOA is a member of the RAS superfamily of small GTPases recognized as a master regulator of the actin cytoskeleton, thus driving multiple cellular processes, such as cell contraction, migration, proliferation, and gene transcription. While basal and controlled RHOA activity is required for homeostatic functions in physiological conditions, its uncontrolled overactivation plays a causative role in the pathogenesis of several diseases, such as cancer, neurodegenerative, or cardiovascular diseases . RHOA possess two hinge regions, a loop called switch I (29–42) and an helix called switch II (62–68), which are described to be more flexible than the core β-sheet  linked by helices and loops .A BLAST sequence search on the Non-Redundant (NR) database with RHOA or ARHGEF1 sequence as query was performed in June, 2018. The resulting sequences were aligned using clustal Omega   is a webserver for quantifying the strength of a protein-protein interface      ATTRACT ff2g (http://research.bmh.manchester.ac.uk/bryce/amber) and converted to GROMACS format files using acpype  on recovering existing crystallographic structures of RHOA bound to a GEF, a process called re-docking, and (ii) on assembling the bound RHOA from a given crystallographic with a GEF from another crystallographic structure, this process being known as cross-docking.a priori which method will produce the most reasonable starting complex for further studies. We assessed the top five performing docking software from CAPRI assessment, briefly introduced in As docking strategies are based on different methods, it is difficult to determine We evaluated the performance of each software according to its ability to recover the existing structure of bound RHOA and ARHGEFs. This method called re-docking allows to discriminate the accuracy of the algorithm studied on our system. The web servers define the first input protein given as the receptor, so it stays fixed (F) and considers the second protein provided as the mobile protein (M). We performed our analysis with the small GTPase or the GEF as (F) or (M). The results are presented in We verified the dependence on the initial structure for the protein-protein binding mode prediction. We applied a cross-docking experiment where each bound RHOA in one crystal structure is evaluated against another GEF partner. We used the free RHOA structure as a sensitivity control for our cross-docking measurements. There is no dependence for the docking result linked to the pdb input for RHOA or GEF. The results are available in Considering the results of the re/cross-docking, we selected ZDOCK as the best method to predict the more favorable binding mode between RHOA and ARHGEF1 .Since no experimental structure of the RHOA-ARHGEF1 interface is available, we used the crystallographic structures of other GEF paralogs bound to RHOA to determine which amino acids are shared in all complexes. The amino acids mapping to RHOA and ARHGEF1 was done using use multiple sequence alignments comparison (data not shown). Four residues are conserved in GEFs interfaces, namely, E423, Q563, R551, and N603 in ARHGEF1. As can be seen in As done for ARHGEF1, we also analyzed the interface residues of the other GEFs present in each crystallographic structures in the complex with RHOA and assessed their amino acids conservation using multiple sequence alignments. Four residues of ARHGEF1 are present at the interface, and 10 for RHOA. As can be seen in http://redshift.med.unc.edu/chiron/)  hydrogen bonding (Ehyd), (ii) inter-chain van der Waals interactions (Evw), and (iii) inter-chain electrostatic interactions (Eele). The total stabilizing energy is then divided by the total number of interface residues to obtain the energy per residue. No significant deviation requiring further refinements with the Chiron webserver was present in both models.Since there are some experimental structures of RHOA bound to ARHGEF1 homologs, we also predicted the bound structure of both proteins with a simpler approach, based on structural superimposition in PyMOL. We first used a rhoA-bound crystal structure and superimposed the free ARHGEF1 on all the homologous GEFs. By doing so with rigid models, we could not take into account the concerted induced-fit required for finely tuning the interaction. We used a webserver from Dokholyan Team named Chiron which allows to relax the most important steric clashes . Starting with two different models, we observe an augmentation in protein surface contact driven by local adjustments. When considering individually each protein at the RMSD level, there is a higher deviation for RHOA than ARHGEF1, implying that RHOA undergoes most of the conformational changes, as we will see in details below.Both complexes were modeled using molecular dynamics simulations to determine if the interface could be refined during this procedure. The initial surface area in complexD, 2,830 Åmulation . The molTo understand the evolution of interface complex during the simulations, we analyzed the hydrogen bonds between the two partners. The result is shown in Since no experimental structure of RHOA-ARHGEF1 was available from X-ray studies, NMR, or EM studies, we had to model it. Using the protein sequences of Rho and GEF families, and the existing protein structures of bound homologs RHOA-ARHGEF8  selected from ZDOCK contains 5 out of 10 shared amino acids in RHOA and 3 out of 4 shared amino acids from ARHGEF1, with an interface surface area of 2,830 Å2.The prediction of protein-protein interface using docking methods is still an important field of research  allowed to correctly find the position of 2 out of 10 amino acids in RHOA and 2 out of 4 amino acids from ARHGEF1.As the docking experiments are time-consuming and contain also uncertainties, we did also a more crude approach using PyMOL. We analyzed the existing structures of RHOA bound to other members of the ARHGEF family to find the best starting template for our structural comparison. A superimposition of ARHGEF1 (3ODO) on RHOA-ARHGEF11 (3T06) was then performed in PyMOL and further refined using Chiron . This alternative positioning of RHOA in comparison to other members of the GEF family is also present in crystallographic structures. Both complexT and complexD seemed therefore reasonable starting complexes, with a comparable building time of 1 day for both protocols: instant for PyMOL superimposition plus 1 day for removal of clashes in Chiron and 1 day for ZDOCK prediction.Both methods allowed to define comparable starting complexes of the RHOA-ARHGEF1 interface from rigid templates. Major steric clashes were carefully examined using Chiron  in comparison to the initial complex . We identified amino acids conserved in all RHOA-ARHGEF complexes by combining structural sequence analysis  , ComplexD and complexT only have a global RMSD difference of 1.4 Å between them, but at the end , the RMSD rises to 3.7 Å. Since the RMSD is a global measure of the movements, i.e., both proteins moved, it is important to understand how proteins evolved independently during the simulations.Both complexT and complexD lead to a similar RHOA-ARHGEF1 interface at the end of the simulation. At the beginning of the simulation  from its initial structure than complexT (2.9 Å). This apparent difference comes mostly from a larger movement of the PH domain in the complexD simulation since the RMSD between the initial structure and the end of the simulation concerning only the ARHGEF1 protein is 18 Å. This apparently large difference in ARHGEF1 position for complexD is the consequence of two movements: (i) the local rearrangements of the DH domain (the core RHOA binding domain of GEFs), which is similar in both complexD and complexT (6 Å), and (ii) a larger movement of the PH domain which may be involved in the nucleotide exchange. When aligning the DH domain in both trajectories, we, therefore, see a more important rotation of RHOA relative to ARHGEF1 for complexD (6 Å) than for complexT (3.5 Å) as illustrated in Both complexes are refined after molecular dynamics simulations, many important amino acids saw an increase in contact frequency and the position of RHOA relative to ARHGEF1, either inherited from the superposition onto ARHGEF11 or from the docking studies with ZDOCK, led to a strong convergence of the interaction. This study confirms the interest of using molecular dynamics simulation to increase model quality.2, the mean surface area for all complexes of RHOA/GEFs available so far. We could identify some specific contacts for RHOA-ARHGEF1 from the complexT, namely D59-K567, Q63-T566, which were not described previously  contacts and for the complexD 4 over 10 (mostly with E423). After simulation for complexT, we can see 8 over 10 conserved contacts and for complexD, there are 6 over 10 contacts during a short time frame where the interface SAS is the highest. Some contacts are seen only thanks to the simulations and one question rises, what is more important to select for qualifying the best model? Its higher number of contacts or the presence of conserved/important contacts? In our case, both models show conserved/important contacts and new contacts specific to each model. The amino acids detected as hotspots are not conclusive since they are present in both trajectories. During the simulations, even if starting from somewhat different structures, the interaction between ARHGEF1 and RHOA converges. Our model building strategy clearly indicates that a molecular dynamics simulation, starting from rationally designed PPI, improves the initial models.2, slightly better than complexD model before refinement , but far from the refined interface obtained after molecular dynamics simulations . Even if it is possible to build a reliable model by integrating various data in HADDOCK, a long molecular dynamics simulation, with a simulation time above 250 ns is still required to enhance the quality of a PPI  for producing the initial interaction mode. In our study, ZDOCK was better if precision, robustness, and time are taken altogether into consideration. When possible, we recommend to perform long molecular dynamics simulations to enhance the network of interaction between both proteins and to get a better overview of the lifetime of each interaction. More generally, we expect these strategies will be successfully applied to a variety of targets where a partial structural coverage of both partners is known, provided the complex to model has characteristics comparable with the two proteins described in this article.Our study aims at exemplifying how one can model a protein-protein interaction when sufficient experimental structures are present, but only experimental data for close homologs are available. We set up two different strategies summarized in The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ST and GL obtained the funding. EG did the experiments and analysis. BO and GL supervised the work. EG and ST wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


In recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells. Therefore they have become one of the main research and development topics in cell biology of recent years3. Nevertheless, the clinical translation of these models has been very limited due to a lack of reproducibility of the results4. Even though some spheroids have found their way to clinical trials, especially for applications such as bone regeneration, it has been more controversial for softer tissues5. Most protocols rely on the use of poorly defined hydrogels which affect cells with their chemical and biomechanical properties6. Furthermore, the use of recombinant growth factors or products from animal origins in these hydrogels is a major issue for human clinical application of these spheroids.3D cell cultures have shown closer biological properties to cells in a physiological context than 2D cell cultures7, non-adhesive surface8 or rotating bioreactor9, have shown spheroid fabrication as 3D cell culture models. Some disadvantages such as variation in spheroid size and shape still remain unsolved10. However, several methods create homogeneous spheroids, including liquid overlay technique, agarose mold wells and ultralow attachment spheroid microplates. Spheroid formation by these methods depends on the contractile properties of the cells and may be less convenient for low contractile cells11.Alternatively, several scaffold-free methods, such as hanging drop12. Besides the various applications of cell focusing or separation13, acoustic methods have been used for tissue engineering and in vitro techniques14. Many works16 studied cell behavior with a short-time ultrasound exposure, classically around 1 h and used it to instantaneously shape complex geometries like layer17, multi-layer18 or spheroid19 in order to culture it in usual way.Thanks to its excellent biocompatibility and versatility, acoustic approaches are promising tools for contactless manipulation of cells20 by clustering and trapping them in multiple disk-like layers in acoustic levitation, located at the different pressure nodes of a resonant cylindrical cavity21. After a self-organization step, the spheroids could be maintained and cultured in levitation, with a system of perfused culture medium.Here, we report the fabrication and the culture of several spheroids over a 24-h period in an acoustofluidic chip with a well-controlled cell culture environment. The acoustic forces act on the suspended cells22. It is also transparent, allowing optical accessibility and is easy to process.We designed an acoustofluidic chip made of a PDMS (Polydimethylsiloxane) body bonded to a microscope cover-glass. We chose PDMS material because it is highly biocompatible and permeable to the air, making it ideal for cell cultureA 2 MHz ultrasonic transducer Fig. a was insWe first demonstrated the functionality of our system using a suspension of mentclass2pt{minimWe then evaluated our system for culturing human cells. MSCs are primary cells that can been isolated from various tissue sources including the umbilical cords, adipose tissue and the bone marrow. They are characterized by their ability to adhere to plastic, by the expression of certain surface markers, as well as their capacity to differentiate into adipocytes, osteoblasts and chondrocytes. These cells are used in many clinical and tissue engineering applications. We decided to use this cell model as a proof of concept for our system. We isolated these cells from human adipose tissue.For the acoustic levitation experiments, the cells were injected in the cavity at a concentration of 1.5 million cells/mL and cultured for 24 h. The setup has been optimized to allow a constant flow of nutrients. The cell culture medium was injected at a rate of The dynamics of the self-organization into spheroids was studied through image processing of the video time series. The axial mentclass2pt{minim23.We evaluated the viability of the cells after a 24 h culture in acoustic levitation using calcein and propidium iodide, which showed that the majority of the cells were alive at the surface of the spheroid Fig. d. HoweveThe spheroids were then reseeded in culture plates Fig. a, and th12. Only a few studies pushed the culture time beyond 24 h. However, either the device was limited to a single aggregate24 or the method produced small aggregates with no levitation  device allows the production and the culture of hundreds of large spheroids with a reproducible size. The CCMAL setup can be easily scaled up to culture thousands of spheroids in acoustic levitation in parallel. It is well adapted for MSC cultures, preserving their viability, proliferation and characteristic surface markers. It also increases their potential of differentiation. It has been previously shown that high density of MSCs in 2D culture, as well as spheroid formation in low attachment dishes26, or the application of ultrasound27 enhances their osteoblastic potential. In our system, the rapid formation of spheroids mimics both high confluency of 2D cultures and spheroid formation owing to the acoustic confinement . Unlike spheroid formation methods which rely only on the contractile properties of cells, our system has the advantage of trapping cells in acoustic traps and forcing them to interact which facilitates cell sheet and spheroid formation. This approach opens the path to a better understanding of these phenomena and application to many other different types of cells. It also demonstrates the use of acoustic levitation as a new method of long-term, reproducible cell culture process for scaffold-free tissue engineering purposes such as organoid formation and multicellular co-cultures.We hereby demonstrate the development of a robust and reproducible cell culture system based on acoustic levitation using a PDMS chip and an ultrasound transducer, with the possibility of a constant flow, which allows culture medium renewal. Even though previous studies have shown the possibility of using acoustic levitation for cell sheet and aggregate formation, these studies were limited to a very short treatment period usually less than 1 hFor each experiment, we ran four cell culture in multi-trap acoustic levitation (CCMAL) devices in parallel in order to increase the number of spheroids and maximize the cell preparation. One has to control the acoustic levitation process, the optical observation, the fluid perfusion as well as the biological part from the cell preparation to the post-levitation analysis and the collect of all samples.iePie Engineering, Sneek, Netherlands) monitored by a computer. Each output of the signal generators supplied two ultrasonic transducers  with a sinusoidal waveform of amplitude The ultrasonic waves were generated by a transducer driven by an arbitrary waveform generator  body bonded on microscope cover-glass and a mono-element ultrasonic transducer. The geometry of the PDMS chip is designed to allow the insertion of the ultrasonic transducer inside the PDMS body. The transducer closed the acoustofluidic cavity. One side of the resonant cavity is ultrasound emitter, in contact with the fluid, avoiding acoustic losses through various interfaces, while on the other side the microscope cover-glass acts as a powerful acoustic reflector and allows an optical access. The overall acoustic resonant cavity is simple and efficient.The chip is composed by a PDMS . Then the PDMS is poured in and put in the oven at To shape the PDMS body, we use a negative mold made with a 3D printer  in a withdrawal mode with a flowrate of The culture medium was continuously perfused in the chip with a syringe pump  for each spheroid. Then an ellipsoid is fitted on the binarized image. The section of the original image passing through the ellipse center is selected in the axial and radial directions. We stored the width of the 70% thresholded curves of the X and Y sections . The adipose tissue was placed in 40 mL of collagenase NB6 diluted in hermofisher, Nunc, Waltham, United States) for expanding in MSC culture medium composed of ibco, Thermofisher, Waltham, United States) supplemented with 10% fetal bovine serum  and 1% antibiotic/antimycotic mix .MSCs were isolated from adipose tissue of the thigh of a healthy donor, after a signed consent, according to the French regulations. MSCs were obtained after the digestion of adipose tissue with collagenase NB6 , CD73-PE , CD90-FITC , CD105-PE , and negative markers including CD31-FITC  and CD34-APC . Unstained cells and cells stained with control isotypes have been used as negative control.At the second passage, cells were characterized by flow cytometry using a panel of MSC positive markers including CD29-PE  diluted in PBS . They were then embedded in a fibrin gel to facilitate manipulation for preparing samples. The fibrin gel containing the spheroids was included in paraffin and gilent-Dako, Santa Clara, United States) 3-step immunoperoxidase technique.After 24 h of acoustic levitation, the spheroids were collected and immediately fixed overnight in paraformaldehyde 4% . Cell viability was further evaluated with a LIVE/DEAD Viability/Cytotoxicity Kit . The components  were diluted in PBS according to the provided instructions of the kit and the spheroids were incubated in the solution for 30 mins in an Ibidi dish prior to observation with a confocal microscope .To evaluate the capacity of MSC spheroids, which also shows their viability, they were placed in 24 well dishes and were observed during 24 h in an Ittune flow cytometer .After 24 h of acoustic levitation, MSC spheroids were collected and reseeded in The potential of differentiation of the aggregates into adipocytes and osteoblasts was also evaluated. For this purpose, the spheroids were collected and reseeded in a 24 wells plate at a density of one spheroid/well in MSC expansion medium. A 2D culture of cells at the same passage that had not undergone levitation was also used as control. These cells were about temXVivo, R&D systems Inc., Minneapolis, United States). MSC expansion medium was also used as the control condition. After a 2-weeks culture, cells were fixed with 4% paraformaldehyde. Adipocyte differentiation was permeabilized with isopropanol for 5 mins and the lipid vesicles were stained with oil red-O . Osteoblasts were stained with alizarin red (Sigma-Aldrich).After allowing the cells to adhere and expand for 3 days, the cell culture medium was replaced by adipocyte differentiation medium or osteoblast differentiation medium  governing scientific research on human subjects and tissue samples, the surgical leftover can be used for scientific research without the prior approval of an ethical committee.Supplementary Information.Supplementary Video 1Supplementary Video 2Supplementary Video 3Supplementary Video 4Supplementary Video 5Supplementary Video 6Supplementary Video 7


Several countries have targeted adolescents for immunization against SARS-CoV-2 to mitigate COVID-19 spread. In Italy, immunization for children ≥ 12 years has been available starting from June 2021. We conducted a cross-sectional study to investigate the knowledge, attitude and intention to vaccinate children < 18 years in Italian families. We used a multinomial logistic regression model to investigate factors associated with intention to vaccinate. We collected a total of 1696 responses. Among the 491 families of children ≥ 12 years, 41.2% would not vaccinate their children and 21.2% were uncertain, while among the 1205 families of children < 12 years, 36.1% would not vaccinate and 33.8% were uncertain. Determinants of intention to vaccinate both age groups were perceived safety and efficacy of vaccines and perceived risk of transmitting infection to adults. For children < 12 years, additional determinants were perceived risk of being infected and being hospitalized because of COVID-19. In view of the expanding strategy to vaccinate adolescents and the availability of immunization for children < 12 years, our results call for a communication strategy targeted at families of children focused on the safety and efficacy of COVID-19 vaccine in children and on the dynamics of infection spread across different age groups. As perceptions in families are volatile and may change rapidly over time, repeated surveys for measuring attitudes to vaccinate would be advisable. The World Health Organization estimates that almost 230 million cases of COVID-19 have occurred worldwide as of September 2021, with more than 4.5 million deaths [COVID-19 severity and mortality increases with age, with the highest figures seen in >85 years and in younger individuals with co-morbidities . In chilDuring the first phase of the pandemic in Italy, incidence rates were higher in adults and in the elderly. After a severe restriction in social contacts from March 2020, including school attendance, travels and social gatherings, limitations were gradually lifted in summer 2020 . StartinImmunization of the elderly and adults started in Italy at the beginning of 2021, while COVID-19 vaccines have been actively offered to teenagers 12–18 years since June 2021. Although the role of children in sustaining the circulation of SARS-CoV2 seems limited ,14, immuMonitoring intention to immunize children may be important to predict immunization rates in this age group and in children less than 12 years old who will possibly be shortly targeted by the immunization campaign .Variable levels of vaccine hesitancy for COVID-19 childhood immunization have been observed in different countries, including the US and Italy ,17, at dAn attempt to monitor vaccine intention over time has been developed by Johns Hopkins Center for Communication Programs in collaboration with Facebook, which is updated every two weeks . This inOn the other hand, no surveys have been conducted recently to gather information on vaccine intention and vaccine confidence toward COVID-19 children’s immunization and their determinants, since vaccines became approved in Italy for children older than 11 years.For these reasons, in July and August 2021 we conducted a survey on parents of Italian children < 18 years old to investigate their intention to vaccinate their children and the factors associated with their attitude.® , after providing informed consent online. Bambino Gesù Children’s Hospital is a 607-bed tertiary care academic hospital located in the Lazio Region, Italy. It is one of the largest pediatric hospitals in Europe, with more than 2 M healthcare services provided yearly to a wide population of children and adolescents. The survey was active from 22 July to 31 August 2021. Participants were asked to promote the study through their contacts, including through social networks. The only inclusion criterion was having at least one child younger than 18 years of age.This is a cross-sectional study, conducted in July and August 2021, investigating intention to immunize their children and confidence in the COVID-19 vaccine in a population of parents of children < 18 years. Parents accessing the Emergency Room of the Bambino Gesù Children’s Hospital, IRCCS , were invited to fill in an online, anonymous survey via SurveyMonkeyThis study was approved by the Ethics Committee of the Bambino Gesù Children’s Hospital in Rome (protocol 1059).Data were collected using a structured questionnaire, which included 21 items divided into 4 sections.In the first section, socio-demographic and other general information were investigated through the following items: age; sex; region; study title; number of household members (including the respondent); number of children; age and school attendance for each child; presence in the household of individuals affected by one of the underlying diseases for which the COVID-19 vaccination is recommended; having received the influenza vaccine in the 2020/2021 season; respondents and/or other household member’s history of COVID-19 ; having received a vaccine against COVID-19 .The second section investigated perception of COVID-19 and COVID-19 vaccination: perceived risk of getting COVID-19 in children < 18 years; perceived risk of COVID-19 hospitalization in children < 18 years; perceived risk of children affected by COVID-19 transmitting the virus compared to adults; perceived effectiveness of COVID-19 vaccines; perceived risks of COVID-19 vaccines .The third section investigated attitude towards children’s COVID-19 vaccination, i.e., intention to vaccinate children when vaccine is available for their age range . Those who declared that they would vaccinate or that had already vaccinated their child were asked to provide an explanation of their choice from the following : vaccine against COVID-19 is the quickest way to have my child back to normal life; it will protect my child from COVID-19 and its complications; it will contribute to preventing COVID-19 in our family; it will contribute to herd immunity; I unconditionally trust vaccines. Those who declared that they would not vaccinate their child or that they were undecided were asked to provide an explanation of their choice from the following : I have fear of side effects; vaccines have not been studied enough in children; the risk of getting COVID-19 in children is low; the risk of developing COVID-19 complications in children is low; the COVID-19 vaccine has a low efficacy; I don’t trust vaccines in general; I do not know.The sample size for the survey was calculated according to the formula adopted in the Raosoft software . SettingCategorical variables were summarized as frequencies and percentages while continuous variables were presented using median and interquartile range (IQR), since the variables were non-normally distributed. Normality was assessed both graphically and using the Shapiro–Wilks test.We carried out multinomial logistic regression to investigate the association between the socioeconomic characteristics, the other variables investigated in the questionnaire and the outcome. The studied outcome was the intention of parents to immunize children categorized into three groups: (1) those who declared a positive intention to vaccinate or those who had already vaccinated their children; (2) those who were uncertain about their children’s vaccination; and (3) those who would not have their children vaccinated. We stratified the model by children’s age (<12 years vs ≥12 years). To define this category, we considered the age of the eldest child .We carried out multiple imputations with chained equations  to generData analysis was performed with STATA 14.1 SE .A total of 2808 persons accessed the online questionnaire via the provided link. Of these, 425 were excluded as they were not eligible as they did not have children, 610 accessed the questionnaire link but did not give consent to participating in the study and therefore were not included, and 77 did not complete the questionnaire. A total of 1696 participants were thus included in the study. Of these, 491 were parents of at least one child ≥ 12 years, while the remaining 1205 (71.0%) only had <12 year-old children.A total of 1381 respondents (81.6%) were mothers and 312 (18.4%) were fathers, with a median age of 42 years (IQR 37–47). Most respondents were from Central Italy (72.9%), followed by Northern Italy (20.6%) and Southern Italy (6.5%). Most parents  had a university degree or a higher level of education, one quarter of participants had a high school certificate  and only 2.9% (48/1696) had a primary or secondary school certificate.Most families had three  or four  household members. In parallel, most families (50.7%) had one child or two children (40%). Seventy-four (15.4%) families had at least one person with an underlying disease.Among the responding parents, 30% had received the influenza vaccination for the 2020/2021 season. A total of 278 (16.8%) had been affected by COVID-19 in the previous months. Most of the responding parents  had received the COVID-19 vaccine or had scheduled their vaccination, 17 (1%) intended to get the vaccine but had not received it yet as they had recently had COVID-19. Almost one fourth of the respondents  stated they did not want to receive the vaccine, while 7.0% (115/1696) were undecided.As for children ≥ 12 years, most parents stated they would not vaccinate their children . The proportion of parents with a positive vaccination intention  was 37.61% (95% CI 33.2–42.1). Undecided parents represented 21.22% of the sample (95% CI 17.63–25.17).For children < 12 years, most parents declared a negative vaccine intention . In this age group, the proportion of parents of children already vaccinated was extremely low (0.26%), as the vaccine has been offered only to children <12 years with underlying diseases, with a very high risk of COVID-19. Overall, the proportion of parents of children <12 years who declared a positive vaccine intention was 30% (95% CI 27.4–32.8). Undecided parents represented 33.83% of the sample (95% CI 31.1–36.6).A total of 363 (22.5%) persons stated that the available vaccines were almost completely ineffective. A very high proportion of parents stated that vaccines could cause serious side effects in children  and 21.9% (372/1696) believed that vaccines put children’s lives at risk .Among parents of children < 12 years, those with a positive intention to vaccinate  provided as the two main reasons for their positive vaccination intention that the vaccine would protect their child from COVID-19 and its complications (73%) and will contribute to herd immunity (71.2%). More than a half (57.3%) stated that the vaccine was the quickest way to have children back to normal life, and for slightly less than a half (48.8%) the child’s vaccine would protect the rest of the family. In the same age group, among those with a negative intention to vaccinate and the undecided, the most frequently reported reason for not vaccinating or being hesitant was that the vaccines had not been studied enough in children (73.4%). Fear of side effects was reported by 46.3%. Only 8% of respondents stated they did not trust vaccines in general.Among parents of children ≥ 12 years, those with a positive intention to vaccinate  provided as the main reasons for their positive vaccination intention that the vaccine will contribute to herd immunity (71.5%). A total of 64.8% stated that the vaccine was the quickest way to have children back to normal life, and 59.8% stated that the vaccine would protect their child from COVID-19 and its complications. In the same age group, one third of those with a negative intention to vaccinate and the undecided reported that the vaccines had not been studied though in children (66.3%). Fear of side effects was reported by 53.9%. Almost 30% reported that the COVID-19 vaccine had a low efficacy, and 13.5% of respondents stated they did not trust vaccines in general.According to the multinomial analysis (reported on For parents of children ≥ 12 years, fathers significantly more frequently stated they refused to vaccinate their children compared to mothers. Other factors significantly associated with a negative intention to vaccinate were believing that children had a low risk of transmitting the virus to others compared to adults, believing that the vaccine efficacy was lower than 70% and believing that vaccines were dangerous. For the same age group, factors significantly associated with being undecided were: believing that children had a lower or absent risk of being hospitalized by COVID-19 compared to adults, believing that the vaccine efficacy was lower than 70% and believing that vaccines are dangerous.After several disease waves and a very high toll in terms of deaths and disabilities, we now have efficacious vaccines available to combat the global spread of COVID-19. Most countries, after a vaccination campaign targeting the elderly, patients with chronic diseases and, subsequently, adults of other age ranges, have started focusing on younger persons to slow down the virus circulation. The rapid introduction of a novel vaccine for children should be accompanied by a comprehensive communication strategy, which may not be able to be fully accomplished during a pandemic, due to time and resource constraints. Studying the relationship between concerns and vaccine behavior of target populations and measuring their level of confidence toward SARS-CoV-2 vaccines is crucial to tailor information strategies and to prevent low immunization coverage.Our survey shows alarming results regarding the intention of families to immunize their children, which is lowest in parents of children below 12 years of age. When reviewing the current literature on this topic we found similar results in several other studies performed in other countries at different times, including the US, Germany, Australia and China ,24,25,26On one hand, it is expected that intention to vaccinate is lower for novel vaccines compared with already available ones. On the other hand, this should prompt public health agencies to rapidly implement tailored information strategies based on current data on public information needs.As intention to vaccinate and vaccine confidence may vary over time because of different circumstances, we conducted a survey immediately after the start of immunization campaigns for 12–18-year-old children, when SARS-CoV-2 vaccines were not yet available for those under 12. The actual monitoring of immunization in children 12–18 reports that nearly 60% of the target population in this age group received two doses of the vaccine as of September 2021 . AlthougTogether with intention to vaccinate, we explored reasons for vaccine confidence and vaccine hesitancy as reported by interviewed families. In essence, there is room for improvement of families’ knowledge about the role of children in sustaining the circulation of infection, the safety of vaccines, and, most importantly, the way vaccines are developed and tested in children, which is the main reason for a negative intention to vaccinate and for vaccine hesitancy in our sample. These areas of knowledge need to be better addressed to create a solid foundation for public trust in COVID-19 vaccines.In the multinomial variable model, we considered the uncertainty regarding children’s vaccination and the more definite refusal of children’s COVID-19 vaccines as two different outcomes, as these behaviors likely require different communication approaches.Being against immunization of children younger than 12 or being uncertain about the vaccine for this age group were associated with three main beliefs. First, parents who did not receive the flu vaccine for the current season were more likely to be against COVID immunization for children. Uncertain parents were also more likely to have received a COVID-19 vaccine themselves. This finding suggests an association with general concerns about vaccines. Secondly, parents who believe that COVID-19 vaccines may cause major side effects and have suboptimal efficacy were more likely to refuse immunization of children or being uncertain, underlining an information gap. Thirdly, parents who are against immunization of children or are uncertain perceived that COVID-19 in children is unlikely and that the disease does not cause major complications in this age group, also suggesting an area for improving information.When looking at parents of children older than 12, only perception of a low efficacy of vaccines and concerns about safety were associated with refusing immunization. Uncertain parents were more likely to have received a COVID-19 vaccine, perceived a low efficacy of vaccines, and believed that the risk of complications in children is low. Of note, being a father and a decreasing age of parents were associated with refusing COVID-19 immunization for their children in this age group.A perceived low risk of transmission from children to other individuals was associated with refusal of COVID-19 vaccines in children of both age groups, but not in uncertain parents.This information may be useful for developing communication strategies to promote COVID-19 immunization with specificities. For instance, communication strategies are generally focused on families but not specifically on fathers, and there are no strategies focused on adult recipients of the COVID-19 vaccine to promote immunization to their relatives. Moreover, addressing concerns on safety and efficacy of vaccines in children should be complemented with information about the development and testing of immunization products for this age group.This work has strengths as it was conducted in a very specific time frame which may be important to understand the relationship between the public perception and their behavior in immunization decisions and included a large population. Moreover, the data analysis was conducted to address the determinants of opposition and vaccine hesitancy separately, offering specific information for communication strategies.This survey also has limitations. As we used a convenience sample, we may have recruited respondents in the emergency room more interested in the discussion about vaccines and amplified the negative perceptions when compared to the general population. Families in the emergency room may also have been distracted by their actual child’s health problem. Moreover, we did not investigate the cause for ER access of participants and we could not adjust the analysis for this factor. Indeed, only a small fraction of the respondents had had their child already vaccinated compared to the general population. On the other hand, we were able to properly investigate the determinants associated with vaccine behaviors in a multivariable model which adjusted for confounding for the several variables we included in the model.In conclusion, although many of the public concerns may be temporary and may not fully predict vaccine behaviors, tools for constant monitoring of information needs of the population at large during immunization strategies represent a necessary means to support vaccine confidence and uptake, reinforce trust in immunization and sustain vaccination strategies in the long term. As, based on the multivariable analysis results, fathers seem to be more frequently hesitant compared to mothers, this suggests the opportunity to assess the potential effectiveness of vaccine promotion campaigns tailored to fathers for the >12 years group. Moreover, vaccine promotion campaigns may take advantage of contacts at the time of adult immunization to promote COVID-19 vaccine for children. Moreover, a wider spread of knowledge regarding COVID-19 development process and safety should be pursued.The balance between the perceived epidemiology of vaccine-preventable diseases and perceived vaccine efficacy and safety are of paramount importance for the success of immunization strategies and require efforts to address the concerns of the public. Misconceptions and perception of high risks associated with vaccination in children may be a cause for concern in parents of child candidates to receive a SARS-CoV-2 vaccine, despite the benefits clearly outweighing the risks even in young children . Parents


The coefficient alpha indicated an acceptable level of internal consistency . Test-retest reliability for total FS intake assessed by the FFQ was positively high (rs = 0.82). A slight agreement between FS intake assessed by the FFQ and the 24-h dietary recall was seen among the study sample . The newly developed FFQ was found to be reasonably valid in assessing children's habitual intake of FS in Saudi Arabia. Validating the instrument among older population in Saudi Arabia is warranted.The World Health Organization emphasizes the urgency to assess and limit the intake of free sugar (FS) among individuals in order to prevent several non-communicable diseases. However, data regarding intake of FS are lacking in Saudi Arabia and in the Middle East. A reliable valid tool is needed to assess children's habitual intake of FS. Thus, we aimed to develop and validate a food frequency questionnaire (FFQ) that assesses children's habitual intake of FS in Saudi Arabia. In this cross-sectional study, 424 healthy Saudi children ages 6–12 years were included using river sampling method. Sociodemographic data and contact information were collected from mothers using an online survey. Dietary data were collected using 24-h dietary recalls (reference method) and a semi-quantitative FFQ through phone interviews. Items and food groups included in the initial draft of the FFQ were adopted from previous work. Content validity was done to the FFQ with total of 12 food groups and 41 food items. Next, a pilot study was conducted to estimate the sample size needed for the study and to ensure that all items reported in the 24-h dietary recalls were included in the final draft of the FFQ. Finally, data were collected to assess the validity and reliability of the FFQ at the population level. Mean intake of FS assessed by the FFQ was significantly higher than that assessed by the 24-h dietary recalls. Spearman's correlation between total FS assessed by the FFQ and 24-h dietary recall was positively low ( The global increase in non-communicable disease epidemics and the documented association with excessive free sugar (FS) consumption had led many countries to establish fiscal policy interventions to limit intake of sugary foods, and it became a global health agenda , 2. The The term “free sugar” is used to describe mono- and disaccharides that are naturally present in syrups, honey, and fruit juices, and sugars added to food by consumers or manufacturers, whereas “added sugar” does not include sugar occurring naturally in foods , 12. DesLimiting FS intake among children has been a priority for many intervention programs given that children are one of the most vulnerable groups for excessive sugar intake . Anotherr = 0.31), a minimum of 85 children was needed with alpha = 0.05 and power of 80% . The online survey was distributed for a duration of 3 months then the data collection was stopped. A text message was sent to all mothers who provided consent for their children to be part of the study to arrange for a phone interview for dietary data collection. The phone interview was conducted by trained dietetic professionals with mothers to collect the dietary data with the presence of the child and the person responsible for child's eating. Dietary data were assessed by 24-h dietary recalls and a semi-quantitative FFQ. During the phone-administered interview, the FFQ was collected followed by the 24-h dietary recall. All mothers received the portion size guide to help them estimate the portion size of foods consumed. The portion size guide included pictures of standardized serving sizes of all food groups . Pictures of standardized portion sizes of drinks, candies, and sweets were also included in the portion size guide.https://www.nutritics.com/p/references  (data source: Nutritics: Middle East/Arabic-Arabic foods/popular Gulf recipes). The Nutritics software provides data concerning content of FS specifically in addition to total sugar and several other nutrients (macro and micronutrients). When amount of FS was not specified for a local food, quantities of FS were estimated based on standardized recipes that were created in the software. The primary investigators trained the data collection team, validated the ingredients of all recipes before inserting them into the software, and monitored the data collection to ensure quality of the data collected. The dietary data were later imported into an Excel sheet in order to calculate the average of the 3 days obtained from the subsample and to prepare the dietary data of all children to be analyzed.The 24-h dietary recall was used as a reference method to assess the intake of FS. Single 24-h dietary recall from all children was collected; two additional 24-h dietary recalls (for non-consecutive days) were collected from a subsample of 165 children (38.9%) to adjust for within-person variation. Mothers were requested to remember and report the food intake of children during the past 24 hours. The data obtained from the 24-h dietary recalls were recorded on paper then entered into the Nutritics software The semi-quantitative FFQ was developed to assess the habitual intake of FS. Items of the FFQ were initially adapted from a study that identified the top food sources of added sugar among American children, of which included 17 food groups and 78 food items , we estiNext, a pilot sample of 36 children were recruited to ensure that all FS-containing food items reported in the 24-h dietary recalls were listed in the newly developed FFQ. Accordingly, a total of 12 food groups and 41 food items divided into liquid food sources and solid food sources of FS were included in the final version of the FFQ.The final version of the semi-quantitative FFQ has been later utilized to collect data of children for the present study. During the phone-administered interview, the FFQ was collected followed by the 24-h dietary recall. Firstly, mothers received the portion size guide to help them estimate the portion size of items included in the FFQ. Additionally, a table that includes the frequency response options of the FFQ has been sent to the mothers to ensure accuracy of reporting through showing the response options as follows: per day , per week , per month (< once or 1–3 times). Responses to FFQ for each child were recorded on a printed copy of the FFQ then entered into an Excel sheet that calculates the FS content. A separate Excel sheet was used for each child to calculate the total FS intake; the total intake was then inserted in the main Excel sheet that include data for all children. To assess test-retest reliability of the FFQ, data were collected one more time for a subsample of 55 children (13.0%) within 1 month of collecting the first FFQ.t-test was used to compare the difference between intake of FS assessed by the FFQ and 24-h dietary recall. All tests used in this study were two-tailed with a significant level of 0.05. Statistical Package for Social Sciences (SPSS) version 20 was used to analyze data presented in this study .Descriptive data for continuous variables shown in this study are mean ± standard deviation (SD) or median [interquartile range (IQR)], while data for categorical variables are presented as frequency [percentage (%)]. To validate the semi-quantitative FFQ, Spearman's correlation test was conducted to (1) Investigate the correlation between free FS assessed by FFQ and the reference method ; (2) to assess the reproducibility of the newly developed FFQ. Children were grouped into quartiles  to test the agreement in ranking children according to estimated FS intake from both methods. Weighted Kappa statistics was used to assess the interrater reliability across the two dietary collection tools. To evaluate internal consistency of items included in the FFQ, Cronbach's alpha test was used. To assess internal consistency of items included in the FFQ, frequency of consumption of each item was coded between 0 to 8. Responses were coded following the criteria: 0 if the response was less than once a month; 1 if the response was 1–3 times a month; 2 if the response was once a week; 3 if the response was 2–4 times a week; 4 if the response was 5–6 times a week; 5 if the response was once a day; 6 if the response was 2–3 times a day; 7 if response was 4–5 times a day; 8 if response was 6 or more a day. The Bland–Altman plot was used to assess the difference in FS intake reported by 24-h dietary recall and the FFQ compared to the mean intake of the two measures. However, a significant difference was observed between the two methods; therefore Bland–Altman plot was not constructed (n = 36) and children with incomplete dietary data . Characteristics of children and mothers included in this study are described in n = 144); children aged 8–9 years was 29.7% (n = 144); and children aged 10–12 years was 36.3% (n = 154). Approximately half of the study sample were girls (n = 214). Twenty-five percent of mothers had a high-school degree or less (n = 105). Fifty-four percent of mothers reported family income of 10,000 Saudi Riyals (SR) per month or more.The final analysis included data of 424 children after excluding children with chronic diseases and allergies  .Median intake FS assessed by FFQ was 83.5 g/day (58.0–114), while median intake FS assessed by 24-h dietary recall was 42.0 g/day (26.0–60). Mean intake of FS assessed by the FFQ was significantly higher than mean intake of FS assessed by the 24-h dietary recall  see . The corn = 312) of children were classified into the same and adjacent quartiles, whereas 26.4% (n = 112) of children were misclassified. The interrater reliability for the FFQ and the 24-h recalls was found to be κ = 0.21 , p < 0.001.Agreement of quartiles between FFQ and 24-h dietary recall is shown in rs = 0.82, p < 0.001).The value of Cronbach's alpha (α = 0.74) suggests good reliability for the FFQ among the study sample. To assess test-retest reliability, Spearman's correlation between FS intake from initial FFQ and repeated FFQ indicated positively high correlation . Utilization of the FFQ can offer the opportunity to investigate the associations between FS intake and related diseases. However, validating the FFQ among the different age groups is warranted. Limitations of this study include that the developed FFQ provides dietary data pertaining FS exclusively, whereas information on energy intake and the intake of other nutrients cannot be determined. In addition, the FFQ included food items that contain quantities of FS only. Thus, percentage of FS from total energy intake cannot be calculated.In conclusion, the newly developed FFQ can be used to estimate the habitual FS intake among children in Saudi Arabia. Future direction may include investigations on possible predictors of excessive FS consumption and relevant food sources. Additionally, validation of the FFQ among different age groups would provide an insight into the relationship between habitual intake of FS and non-communicable diseases.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethical Committee of the College of Applied Medical Sciences, Taibah University (certificate# 2020/55/202/CLN). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.WM and HK conceived the study, developed the tool, conducted the analyses, interpreted the results, and wrote the paper. All authors critically reviewed the manuscript and approved the final version submitted for publication.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.