During the first wave of the COVID-19 pandemic various ambulatory health care models  were organised in a short period in Baden-Wuerttemberg, a region in Southern Germany. The aim of these SARS-CoV-2 contact points was to ensure medical treatment for patients with (suspected) and without SARS-CoV-2 infection. The present study aimed to assess the beliefs and practices of primary care physicians who either led a Subspecialised Primary Care Practice or a Primary Care Practice providing care as usual in Baden-Wuerttemberg during the first wave of the COVID-19 pandemic.This cross-sectional study was based on a paper-based questionnaire in primary care physicians during the first wave of the pandemic. Participants were identified via the web page of the Association of Statutory Health Insurance Physicians Baden-Wuerttemberg. The questionnaire was distributed in June and July 2020. It measured knowledge, practices, self-efficacy and fears towards SARS-CoV-2, using newly developed questions. Data was descriptively analysed.p < 0.001). The same was found for the confidence level regarding decision-making concerning the further treatment (p < 0.001). Several prevention measures to contain the spread of SARS-CoV-2 were adopted. Subspecialised Primary Care Practice had treated on average more patients with (suspected) COVID-19 (mean 408.12) than primary care practices (mean 83.8) (p < 0.001).One hundred fifty-five participants (92 leads of SARS-CoV-2 contact points/ 63 leads of primary care practices) completed the questionnaire. Out of 92 leads of SARS-CoV-2 contact points 74 stated to lead n Subspecialised Primary Care Practices. About half participants of both groups did not fear an own infection with the novel virus (between 50.8% and 62.2%), however about 75% feared financial loss. Knowledge was gained using various sources; main sources were the Association of Statutory Health Insurance Physicians (between 82.5% and 83.8%) and the German Society for Hygiene and Microbiology (RKI) (between 88.9% and 95.9%). Leads of Subspecialised Primary Care Practice felt more confident to perform anamnestic/diagnostic procedures .The online version contains supplementary material available at 10.1186/s12875-021-01433-9. The novel coronavirus SARS-CoV-2) which causes the disease COVID-19 was first recognized in the Chinese province of Wuhan, Hubei in December 2019  which ca. The effIn Germany the primary healthcare sector consists of all ambulatory care services  that areAfter the first case of COVID-19 was detected in Baden-Wuerttemberg, Germany on 25 February 2020, the Association of Statutory Health Insurance Physicians Baden-Wuerttemberg (Kassenaerztliche Vereinigung Baden-Wuerttemberg (KVBW)) asked all patients with suspected SARAS-CoV-2 infection to contact their primary care physician by telephone prior to visit a doctor . TherefoIn BW a variety of ambulatory health care models (SARS-CoV-2 contact points), besides the regular primary care practices, were established in a short period during the first wave of the SARS-CoV-2 pandemic as part of a crisis management: 204 Subspecialised Primary Care Practices, 51 Fever Clinics, and 16 Testing sites (Status: June 2020) by primary care physicians in cooperation with the public health sector and the Association of Statutory Health Insurance Physicians (German: Kassenaerztliche Vereinigung Baden-Wuerttemberg (KVBW)).Subspecialised Primary Care Practices, are primary care practices who offer appointments for patients with potential SARS-CoV-2 infection or other infectious diseases.Fever Clinics, which are usually located centrally or decentralised in local buildings  and are organised by primary care physicians and the KVBW.Special Points for Corona Testing, are available in some regions and are organised by the primary health care sector in collaboration with health authorities.Primary care practices should be, next to the public health organisations, the first contact point for patients if they are worried they may be infected with SARS-CoV-2 and show symptoms of the COVID-19 disease. In BW, three types SARS-CoV-2 contact points were established to support primary care physicians:The SARS-CoV-2 contact points allowed primary care physicians to refer patients with (suspects) SARS-CoV-2 infection in order to fulfil the requirement of patient separation Fig. .Fig. 1OrThe newly implemented SARS-CoV-2 contact points are claimed to have an important role in the relatively moderate spread of the disease and the relatively low mortality by COVID-19 in Baden-Wuerttemberg, Germany during the first wave of the pandemic . Little The physicians` involvement in active participation in contact points was on a voluntary base, however incentivised by supply PPE in a time of extreme shortage in this regard. Therefore, the implementation of these ambulatory healthcare models was not systematically planned and the SARS-CoV-2 contact points were not equally spread over the country. The knowledge and beliefs of primary care physicians may influence the effective organisation of the different ambulatory health care models.Fever Clinics and Special Points for Testing) were excluded from the analysis due to the different settings and aims of those ambulatory models.To get insights for further pandemic management, the present study aimed to assess beliefs such as the self-efficacy, practices, knowledge and fear among primary care physicians who decided to become a Subspecialised Primary Care Practices in comparison to primary care physicians who continued “usual” primary care. The other two SARS-CoV-2 contact points . Informed consent to participate was obtained from all participants when they posted the completed questionnaire to the research team. The research conducted in this study was performed in accordance with the Declaration of Helsinki.All 271 SARS-CoV-2 contact points in BW were identified via the web page of the Association of Statutory Health Insurance Physicians Baden-Wuerttemberg (Kassenaerztliche Vereinigung Baden-Wuerttemberg (KVBW)) and invited to participate in this survey in June 2020. Included were all primary care physicians who led one of the SARS-CoV-2 contact points. The aim was a full census of primary care physicians leading a SARS-CoV-2 contact point in BW during the first wave of the SARS-CoV-2 pandemic (March—June 2020). No inclusion criteria were set regarding the time point of the establishment of the SARS-CoV-2 contact points. They only had to have operated during the first wave of the pandemic. Furthermore, a random sample of 400 other primary care practices, also identifies via the web page of the KVBW, was invited to participate in the study. The invitation was addressed to the practice owner of the primary care practice. In case of an group practices, physicians were given the option to complete the questionnaire together or to decided who would be best to complete the questionnaire. Primary physicians of those were excluded from the analysis, if they indicated that they also worked for a SARS-CoV-2 contact point.in categories). The second part of the questionnaire covered the structure and organisation of the SARS-Cov-2 contact points/ primary care practices including questions such as number of staff , opening hours, changes to opening hours, remuneration, spatial conditions, diagnostic possibilities, treatment offers, the financing of the SARS-CoV-2 contact, and type of support which enable the implementation (in categories). The third part comprised patients contacts and treatment capacities . Part four covered questions regarding the nature of the medical documentation and medical history used, the main content of medical documentation used, and the satisfaction with these documents . This part also included questions concerning collaboration/cooperation with other healthcare facilities (in categories) and satisfaction with it .The questionnaire , German in categories) and level of knowledge . The last part focused on PPE and disinfection methods (in categories), other prevention measures, the utilisation of their SARS-CoV-2 contact point/ primary care practices and how healthcare during the second wave of the SARS-CoV-2 pandemic should be managed  in BW, Germany. The last question was an open-end question which gave the participants the opportunity to share their personal thoughts regarding the pandemic.The main part of the questionnaire, which is reported in this paper, covered questions regarding fear for an infection, self-efficacy and practice, sources  (responds rate 33.9%). Of the 400 invited primary care physicians 79 participated (responds rate 19.7%), 16 of those were excluded from the analysis since they indicated that they also worked for a SARS-CoV-2 contact point.2 = 4.482, p = 0.034). Other sociodemographic factors such as age or professional qualification were not significant . Primary care physicians, stated to be worried for various reason like transmitting the virus to the private environment (42.9%) or the professional environment (46.0%). In comparison, primary care physicians who led a Subspecialised Primary Care Practices tended to feel less anxious to fall sick themselves (62.2%) or to spread the virus within their private environment (37.8%) or their professional environment (45.9%). The vast majority of participants of the regular Primary Care Practices and Subspecialised Primary Care Practices (between 75.7 and 84.1%) feared financial loss during the pandemic because of a possible closure of practices and therefore loss of income. However, none of the differences between the two groups were significant , after four weeks , and present . This result suggests that primary care physicians who led a Subspecialised Primary Care Practice felt significantly more confident. The same is true for the confidence level regarding the decision-making process regarding further procedure. At the beginning of the pandemic and after four weeks there was a difference between the both groups . There was no significant difference regarding deciding how the further procedure of patients with COVID-19 should look like when the participants completed the questionnaire (June/July 2020) (Table Another part of the survey was the self-efficacy and practice of participants regarding anamnestic and diagnostic procedures related to SARS-CoV-2 and the decision-making process regarding the further procedure. The results show that in both settings the confidence to treat patients with (suspected) SARS-CoV-2 infection increased over time. There was a difference in the confidence level between the two groups in performing anamnestic and diagnostic procedures for patients with COVID-19 at the beginning of the pandemic . The vast of majority of all participants stated that physical and temporal separation of patients’ groups was done. However, there was a significant difference regarding physical separation, suggesting that leads of Subspecialised Primary Care Practice points tended to feel like introducing physical separation was implemented easier . Physical separation was ensured by using different waiting or treatment rooms, waiting times in cars or in front of the facility. Temporal separation was ensured by appointment allocation  was possible . However, the difference was not significant (xp < 0.001). The maximum treatment capacity of Subspecialised Primary Care Practice can be increase immediately on average by 25.32 patients, if personal resources are increased by 25.70 patients, or if other actions are adapted by 43.41 patients. Noticeable is the range of variation in treating patients between individual participants during the first wave of the pandemic in both groups  COVID-19 (mean 7.70 (0–50)) than primary care practices (mean 5.70 (0–120)). However, the difference was not significant. In total Subspecialised Primary Care Practice treated on average 408.12 0–3846) patients since they opened, whereas on average 83.8 patients were treated by primary care practices (F (151.447 – 501.469) = 19.614,  patientsThe main goal of this study was to describe beliefs such as knowledge, practice, self-efficacy and fears among primary care physicians leading a Subspecialised Primary Care Practice compared to primary care physicians who “continued usual primary care practice” during the first wave of COVID-19 pandemic in Baden-Wuerttemberg, Germany. A quarter of all participating primary care physicians did not fear an infection with SARS-CoV-2 but were afraid of transmitting it to members of their families or colleagues. The majority of all participants was afraid of financial loss due to lost revenues. Acquiring knowledge about the new coronavirus disease was challenging but this changed during the pandemic and extending knowledge. Sources of knowledge varied but the Association of Statutory Health Insurance Physicians, the German Society for Hygiene and Microbiology, and medical journals represented the most important sources of information. The majority of primary care physicians of both groups felt confident in anamnestic and diagnostic procedures. Primary care physicians who led a Subspecialised Primary Care Practice were more confident compared to their peers. Hygiene regulations were implemented in all healthcare facilities. Physical separation was ensured mainly by different treatment rooms and waiting in front of the facility by both groups but the results of this study showed that leads of Subspecialised Primary Care Practice tended to introduce physical separation easier. Temporal separation was ensured via appointment allocation, overall fewer patients in Primary Care Practices or special consultation hours for patients with suspected infection. Subspecialised Primary Care Practice treated on average significant more patients with (suspected) COVID-19 compared to Primary Care Practices.The results of this study showed that most participants did not fear an infection with the novel virus but expressed concerns regarding the possibility to spread it within their professional and private environment. It is unclear why some decided to lead a Subspecialised Primary Care Practice and some continued usual primary care practice. Interestingly, the majority of physicians who led a Subspecialised Primary Care Practice identified as male and were between 51 and 60 years old and therefore more at risk for a severe infection . In GermThe majority of both groups feared financial lose. This could be explained by the concept of fee-for-service, particularly physicians who continued to work in their own practices had overall less patients which may had an impact on the fear of financial lose. These results agree with those by Huston et al. . In AustIn order to separate patients with potential infections they introduced special consultation hours  and waiting times e.g. in front of the practice. Offering special consultation hours may had impact on their workload that was not adequately remunerated. In addition, waiting hours in front of the practice may had an impact on the decision of patients to consult their physicians at all. The specific concerns of primary care physicians should be taken into consideration since primary healthcare systems and public health systems rely on the mental and physical health of primary care physicians and their economic efficiency .The majority used the Association of Statutory Health Insurance Physicians and the German Society for Hygiene and Microbiology as source of knowledge which provide an easy and accessible way of getting valid and processed information regarding a growing and changing knowledge base. Interestingly, almost half of the participants (between 36 and 42%) stated to listen to a podcast even though the majority of participants were in the age groups between 41 and 60 years. Podcasts or broadcasts are primarily used by the younger generation (between 14 and 29 years) . AnotherThe vast majority of primary care physicians in this study used official information provided by the Association of Statutory Health Insurance Physicians and the RKI. This may can be interpreted, as trust in official information channels by the participants. Nevertheless, various other sources of information were used additionally. This poses the questions if the participants probably felt like information of official channels may be not enough or if those sources may not suitable for the primary care sector. In addition, gathering knowledge was difficult for more than half of the participants at the beginning of the pandemic and was still evaluated as challenging by a third during the pandemic. This may made it difficult to be always up-to-date. The diversity of potential information resources and the need of being up-to-date probably had an impact on the perceived workload of primary care providers. Moreover, primary care physicians are in direct contact with patients with (suspected) SARS-CoV-2 infection and are therefore at high risk for nosocomial infections . During Self-efficacy regarding anamnestic and diagnostic procedures related to SARS-CoV-2 of primary care physicians increased over time during the first wave of the pandemic. This can be explained by the experience they gained during the first month of the pandemic. Physicians had to acquire knowledge on the novel virus first under the permission of the dynamic situation of the pandemic. For a second wave of SARS-CoV-2 pandemic primary care physicians may be better prepared since scientific as well practical knowledge improved during the first months of the pandemic. The findings of this study indicated that although self-efficacy increased over time in both groups, leads of Subspecialised Primary Care Practice felt more confident. This may be important in order to decide which structure of medical care and treatment is the most favourable during the second wave of the SARS-CoV-2 pandemic or other pandemics.The results of our study showed that key challenges which were difficult to tackle during the 2009/A/H1N1 pandemic in Australia, Israel, and England such as segregation of patients with (suspected) infection  were easFirst evaluations on accounting data have shown that seven out of eight COVID-19 patients (about 85%) were treated in an ambulatory setting . In our Although the results of our study cannot be generalized, they may give a first impression on the workload primary care physicians had to manage in Baden-Wuerttemberg, Germany. As requested and expected more patients with (suspected) COVID-19 were treated in Subspecialised Primary Care Practice than in Primary Care Practices. The implementation of Subspecialised Primary Care Practice as well as the prevention measures in both settings may have contained the spread of the virus.Although this study gives first impressions and important information on challenges primary care physicians had to face during the first wave of the SARS-CoV-2 pandemic, limitations on the study have to be considered. The analysis is based on self-disclosures, so biases cannot be excluded. The response rate is similar to other studies in German physicians, but it may imply selection bias. Furthermore, it is unclear whether the results can be generalized to Germany as the study concerns one region.The results of this study showed that the Subspecialised Primary Care Practice implemented during the first wave of the SARS-CoV-2 pandemic in Baden-Wuerttemberg, Germany were a strategy to face the pandemic. Particularly, since leads of Subspecialised Primary Care Practice indicated that implementing physical separation of patients with potential SARS-CoV-2 infection was easier compared to those who continued working in their own practice. In addition, primary care physicians who lead a Subspecialised Primary Care Practice felt in general more confident in dealing with patients with COVID-19 infection. Although, primary care physicians rose to the challenges of the SARS-CoV-2 pandemic, addressing their fears is essential. Interventions such as adequate payment for COVID-19 care or loss of revenue due to the pandemic combined with measures that support the implementation of prevention measure not only in Subspecialised Primary Care Practice but also Primary Care Practices should be introduced to support the primary health care sector.Future research should focus on strategies how primary care physicians can be better prepared for a pandemic and how to address mental, physical and financial challenges. This could be for example the expansion of adequately financially compensated points of care, support by tools for managing testing, communication with and care of patients, setting up paths of cooperation between primary care, hospital care, rescue service and health authorities. Primary care physicians will be stressed by the challenges of a prolonged response to the SARS-CoV-2 pandemic. Strengthening primary healthcare, particularly primary care physicians, is therefore of great importance.The results of this study indicated that primary care physicians who continued with “care as usual” did fear an infection with SARS-CoV-2 compared to physicians who lead a Subspecialised Primary Care Practice. Although the difference was not significant, for the second wave of the pandemic or other pandemics a list with primary care providers who are willing to open a Subspecialised Primary Care Practice and feel more confident in doing so should be developed. Additionally, Primary Care Practices that can implement prevention measures or already implemented them should be systematic listed and functioning as pandemic response practices. An information system with the latest state of scientific knowledge regarding the pandemic should be develop based on the preferences of primary care providers in order to support them to practice safely and to be up-to-date without an increased workload.Additional file 1


There is limited evidence regarding socioeconomic inequalities of exposure to the food environment and its contribution to childhood obesity.We used data from 4235 children from the Generation R Study, a large birth-cohort conducted in the city of Rotterdam, The Netherlands. We included 11,277 person-observations of body mass index (BMI) and 6240 person-observations of DXA-derived fat mass index (FMI) and fat-free mass index (FFMI) when children were between 4 and 14 years. We applied linear regression models to evaluate changes in the relative and absolute exposure of fast-food outlets, and the healthiness of the food environment within 400 m from home by maternal education. Furthermore, we used individual-level fixed-effects models to study changes in the food environment to changes in BMI, FMI and FFMI.Children from lower educated mothers were exposed to more fast-food outlets at any time-point between the age of 4 and 14 years. Over a median period of 7.1 years, the absolute (0.6 fast-food outlet (95% CI: 0.4–0.8)) and relative (2.0%-point (95% CI: 0.7–3.4)) amount of fast-food outlets increased more for children from lower as compared to higher educated mothers. The food environment became more unhealthy over time, but no differences in trends were seen by maternal education level. Changes in the food environment were not associated with subsequent changes in BMI, FMI and FFMI. For children from lower educated mothers not exposed to fast-food at first, we found some evidence that the introduction of fast-food was associated with small increases in BMI.Our findings provide evidence of widening inequalities in exposure to fast-food in an already poor food environment. Access to more fast-food outlets does not seem to have an additional impact on BMI in contemporary contexts with ubiquitous fast-food outlets. Childhood obesity is a major public health concern due to its widespread prevalence and rapid increase in the past decades , 2. The Changes in the food environment and their impact on obesity have been hypothesised to differ between individuals from higher and lower socioeconomic groups. A systematic review of 21 studies showed that fast-food access was higher in more-deprived compared to less-deprived areas . IndividThe Generation R Study is a birth-cohort study in which four objective measures of BMI and two indices of fat mass obtained from dual-energy x-ray absorptiometry (DXA) scanners were available for children between the age of 4 and 14 years. Linkage with yearly updated food environment measures created the unique possibility to evaluate whether changes in the food environment were associated with changes in body composition. We hypothesised that children with parents of lower SEP lived in neighbourhoods characterised by a higher number of unhealthy food outlets, and that over time, this resulted in unfavourable changes in body composition. First, we evaluated if the exposure to food environment around the home address differentially evolved over time for children from lower and higher educated mothers using up to four repeated measures between the age of 4 and 14 years. Second, we studied if the impact of changes in the food environment on changes in measures of body composition differed between children from lower and higher educated mothers.This study used data collected from the Generation R Study, a prospective birth-cohort study in the city of Rotterdam, in The Netherlands . We inclInvitations to participate in the Generation R Study were sent out to all pregnant women who had an expected delivery date between April 2002 and January 2006 and who lived in the study area  at time of delivery . The Medn = 5418) were included. Younger siblings from the same mother (n = 397), and children from which information on maternal education level were missing (n = 441) were excluded. In total, 4594 children were eligible for the present study. We included observations for the address where the child lived for the longest period between the age of 4 and 14 years, and children without two consecutive observations on one address (n = 359) were excluded. The sample for the main analysis included 4235 children with 11,277 person-observations for BMI, and 6240 person-observations for fat mass  and type of food outlet were obtained from Locatus, a commercial company that collects information on food retailers in The Netherlands by means of yearly field audits. A validation study using field audit data found an overall ‘good’ to ‘excellent’ agreement for both the location and classification  and age- and sex-specific standard deviation scores (SDS) for BMI were obtained from Dutch reference growth charts   and fat-free mass index (FFMI) [fat-free mass (kg)/height (m3)] according to standard procedures [Body composition was measured at the research centre at the age of 6 and 10 years using a DXA scanner  using enCORE software version 12.6. Children were placed without shoes, heavy clothing and metal objects in supine position on the DXA table. We calculated fat mass index (FMI) [fat mass (kg)/height , mid-high , mid-low  and low ) .Sociodemographic characteristics obtained at baseline and during follow-up visits by means of questionnaires included age, sex, ethnicity and net household income. In accordance with Statistics Netherlands , a childCharacteristics of the children during the first and last measurement when living on the same address were presented. Number of missing values were presented in the supplement Appendix . The medFixed-effects linear regression models with repeated measurements were used to study within-person changes in the food environment and the association with within-person changes in body composition measures. We tested for interactions with maternal education level to assess whether the association between changes in the food environment and changes in body composition differed by level of maternal education. To retain statistical power, maternal education was categorised into lower  and higher  education. We used up to four measures of BMI, and two measures of FMI and FFMI. This method allowed to control for measured time-variant variables and for unmeasured time-invariant variables . All modResults were presented as a relative increase by 10%-point in fast-food outlets, an absolute increase by one fast-food outlet, and per 0.5 point increase in healthiness score of the food environment . Units were based on the average change for those with changes in the population between time-points  may have stronger effects than similar changes observed at the higher end of the distribution (e.g. from 5 to 7 outlets). Hence, we also performed analyses restricted to children without any fast-food outlets during the first measurement round, and explored if the introduction of fast-food outlets mattered for the subsequent changes in outcomes.n = 222). Income did not change between time-points for 77.5% of the children. We imputed income measured at the nearest time-point assuming that income did not change (12.7% imputations). Second, we additionally included changes resulting from residential moves. In this analyses, we included 4594 children with 13,528 person-observations of BMI, and 7856 person-observations on fat mass.Two sensitivity analyses were conducted. First, we controlled for the time-varying factor net household income, since having a higher income may contribute to healthier dietary behaviours . Income P values <0.05 were considered statistically significant. R scripts are available for researchers upon authors request.All analyses were conducted in R version 3.4.1, using the plm package for the fixed-effects analyses. Clustered sandwich estimators were used to allow for within-child correlation between error terms. Two-sided At all time-points, children from lower educated mothers were exposed to more fast-food outlets, for both the relative and absolute exposure to fast-food outlets. These children were also exposed to unhealthier food outlets, and had a higher BMI and FMI than children from higher educated mothers Table . Some chAt their first measurement round, 39.7% of the children from low educated mothers and 20.7% of the children from high educated mothers were exposed to four or more fast-food outlets in the direct vicinity of their home. Subsequent changes in fast-food outlets, both in relative and absolute terms, and the healthiness of the food environment were not associated with changes in body composition , but also factors influencing body composition  occurring for children that also experienced changes in the food environment. Instrumental variable (IV) approaches may further eliminate bias from unobserved time-varying confounding, and two studies found that associations between the food environment and diet quality or BMI were stronger in magnitude using IV models compared to fixed-effects analyses , 14. IdeThe current evidence base defined and observed inequalities in the fast-food environment in different ways. A review reported that fast-food locations were more prevalent in low-income neighbourhoods and neighbourhoods with high concentrations of ethnic minority groups . AlthougThe counterintuitive finding of a US study, suggesting that BMI decreased following the increase in fast-food outlets, was not found in our study . No chanOur results gave some indications that fast-food exposure was associated with small increases in BMI for children of lower educated mothers that were not exposed to fast-food during the first measurement round, but not for children from higher educated mothers. DXA scans suggested that especially fat mass increased following the introduction of fast-food outlets. We are not aware of any other study looking at changes in fat mass in relation to the built environment. In sensitivity analyses, no associations between the food environment and body composition were found when additionally including changes resulting from residential moves. This could suggest that other factors that change during residential relocation, such as the impact of changing schools, may contribute to body composition to a larger degree.Other longitudinal studies using a fixed-effects approach reported that fast-food outlets were associated with higher fast-food intake among young adults from low-income families, but not among higher-income families . AnotherThe high and increasing number of fast-food outlets, and the larger exposure to them of children from lower socioeconomic groups provide support to policies aimed at ‘shifting the curve’. To date, strategies to improve dietary habits mainly rely on nutritional education rather than on structural interventions to modify the food environment. That changes in BMI were small following the introduction of fast-food outlets around home between a 2- and 4-year period should not limit efforts to do so. The cumulative effect of the food environment over time and across various daily activity settings was not explored, but may steadily impact on children’s body composition. Unravelling causal mechanisms how fast-food outlets influences body composition, for example by having information on fast-food consumption or dietary patterns, is essential. Furthermore, the wider context in which food environments change and food choices are made is important to consider. It has been suggested that more food outlets could reflect general investments made to that area, which could positively or negatively influence children’s body composition . More inIn conclusion, this study provided evidence of widening socioeconomic inequalities in the food environment around children’s homes growing up in a large urban area in The Netherlands over a period of 7.1 years. Children from lower educated mothers were at higher risk that the food environment shifted more rapidly towards an unhealthy environment. Exposure to more fast-food outlets does not seem to have an additional impact on BMI in contemporary contexts with ubiquitous fast-food outlets. These findings support the importance of an equity-focus on the link between the food environment and body composition.Supplemental material


The potential risk of coronavirus disease 2019 (COVID-19) transmission from asymptomatic COVID-19 patients is a concern in dental practice. However, the impact of this risk is not well documented to date. This report describes our dental clinical experience with patients who did not exhibit symptoms of COVID-19 but were later confirmed as positive for COVID-19.Of the 149,149 patients who visited the outpatient clinic of KNUDH and the 3291 patients who visited the Oral and Maxillofacial Surgery Clinic of KNUH, 3 were later confirmed as having COVID-1 between 1 February 2020 and 28 February 2021. Owing to close contact with these patients during their treatments, 46 dental and medical staff had to undergo quarantine from the date of the patients’ confirmation of COVID-19 infection.The presented cases showed the potential existence of asymptomatic COVID-19 patients after dental treatment with aerosol-generating procedures. Clinicians should be aware of the infection prevention measures and try to protect healthcare personnel from secondary infection of COVID-19 during dental treatments. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused an outbreak in China in December 2019. On 11 February 2020, the disease was officially named coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO). In March 2020, the crisis was declared a pandemic [Transmission of SARS-CoV-2 can occur directly via inhalation of and exposure to infected secretions, such as saliva and respiratory droplets or aerosol particles, or indirectly through contact with a contaminated surface or with infected individuals . Close cStudies have reported that symptomatic, presymptomatic, and asymptomatic patients can transmit SARS-CoV-2 to others . SymptomDental treatments include a number of aerosol-generating procedures (AGPs), such as those that require ultrasonic scalers, air-water syringes, and air turbine handpieces, for which the possibility of airborne transmission of SARS-CoV-2 in dental practice cannot be ruled out. Virus particles with infective potential can be present in saliva and asymptomatic individuals can possibly transmit the infection. Given the high percentage of asymptomatic carriers of SARS-CoV-2, the possibility of cross-infection during dental practice cannot be excluded . HoweverIn this case report, we describe the circumstances surrounding contact that transpired between asymptomatic COVID-19 patients and healthcare personnel (HCP) during dental practice. The objectives of this study were to share our experience in managing the situation and to determine the necessary precautions for dental treatments involving asymptomatic COVID-19 patients.Patients who visited the Kyungpook National University Dental Hospital (KNUDH) and Kyungpook National University Hospital (KNUH) between 1 February 2020 and 28 February 2021 and were later confirmed to be infected with COVID-19 were evaluated in this study. Of the 149,149 patients who visited the outpatient clinic of KNUDH and the 3291 patients who visited the Oral and Maxillofacial Surgery Clinic of KNUH, 3 were later confirmed as having COVID-19  confirmed that the patient was positive for COVID-19. The patient did not experience symptoms before the positive confirmation and tested negative after 14 days of quarantine.At the time of the patient’s hospital visit, 1 doctor and 5 student hygienists participated in his treatment; they were wearing a surgical mask and latex gloves during the treatment. These 6 staff who had close contact with the patient underwent quarantine from 24 December 2020 to 1 January 2021, and tests performed before the quarantine period ended confirmed that all of them were negative for COVID-19.A 73-year-old woman visited our emergency department (ED) for temporomandibular joint (TMJ) dislocation and underwent TMJ reduction on 1 February 2021. As the TMJ reduction was not successful, the patient visited our dental hospital the next day. The patient was asymptomatic to COVID-19 at the time of her visits and treatment. Ten days later, the patient was diagnosed with COVID-19 at the nursing home where the patient was a resident of. A caregiver at the nursing home initially tested positive for COVID-19. Immediately before her COVID-19 infection was confirmed, the patient had fever and was subsequently admitted to KNUH.At the time of the patient’s visit to the ED, a doctor participated in the treatment. Because 14 days had already passed since the date of the doctor’s initial contact with the patient and the date of the patient’s confirmation of COVID-19 infection, the doctor did not undergo quarantine after testing negative for COVID-19. At the dental hospital, 4 doctors and 2 radiological technologists had close contact with the patient for the treatment. These 6 dental staff also did not undergo quarantine after confirming a negative result for COVID-19 because 13 days had passed since the date of their initial contact with the patient. All the HCP were wearing a surgical mask and latex gloves as personal protective equipment (PPE) during the treatments.A 63-year-old man visited the ED of KNUH for mandibular symphysis, fracture of both condyles, and multiple teeth injury, which were caused by a slip accident on a street, on 15 December 2020. Intraoral bleeding continued owing to a number of broken teeth in the patient’s oral cavity; extraction of fractured teeth was performed using a high-speed handpiece, and intermaxillary fixation screws were placed in the ED. The patient was admitted to the ward after visiting the ED. Although the patient did not exhibit symptoms of COVID-19 infection, the patient underwent a COVID-19 test on 16 December 2020. RT-PCR result confirmed that the patient was positive for COVID-19. The patient was isolated and treated at the negative pressure room of KNUH before being discharged.A total of 33 dental and medical staff, including 11 doctors, 16 nurses, 1 hygienist, 2 student nurses, and 3 medical assistants, were classified as close contacts of the patient. All the HCP were wearing a surgical mask and latex gloves as PPE during the treatments. As of the date of their last contact with the patient, the 33 staff underwent 14 days of quarantine, and all of them were COVID-19 negative based on laboratory tests performed before their release from quarantine.This report describes the cases of 3 asymptomatic patients who visited our hospital for elective and emergency dental treatments and were later confirmed as having COVID-19 infection. A total of 46 dental and medical staff who were in close contact with these patients during their treatments had to undergo quarantine from the date of the patients’ confirmation of COVID-19 infection. None of the hospital staff tested positive for COVID-19 after coming in close contact with the asymptomatic COVID-19 patients, and no further cases had been linked to the patients. During the same period as this study, 8623, 3255, and 89,676 COVID-19–positive patients were identified in Daegu Metropolitan City, nearby province (Kyungpook Province), and South Korea, respectively [COVID-19 infection in asymptomatic patients can be confirmed in 2 ways: One is when a public health center is notified that a COVID-19–positive person had visited the facility, and another is when a patient who visits a hospital is directly tested and confirmed as having COVID-19 infection. In both scenarios, which can happen at any time, dental treatment poses a risk to dental staff.According to the interim guidance released by the US Centers for Disease Control 2020)  and WHO 020  and WHO recommends the use of fluid-resistant medical masks or respirators if an AGP is anticipated, fluid-resistant gowns, gloves, and eye protection (goggles or face shield) as PPE for dental surgery as well as performing proper hand hygiene. A fit-tested N95 or FFP2 respirator (or higher) is recommended when AGPs are performed , 8, 10. A study on the prevalence of COVID-19 among dentists in the USA found that 20 of 355 tested dentists were diagnosed as having confirmed or probable COVID-19 infection . The proThe incubation period, which is the time between exposure to the virus and symptom onset, for COVID-19 is, on average, 5 to 6 days, but it can take as long as 14 days –4. EarlyFrom our experience of treating COVID-19–positive patients who were asymptomatic, additional infections did not occur among dental and medical staff. Practitioners should keep abreast of new scientific knowledge and protection guidelines regarding COVID-19 because they could be treating asymptomatic patients at any given time. Further research, including investigation into the distinction between asymptomatic infection and presymptomatic transmission, is needed to be continued.The current report of cases showed the potential existence of asymptomatic COVID-19 patients after dental treatment with aerosol-generating procedures. Clinicians need to be fully aware of the infection prevention measures and try to protect healthcare personnel from secondary infection of COVID-19.


Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in a global pandemic associated with substantial morbidity and mortality worldwide, with particular risk for severe disease and mortality in the elderly population. SARS-CoV-2 infection is driven by a pathological hyperinflammatory response which results in a dysregulated immune response. Current advancements in aging research indicates that aging pathways have fundamental roles in dictating healthspan in addition to lifespan. Our review discusses the aging immune system and highlights that senescence and aging together, play a central role in COVID-19 pathogenesis. In our review, we primarily focus on the immune system response to SARS-CoV-2 infection, the interconnection between severe COVID-19, immunosenescence, aging, vaccination, and the emerging problem of Long-COVID. We hope to highlight the importance of identifying specific senescent endotypes (or “sendotypes”), which can used as determinants of COVID-19 severity and mortality. Indeed, identified sendotypes could be therapeutically exploited for therapeutic intervention. We highlight that senolytics, which eliminate senescent cells, can target aging-associated pathways and therefore are proving attractive as potential therapeutic options to alleviate symptoms, prevent severe infection, and reduce mortality burden in COVID-19 and thus ultimately enhance healthspan.  In the 1960s, Leonard Hayflick, reported the limited proliferative capacity of cultured human diploid cells, where cells ceased to proliferate after serial passage in vitro. This was the first introduction of the permanent state of cell cycle arrest which is referred to as “cellular senescence” . HayflicSenescence is a very exciting, fast-moving field of research. Life expectancy worldwide is increasing, in parallel with this comes the significantly increased burden of public health and economic challenges due to increased incidence of multiple chronic diseases and disorders within the aging population, despite advances in science and medicine. Aging and the connection with senescence is acknowledged as one of the core risk factors for many of these chronic and life-threatening conditions such as cancer and cardiovascular disease. This is possibly true also for coronavirus disease 2019 (COVID-19) where it is apparent that the older population are at increased susceptibility to infection and more severe infection. Therefore, many scientists across the world are frantically racing to investigate the field thoroughly to prevent healthcare systems becoming overwhelmed.Aging in all tissues is linked with elevated levels of cellular senescence, which is triggered by various compounding extrinsic and intrinsic factors. This stress-induced response process means cells permanently stop dividing and exit from the cell cycle and acquire a pro-inflammatory senescence-associated secretory phenotype (SASP) . The acqThe era of senolytics is still in its infancy and senolytic agents tested to date include Dasatinib , Quercetin , Navitoclax, A1331852 and A1155463  and Fistein . Cardiac glycosides, such as digoxin, which is currently used to treat irregular heartbeat in atrial fibrillation, have also been reported to have broad senolytic activity ,15. SuchEven though aging is a major risk factor for COVID-19, the exact role of senescence in COVID-19 is not fully understood. Further research and studies are required to fully explore disease mechanisms and elucidate biological factors which may drive immunosenescence particularly in the elderly in the context of COVID-19. Furthermore, the timely assessment of COVID-19 severity is critical so that adequate treatments can be administrated rapidly to patients. Currently, there is a lack of definitive parameters which can be utilized to identify patients who are at high risk of requiring admission to intensive care unit (ICU). Many studies are focusing on identifying such biomarkers for this use. Indeed, changes in neutrophil-to-lymphocyte ratio and urea-to-creatinine ratio have emerged to have clinical utility as stand-alone parameters in being able to identify patients with aggressive disease at an early stage . CurrentWhen cells become senescent, they undergo a plethora of significant characteristic changes including permanent, irreversible cell cycle arrest, morphological, metabolic changes, epigenetic, transcriptional as well as chromatin remodeling, metabolic reprogramming, altered gene expression, altered microRNA (miRNA) expression and the adoption of a pro-inflammatory phenotype commonly known as SASP . These pThe main drivers instigating senescent growth arrest are the CDK inhibitors (CDKi) encoded in the CDKN2A (p16INK4a), CDKN2B (p15INK4b) and CDKN1A (p21CIP1) loci. It is well documented that the expression levels of these drivers are known to deviate depending on the phase of senescence and are subject to changes with progression to different phases of senescence ,36,37,38Epigenetic modifications, chromatin rearrangements and global elevations in chromatin accessibility have been frequently reported in senescence ,57,58,59Altered gene and microRNA (miRNA) expression can impact and modulate the senescence process by directly or indirectly targeting key senescence factors including p53, p21CIP1 and SIRT1 ,70. Variβeta Galactosidase  activity which is known to be greatly enhanced in senescent cells due to the presence of elevated lysosomal substance [β-gal activity to distinguish senescence. Ki67, is another frequently used marker to indicate cell proliferation. Senescent cells permanently exit the cell cycle therefore this marker should not be expressed in senescent cells [Finally, one of the most extensively exercised biomarkers of senescence and aging cells, detected in most in vitro and in vivo settings, is senescence associated ubstance ,78,79. Iubstance ,81. Thisnt cells . Howevernt cells , and alsnt cells . Again, β-gal staining, p21CIP1, p16INK4a, heterochromatin (SAHFs & SADSs) and proliferation (Ki-67) markers) must be used in combination as part of a panel [In summary, many biomarkers , which has a fundamental role in regulating gene expression and viral pathogenicity. COVID-19 has resulted in a pandemic associated with substantial morbidity and mortality worldwide. The identification of diagnostic/prognostic biomarkers and therapeutic targets is therefore critical in directing the pandemic. The link, if it exists, between senescence, aging and COVID-19 is an extremely novel area of research, has not been extensively studied and therefore remains an open question.Epidemiological studies to date have shown that COVID-19 has a high mortality especially in patients of advanced age and those with comorbidities such as cardiovascular disease, diabetes, high blood pressure, chronic obstructive pulmonary disease, asthma, and cancer are much more susceptible to developing severe COVID-19 disease ,114,115.Currently, COVID-19 research using proteomics and transcriptomic analyses are focusing on identifying and validating prognostic, diagnostic and prediction markers which are more robust and reliable than current inflammatory markers. Studies are also working to establish if the presence of specific biomarkers make a patient more susceptible to a more severe infection. The identification of such biomarkers could help predict the severity of infection expected and therefore could help determine in advance the level of medical intervention that would be much more beneficial and could be administered much faster to the patient. For example, a recent study has reported that long pentraxin 3 (PTX3) is an independent strong prognostic indicator for predicting mortality in COVID-19 and is a superior biomarker compared to conventional inflammatory markers . ConversIt is therefore likely that a combination of biomarkers will be required as opposed to individual biomarkers for accurately predicting COVID-19 clinical outcomes. Senescence markers could be combined with patient phenotypes to identify patients that might develop severe COVID-19 or predict those who will go on to have post COVID-19 syndrome, referred to as Long COVID. They may also help understand the immune response to both SARS-CoV-2 infection and vaccination. Long COVID is defined as having signs and symptoms post COVID-19 infection which continue for greater than 12 weeks and cannot be explained by alternative diagnoses. It is recognized as an evolving problem with substantial potential to impact healthcare and economics on a worldwide and longer-term basis. Patients with Long COVID have been reported to present with ongoing symptom burden such as persistent breathlessness, cough and fatigue, elevated blood biomarkers such as d-dimer and C-reactive protein (CRP) levels, have changes in lung function and deteriorating chest imaging which could lead to lung fibrosis ,128,129.The global pandemic of COVID-19 was declared on 11 March 2020 by the World Health Organization (WHO). As per the WHO statistics, as of 17 September 2021, there have been 226,844,344 confirmed cases of COVID-19 including 4,666,334 deaths . COVID-1COVID-19 is recognized as having a heterogeneous nature and therefore a diverse plethora of host factors are fundamental in determining the severity and progression of the disease in which a patient may acquire . Some ofChina was the first country to report SARS-CoV-2 infection amongst its population. Early data from China indicated that the case fatality ratio (CFR) of COVID-19 increases with age. It was reported that the CFR for patients aged 40 years or less was lower than 0.4%, the CFR increased to 8% for patients in their 70’s and further increased to a staggering 14.8% in patients aged 80 years and above ,149. ItaIndeed, patient age is reported to contribute to the risk of COVID-19 incidence and severity . NursingOverall, taken together, the current epidemiological data indicates that COVID-19 patients aged >80 years illustrate a higher risk of mortality compared with younger patients ,131,144.As mentioned in previous sections, senescence is the phenomenon which describes the state when cells stop proliferating and enter permanent cell cycle arrest. A multitude of biomarkers must be used to correctly identify senescent cells. A major age-associated hallmark is the accumulation of senescent cells with the acquirement of the SASP. The next sections will endeavor to highlight how senescence and the process of biological age, rather than chronological age, is perceived as a potential mediator in COVID-19 pathogenesis and severity. Studies are required to identify functional mechanisms which link SARS-CoV-2 infection, COVID-19, and biological aging. Addressing this may potentially provide attractive avenues for therapeutic targets to alleviate COVID-19 disease severity, this is of particular interest for the elderly who are most vulnerable and susceptible.During aging and chronic disease, senescent cells are allowed to accumulate and are not removed via immune regulation or apoptosis processes. Aged tissues are also prone to damage and thus are more likely to enter into senescence ,159,160.One of the principal elements of SARS-CoV-2 infection is the consequent reaction of the patient’s immune response to the virus and the subsequent multi-organ inflammatory response. Aging is associated with weak and less effective immune responses; this phenotype is defined as “immunosenescence”. The acquirement of this phenotype in the elderly predisposes them to complications instigated by viral infections . A signiIn brief, it is expected that patients of increased age have a higher probability of accumulating high levels of cellular senescence. The rationale behind this is that the proportion of senescent cells is already increased prior to the time of SARS-CoV-2 infection thus may accelerate paracrine senescence events. Older cells are also more prone to becoming senescence due to the reduced ability to restore impairments. Finally, an aged immune system is less competent in its ability to eradicate senescence cells, this is partially due to a deterioration in immune functionality. This may explain why older patients are more susceptible to SARS-CoV-2 infection and at a higher risk of more severe COVID-19 disease and mortality. It is thought that inhibition of SASP or reducing senescent lung cells via senolytics may be more beneficial in elderly people with COVID-19. This is a hot topic and new research findings may have a significant translational impact on patients by identifying novel senescent biomarker signatures which have the potential to change the way we predict COVID-19 severity. This may provide exciting avenues for therapeutic intervention by targeting senescence-associated mechanisms via senotherapies in COVID-19 patients and may also serve to help enhance vaccination efficacies. SARS-CoV-2 infection activates both innate and adaptive immune responses. In at risk individuals, with diminished anti-viral defenses, an excessive immune response can develop which is associated with aberrant inflammation and increased disease severity ,180. ThiImmunological responses detected post-SARS-CoV-2 infection are highly variable, though appear to be associated with severity and influenced by age and comorbidities. Patients with severe disease tend to have higher antibody titres than those with mild disease, possibly due to higher viral load . Older aTranscriptome wide analyses of blood from severe COVID-19 patients reveals a series of activated immune system genes including those associated with neutrophils, secretory granules, neutrophil extracellular traps (NETS) along with clonal expansion of adaptive immune response and diminished lymphocyte gene expression . The folInitial innate system Type I and III interferon responses are suppressed or disrupted which results in early IL-6, IL-10 and IL-1β enhanced inflammation . It has CD4+ and CD8+ are an important part of the antiviral adaptive immune response to SARS-CoV-2 infection. The quality and breadth of CD4+ and CD8+ T cell response plays a key role in COVID-19 resolution and modulation of disease severity. B and T cell depletion are early indicators of severe disease development and along with markers of liver damage, act as predictors of mortality in severe COVID-19 patients . RegulatIn recovered COVID-19 patients and in terms of resultant protective immunity, an integrated study of SARS-CoV-2 immune memory in a longitudinal cohort over 8 months exhibited distinct kinetics . ImmunogThere is continued debate around the role of the so-called “cytokine storm” in COVID-19 pathophysiology and severity . Albeit Immunosenescence is a distinctive feature of aging and is associated with defective immune responses and thus less effective anti-viral responses. As individuals get older, there is aberrant interruption of both the innate and adaptive immune responses alongside chronic inflammation due to recurrent production of inflammatory mediators and cytokines, also referred to as “inflammaging”, thereby predisposing the elderly to complications during viral infections ,203,204.There are limited studies on the role of cellular senescence and specifically immunosenescence in COVID-19. Recent findings have however shown that there is the presence of an immunosenescent phenotype which appears to be underpinned by elevated neutrophils-to-lymphocytes ratio (NLR) , and higMany hospital programs and services for treatment of health conditions within patients has been detrimentally destabilized due to the COVID-19 pandemic. In particular, cancer services including elective surgeries have been severely disrupted. There is now a tsunami of patients on extremely long waiting lists. Such emergency and elective surgery programs need to be separated from the COVID-19 pandemic and need to instead run in parallel. Multimorbidity (multiple long-term conditions) is defined as two or more diseases  occurring simultaneously in an individual  , necessiCOVID-19 is an emergent disease of aging and poorer clinical outcomes are evident in patients with existing comorbidities ,145,150.The intricate means by which the phenomenon of senescence contributes to multiple long-term conditions is not yet known. We therefore hypothesise that specific sendotypes may indeed act as differentiating factors in multimorbid diseases as well as in COVID-19 and may have potential to act as therapeutic targets for intervention. We also postulate that senescence may be involved in driving more severe COVID-19 especially in elderly patients burdened with multimorbidity. Vaccination against COVID-19 unified scientific communities globally as they raced together to identify therapeutic and preventative solutions including the identification and development of viable vaccine candidates to stem the threat of COVID-19 worldwide. There are now several vaccines which are in use across the globe, including the Pfizer-BioNTech messenger RNA (mRNA) vaccine, BNT162b2 and the Oxford-AstraZeneca adenovirus vector vaccine, ChAdOx1-S, which were approved for emergency use ,221 by tThe COVID-19 vaccines currently available have demonstrated very high levels of efficacy during the clinical trials and these levels of efficacy are being continuously monitored and analyzed as new SARS-CoV-2 variants of concern (VOC) and variants of interest (VOI) emerge. Seropositive SARS-CoV-2 patients are projected to have 89% protection from reinfection , whilst As elderly individuals are most at risk of developing more severe COVID-19, the safety and efficacy of vaccines in the elderly is critical to their success. Studies to date on the efficacy of the vaccines in the elderly individuals have shown promising results. The administration of one dose of either the BNT162b2 or ChAdOx1-S in elderly patients specifically was associated with a significant reduction in symptomatic COVID-19. Furthermore, one dose of either vaccine was 80% effective at preventing hospital admission, and notably, a single dose of the BNT162b2 was 85% effective at preventing mortality in patients with COVID-19 . This hiClinical trials are also ongoing within some countries to investigate and establish if a patient can have a first dose from one vaccine and the second dose from a different vaccine. Preliminary data from a Spanish clinical trial, CombiVacS, of ~600 patients who received AstraZeneca as the first dose followed by Pfizer as the second dose has been reported. Results so far indicate, that the mix and match of two different vaccines was able to elicit an extremely potent immune response and antibody levels increased dramatically upon the second dose with Pfizer . This stAs the vaccines against COVID-19 are relatively new, it is still too early to know the exact duration of the protection of the vaccine and the level of immunity these vaccines offer against new emerging variants and also against reinfection. As mentioned already, current vaccines appear to have excellent efficacy when administered using the two-dose approach and data published early in 2021 demonstrated that immunological memory in 95% of individuals was retained for ~6 months after infection . HoweverCurrent studies are focusing on investigating these waning immune responses. A recent study has analyzed the association between in vitro neutralization levels and the apparent protection from COVID-19 infection using data from seven different vaccines and from different patient cohorts. The study highlighted that neutralization levels are highly predictive of immune protection in COVID-19 infection and that their prediction models show that deterioration in the neutralization titre occurs over the first 250 days after vaccination, resulting in a significant drop in protection from COVID-19, albeit protection against severe infection should remain ,235. OthThere is limited data available on neutralizing antibodies or vaccine efficacy in the elderly, the most vulnerable older group of patients. Increasing evidence shows that both humoral and cellular immunity are both vital in fighting against COVID-19 . A groupCumulatively, these findings affirm that specific measures such as booster vaccination campaigns, in the elderly especially, to enhance immune responses may need to be implemented, particularly where SARS-CoV-2 VOC are imminent. Booster vaccination programs are already underway in some countries, including Israel. Indeed, preliminary findings have shown that a third Pfizer booster shot for those aged above 60 years was able to reduce the risk of infection by 86% and reduced the risk of severe COVID-19 by 92% . Other sStudies highlight the utility of neutralization and immune protection prediction models in developing vaccine approaches to control the future trajectory of the COVID-19 global pandemic. Neutralization antibody levels wane over time, though this parameter alone is not specific as a correlate of immunity and must be considered with the innate/adaptive immune responses and immunological memory. It can be speculated that senescent cells, which are found at higher abundance in older people ,160, mayThe emergence of new viral variants is a persistent concern as they may have increased transmissibility , and theFurthermore, it has been reported that vaccine effectiveness after one dose of the vaccine (BNT162b2 or ChAdOx1-S) was markedly lower in those who subsequently became infected with Delta variant, compared to those with the Alpha variant. This highlights the importance of sustaining efforts to maximize vaccine uptake of both first and second doses especially in high-risk populations such as the elderly . FurtherOverall, the take home message is that the combination of neutralizing antibody titres due to both age and VOC/VOI, must be considered when developing and designing strategies for booster vaccinations and the duration of protection that would be conferred by such additional booster doses and the clinical impact this would have on severe COVID-19. It is also important to consider that this is fundamental in high-risk populations, such as the elderly, who also have high levels of senescence cells. Further studies are also needed to address the transcriptional, epigenetic, and functional reprogramming, termed “trained immunity”, which occurs and drives immunological memory in those who have recovered from COVID-19 ,269,270.The effects ofSARS-CoV-2 infection and resultant severe COVID-19 symptomology is likely driven by the highly destructive pathological hyperinflammatory response which results in a derailed cytokine storm, thrombosis, vascular leakage, organ damage and aberrant antibody immune responses, ultimately leading to high morbidity and mortality in the most vulnerable. Our review highlights that senescence and aging together, potentially play a central role in COVID-19 pathogenesis. As alluded to already, therapeutic invention via senolytics is paving the way forward in the field of aging research. Indeed, we draw upon the potential of utilizing senolytics as viable therapeutic interventions or targets in COVID-19 to prevent severe infection in patients and specifically aged patients. Strong evidence has been reported recently in old mice where senescent cells became hyperinflammatory upon exposure to the SARS-CoV-2 virus. This resulted in enhanced SASP production of pre-existing senescent cells, increased senescence, inflammation, and high mortality rates in these aged mice. Furthermore, this study went on to show that the pharmacological removal of senescent cells via senolytic (Fisetin or Dasatinib + Quercetin) treatment, in these aged mice, was shown to significantly reduce senescence, inflammatory markers and mortality associated with COVID-19 . These fThe COVID-19 pandemic has identified gaps in our current knowledge of the aged immune system and the integral involvement of specific immune cell subsets. Even though aging is considered to be one of the highest risk factors for COVID-19, the biological mechanisms behind this are not fully known. Indeed, the role of senescence and immunosenescence in COVID-19 is not fully known either. Despite the large numbers of scientific articles and preprints which are being published almost daily, the exact mechanisms behind this conceptual axis of senescence-aging-COVID-19, the phenomena of immunosenescence, novel sendotypes  and immune regulation are not fully known. Understanding the biological pathways involved in this disease will ultimately lead to the identification of key genes and pathways which are linked to senescence-aging-COVID-19, thus paving the way to developing novel drug targets and biomarkers for this disease. Indeed, a recent study which combined gene co-expression network analysis with artificial intelligence approaches has identified a potential novel gene signature which may have clinical value in the pathogenesis of COVID-19 .Therefore, extensive studies are required to further elucidate the mechanisms as to why some patients develop more severe COVID-19 compared to others and to identify the specific aspects of the aged immune system such as novel sendotypes (senescent endotypes) which predispose the elderly to more severe clinical outcomes including mortality. It will also be interesting to explore whether senescence is a confounding factor in waning immune responses especially in the elderly and whether intervention via senolytics could mitigate decreases evident in immune responses after vaccination. It would also be interesting to explore if senescence is a factor involved in predisposing patients to Long COVID. Finally, as many people worldwide have been infected with SARS-CoV-2 and as the pandemic is very much still in its infancy and the long-term impacts from COVID-19 are yet to be fully understood and it is likely that COVID-19 may instigate a long-lasting genomic imprint in humans and their associated physiological processes.


Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer. Ferroptosis is a newly recognized process of cell death, which is different from other forms of cell death in morphology, biochemistry, and genetics, and has played a vital role in cancer biology. This study aimed to identify a ferroptosis-related gene signature associated with LUAD prognosis.via Kaplan–Meier, Cox, and ROC analyses. In addition, the correlations between risk score and autophagy were examined by Pearson correlation coefficient. The analyses of GSEA and immune infiltrating were performed for better studying the function annotation of the gene signature and the character of each kind of immune cells played in the tumor microenvironment.Dataset TCGA-LUAD which came from the TCGA portal was taken as the training cohort. GSE72094 and GSE68465 from the GEO database were treated as validation cohorts. Two hundred fifty-nine ferroptosis-related genes were retrieved from the FerrDb database. In the training cohort, Kaplan–Meier and univariate Cox analyses were conducted for preliminary screening of ferroptosis-related genes with potential prognostic capacity. These genes then entered into the LASSO Cox regression model, constructing a gene signature. The latter was then evaluated in the training and validation cohorts A 15-gene signature was found from the training cohort and validated by Kaplan–Meier and Cox regression analyses, revealing its independent prognosis value in LUAD. Moreover, the ROC analysis was conducted, confirming a strong predictive ability that this signature owned for LUAD prognosis. One hundred fifty-one of 222 (68.01%) autophagy-related genes were discovered significantly correlated with risk scores. Analyses of GSEA and immune infiltration exhibited in detail the specific pathways that associate with the 15-gene signature and identified the crucial roles of resting mast cells and resting dendritic cells owned in the prognosis of the 15-gene signature.In this present study, a novel ferroptosis-related 15-gene signature  was built. It could accurately predict the prognosis of LUAD and was related to resting mast cells and resting dendritic cells, which provide potential for the personalized outcome prediction and the development of new therapies in LUAD population. Lung cancer is the leading cause of death from cancer. In the United States, there will be approximately 228,820 newly diagnosed cases and 135,720 deaths in 2020 . Lung cavia ferroptosis-mediated pathways  and GSE68465 (n = 443) were downloaded from the GEO database and taken as validation cohorts to examine the gene signature we trained.We selected the TCGA-LUAD dataset as the training cohort and only included cases that meet the following criteria: (1) gene expression data is available; (2) survival data is available; (3) and follow-up time is greater than 0 days. Finally, 500 LUADs were included, and their gene expression profile, survival data, and clinical characteristics were downloaded from GDC Xena Hub2 is the world’s first manually curated database for regulators and markers of ferroptosis and ferroptosis–disease associations , which contains an exhaustive, up-to-date list of human autophagy-related genes  gene-set collections using GSEA software (v4.1.06) in the training cohort to uncover the functions and pathways in the differentially expressed genes (DEGs) between high- and low-risk groups. Gene sets with | NES | > 1, NOM p-value < 0.05, and FDR q-value < 0.25 were considered significant.Gene-set enrichment analysis was performed based on Hallmark , 391 LUADs were selected for the next analysis. The correlations between 22 kinds of TICs were examined by the Pearson coefficient.The relative proportion of 22 TICs in the training cohort was calculated using the CIBERSORT algorithm . After tP-value < 0.05 was a statistically significant threshold.For identifying the relationship between the 22-TIC proportion and risk score, an integrated analysis of consisting of the Spearman coefficient and Wilcoxon rank-sum was applied. Based on the 22-TIC proportion and survival data, Cox and Kaplan–Meier analysis were deployed to screen 22 TICs with prognostic meaning. P-value < 0.05 was a statistically significant threshold.The LASSO analysis was conducted applying the “glmnet” R package. Kaplan–Meier analysis was conducted using “survival” and “survminer” R packages. Univariate and multivariate Cox proportional hazard regression analyses were conducted using “survival” R package. The ROC analysis was applied using “survivalROC” R package. The flowchart of the present research is shown in P-values < 0.05 in both Kaplan–Meier and univariate Cox analyses was considered having potential prognostic capacity. Finally, 4,138 genes were identified as potential prognostic genes . Consistent with that in the training cohort, in either univariate or multivariate analysis, the 15-gene signature showed excellent ability in the other two independent validation cohorts in predicting overall survival . Also, we utilized progression-free survival data in the training cohort to perform the Cox analysis and found that the 15-gene signature had the ability not only in univariate but also in multivariate models to predict the outcomes . These evidences demonstrated that the 15-gene signature was to be an independent and strong variable of prognosis.Univariate and multivariate Cox analyses were applied in the training and two validation cohorts based on overall survival or progression-free survival, using the available co-variables including risk score, gender, age, race, tumor stage, tobacco smoking history, radiation therapy, KRAS mutation, and EML4-ALK mutation to validate the prognostic capacity and the independence of the 15-gene signature among other clinic-pathologic characteristics . In the We built ROC curves and time-dependent dynamic AUC comparisons with other variates that showed an independent prognostic value in the multivariate Cox analysis in Moreover, we conducted Pearson correlation to evaluate the relationship between autophagy-related genes and the 15-gene signature risk score. Of the 222 autophagy-related genes, 151 (68.01%) were significantly correlated with risk scores, of which 74 were positively correlated and 77 were negatively correlated . As showIn view of the negative correlation between the 15-gene signature risk score level and the LUAD outcomes, GSEA was performed between the high-risk and low-risk groups. As displayed in To better study how the 15-gene signature and the immune microenvironment interact, we used the CIBERSORT algorithm to evaluate the proportion of tumor-infiltrating immune subpopulations and made comprehensive comparisons with the risk score. The relative content distribution of 22 TICs in the TCGA-LUAD cohort and the correlation between 22 TICs are shown in Incorporating the results of difference analysis  and corrFurthermore, the prognostic capacity of each TIC was examined using univariate Cox and Kaplan–Meier analyses. As shown in In this study, we built a ferroptosis-related 15-gene signature for the prognosis of LUAD by comprehensively mining the TCGA and GEO databases. After discovering the potential ferroptosis-related prognosis genes using Kaplan–Meier and univariate Cox analyses in the TCGA-LUAD cohort, the LASSO Cox regression model was applied, and a 15-gene signature was generated which was related to outcome of LUAD. By applying the 15-gene signature in the training and validation cohorts, pronounced statistical differences were seen in Kaplan–Meier analysis, univariate and multivariate Cox regression models, and ROC curves, demonstrating the effectiveness and broadness of the gene signature in predicting LUAD prognosis. In the following correlation analysis, the 15-gene signature was found correlated with most autophagy-related genes. The GSEA and analysis of immune infiltration exhibited important pathways that relate to the 15-gene signature and vital roles that resting mast cells and resting dendritic cells may have played backing the 15-gene signature influencing the outcome of LUAD. Compared with previous studies discovering the prognostic gene signature in LUAD, we are the first to utilize ferroptosis-related genes for training and validation in two independent cohorts (more than 400 cases each). This work we have done aimed to present more hints in future LUAD research.via acetylation of HSPA5 and subsequent activation of ATF4 and DDIT4 to inhibit the mTOR signaling pathway in NSCLC cells. It is reported that RRM2 is involved in the progression of various cancers, including glioma, colorectal cancer, bladder cancer, and NSCLC  can be found below: AZ, JY, and CM organized and wrote the manuscript. FL and HL produced the figures and visualized the data. CM revised the manuscript. All authors reviewed the manuscript and approved the manuscript 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.


Glycogen storage disease type Ia (GSDIa) is an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-α (G6Pase-α). Affected individuals develop renal and liver complications, including the development of hepatocellular adenoma/carcinoma and kidney failure. The purpose of this study was to identify potential biomarkers of the evolution of the disease in GSDIa patients. To this end, we analyzed the expression of exosomal microRNAs (Exo-miRs) in the plasma exosomes of 45 patients aged 6 to 63 years. Plasma from age-matched normal individuals were used as controls. We found that the altered expression of several Exo-miRs correlates with the pathologic state of the patients and might help to monitor the progression of the disease and the development of late GSDIa-associated complications. Glycogen storage disease type Ia (GSDIa) is a rare disease caused by mutations in glucose-6-phosphatase-α (G6Pase-α), an enzyme expressed in the liver, kidney and intestine, which is fundamental in the terminal steps of gluconeogenesis and glycogenolysis. The lack of a functional G6Pase-α results in the ineffective regulation of glucose homeostasis .GSDIa is a metabolic disorder that causes hypoglycemia, hyperlipidemia, hyperuricemia, and lactic acidemia. The accumulation of glycogen in the liver and kidney causes hepatomegaly and kidney enlargement. Additionally, with time, patients develop serious complications, such as osteoporosis, renal failure and hepatocellular adenomas (HCA), which may progress to hepatocellular carcinomas (HCC). The disease can be managed by a very strict diet to prevent hypoglycemia, consisting of continuous nocturnal gastric drip feeding and/or the frequent oral administration of meals containing complex carbohydrates and uncooked cornstarch .Under dietary therapy, metabolic control can be established in GSD-Ia patients and hypoglycemic episodes can be prevented. Moreover, improved metabolic control reduces the frequency of complications, including HCA and HCC development, ameliorating the prognosis for patients with GSDIa ,4. On thIn this respect, the identification of new biomarkers prognostic of the evolution of the disease and diagnostic of tumor formation in livers may serve to develop new pharmacological approaches, especially since GSDIa patients manifest marked variability in the severity of symptoms and long-term complications.Exosomal microRNAs (Exo-miRs) may represent an important source of disease markers and are being extensively studied because of their mirroring of cellular components. The utilization of blood exosomes as surrogate tissues to diagnose, monitor and predict disease evolution and response to therapy would be a minimally invasive, highly desirable alternative to tissue biopsies ,9,10.G6pc−/− mice that we have generated [G6pc−/− mice with adenomas from LS-G6pc−/− mice without adenomas [The use of genetically engineered mouse models can be an efficient way of discovering prognostic markers and can minimize the problems associated with the use of human subjects, such as the low availability of biological samples from patients, especially when rare diseases or children are involved. To this aim, we have previously analyzed the expression of Exo-miRs in the plasma of a liver-specific murine model of GSDIa, LS-enerated . This anadenomas .G6pc−/− mice to derive a common signature that could be specific and prognostic of liver degeneration and connected with the biological pathways associated with tumor development and progression.In the present work, we have analyzed the expression of Exo-miRs in the plasma of GSDIa patients to derive specific biomarkers and prognostic indicators of liver and kidney tissue degeneration and HCA development. We have identified several Exo-miRs that are modulated in patients in comparison with healthy controls and whose altered expression may be correlated with liver disease and tumors. Moreover, we have identified Exo-miRs involved in renal pathology that are deregulated in GSDIa patients, and compatible with the progressive severe kidney disease occurring in GSDIa. In addition, the Exo-miR expression profiles obtained from the patients was correlated with the liver-specific Exo-miR profiles obtained from the LS-We found that several Exo-miRs that are deregulated in GSDIa patients might serve as biomarkers and thus help to monitor the progression of the disease and the development of late GSDIa-associated complications.We compared the Exo-miR expression profiles of 45 GSDIa patients versus 14 control (CTRL) subjects in order to identify those miRNAs specific for the pathologic manifestations of GSDIa and/or for its evolution. The analysis workflow is summarized in The RNA was isolated from plasma exosomes, reverse transcribed, pre-amplified and used to arrange for each sample a human microRNA array card for the measurement of the expression of 384 targets by the qRT-PCR. A visual inspection of the percentage of raw cycle thresholds (Ct) and missing values across all sample profiles indicated a comparable percentage of Ct and missing values across the samples A. Raw dap-value < 0.05, Visual inspection of the distribution of the most reliable expression values across the samples shows a clear heterogeneity among Exo-miR expression profiles , which mMissing values are difficult to handle using standard statistical analysis . We usedIn order to identify specific deregulated Exo-miRs that could represent new potential biomarkers of disease development and progression, we first analyzed the expression levels of Exo-miRs in GSDIa patients and compared them with the expression levels of Exo-miRs in CTRL subjects. Our analysis identified six upregulated and four downregulated Exo-miRs in GSDIa compared to CTRL patients . Violin SIRT1 and is detectable in the serum and urine of patients with diabetic nephropathy [We also found that some of these microRNAs are relevant in kidney injury, failure and chronic disease. In particular, these microRNAs have been associated with diabetic kidney disease, one of the major microvascular complications in patients with type 1 or type 2 diabetes, which represents the primary cause of end-stage renal disease. miR-483-5p has diagnostic value in diabetic nephropathy and can protect human proximal renal tubular cells from the apoptosis and inflammation induced by high glucose . The serhropathy .The finding of Exo-miRs involved in renal pathology is very relevant considering that progressive severe kidney disease is a major long-term pathological manifestation in GSDIa. In fact, the pathological manifestations of the life-threatening kidney disease in GSDI overlap with diabetic nephropathy. Both GSDI and diabetic nephropathy start with a long period of silent glomerular hyperfiltration, followed by the development of microalbuminuria, proteinuria and, eventually, renal failure . DiabetiKI67 and CAPNS1 and its overexpression reverses the epithelial–mesenchymal transition induced by hepatectomy through the targeting of IL-1Β, SNAIL1 and TWIST1 [We then compared the Exo-miR expression levels in GSDIa patients with HCA with the Exo-miRs expression levels in GSDIa patients without HCA. Differential expression analysis identified three upregulated and two downregulated Exo-miRs in GSDIa patients with HCA versus GSDIa patients without HCA . Violin d TWIST1 . Anotherd TWIST1 . Moreoved TWIST1 , a primad TWIST1 .Therefore, the altered expression of several Exo-miRs may be correlated with pathologic liver conditions and might help to discriminate between affected patients during the progression of the disease and the development of HCA and HCC.IGF1R, which is highly expressed in tumor tissues and a target of miR-16-5p [IGF2 in patients with hepatitis B virus-related HCC [LRP6 and PIK3R2 [The age-dependent modulation of Exo-miRs might be instrumental to study the evolution of the disease, and to find biomarkers prognostic of HCA and HCC and the onset of GSDIa. Patients were assembled into three groups according to their age at the time of sample collection  to reflect the different stages of disease progression. The levels of expression of the 103 Exo-miRs were examined in the GSDIa patients across the age groups using the BETR method. For each Exo-miR, BETR calculated a numeric value indicating the probability of the differential expression of Exo-miRs in the dataset . Exo-miRiR-16-5p ,50. Simiated HCC ,52. miR-d PIK3R2 . The assd PIK3R2 . Interesd PIK3R2  and hyped PIK3R2 .These findings indicate an age-dependent modulation of Exo-miR expression as the GSDIa patients became older and suggest that alterations of these microRNAs are compatible with their involvement in the long-term complications associated with GSDIa.p-value < 0.05; SIRT1, FoxO3a, AMPK, and PPAR-α, and most of these genes are targets of the deregulated microRNAs we identified. Moreover, a study by Rossi et al. [SMAD, FoxO, mTOR and Notch signaling pathways  processes and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway ontologies. Pathway analysis was carried out for each significant Exo-miR using the MirWalk tool . Each sii et al.  suggestspathways . Finallypathways . TherefoG6pc−/− mouse [To identify potential biomarkers of the pathophysiology of the GSDIa-affected liver we have recently analyzed the plasma exosomes of a murine model of GSDIa in an LS-/− mouse  to uncov/− mouse , as well/− mouse .On the basis of those findings, we have here evaluated whether the Exo-miR modulation of expression found in GSDIa patients would overlap with that obtained in mice and thus allow us to derive a common signature of Exo-miRs that could be specific and prognostic of liver degeneration and HCA development in this disease.G6pc−/− mice with tumors, is involved in the signaling pathways associated with HCC, including Wnt, TGFβ, and Ras, interacts with circular RNA in HCC [G6pc−/− mice in comparison to wild-type mice.We first compared the Exo-miRs found modulated in humans with those modulated in mice. miR-145-p and miR-203-3p, biomarkers of HCC and involved in HCC growth ,61, wereA in HCC  and is oA in HCC . miR-203A in HCC , and wasG6pc−/− mice livers and associated with specific biological pathways [We then evaluated whether the Exo-miRs modulated in patients would regulate genes expressing the proteins previously found differentially represented in the LS-pathways .G6pc−/− mice into human genes and then used the miRGate database [G6pc−/− mice, including protein sets related to response to hypoxia, glucose and lipid metabolism, and the inflammatory and immune responses .g for 10 min at room temperature and was stored at −80 °C for exosomes isolation. Exo-miRs were prepared from 500 μL of plasma, after centrifugation at 16,000× g at 4 °C to eliminate cellular debris, utilizing the ExoRNeasy Midi kit , according to the manufacturer’s instructions. Quality control and microRNA evaluation were determined with the Agilent 2100 Bioanalyzer, using the small RNA assay . To collect intact exosomes, the exoRNeasy Serum/Plasma Midi kit protocol was used: in the last step of the process, QIAzol was substituted with 150 μL of Buffer XE and the particle size was evaluated using the zetasizer nano ZS90 particle sizer . As determined by dynamic light scattering analysis, isolated vesicles had the typical size range of exosomes, ranging between 30 and 120 nm . cDNA was pre-amplified with TaqMan®PreAmp Master Mix and MegaplexTM Pre-Amp primers Human Pool A. The pre-amplified product was diluted according to the manufacturer’s instruction and mixed with 450 μL of TaqMan®Universal Master Mix II, No UNG , and 441 μL of nuclease-free water. The microRNA profiling was performed with the ViiATM 7 Real-Time PCR System on the TaqMan®Array human microRNA A card , enabling the quantification of 381 human microRNAs.Exo-miRs were analyzed using the TaqMan Array Card Technology. Briefly, 50 ng of RNA were reverse transcribed with the TaqMant-test. Statistical analysis and plots were carried out using GraphPad Prism version 8.0 for Windows). miRGate, a curated database of human, mouse and rat miRNA–mRNA targets [−/−-G6pc mouse livers [https://www.genenames.org/ (accessed on 21 October 2021)). Candidate microRNAs were compared with the significantly modulated Exo-miRs in the GSDIa patients identified in this study to assess the association between liver degeneration and Exo-miR deregulation in GSDIa patients.RT-qPCR data processing, categorization, normalization, filtering, imputation and differential expression were performed using the PIPE-T Galaxy tool . The Ct  targets , was usee livers  to idente livers  (https:/In our study, we investigated microRNA expression profiles in the circulating exosomes of GSDIa patients in order to identify the Exo-miRs that are relevant as biomarkers of the pathological manifestation and progression of disease. The results of our studies delivered several microRNAs relevant as biomarkers to various statuses of the disease. We could identify the microRNAs associated with liver disease and the metabolic alterations of glucose and lipid pathways. We also found the deregulation of microRNAs relevant in liver tumor development and, finally, several microRNAs whose altered expression has been associated with diabetic and chronic kidney diseases.These results are important because they highlight the potential of plasma exosomes to be surrogate tissues to study the development of the disease and the onset of serious complications. The existence of a communication network between pathologic tissues and their environment through the shedding of exosomes has been shown in several diseases, including cancer, rare diseases and tissue degeneration. For example, exosomes can be readily detected in higher concentrations in the serum and plasma of cancer patients. Thus, the utilization of blood exosomes as surrogate tissue to diagnose, monitor and predict disease evolution and response to therapy constitutes a less invasive and highly desirable alternative to biopsies.−/−G6pc mouse has proven to be essential in the present and previous studies, since it reproduces all the pathological characteristics of the GSDIa liver, including hepatomegaly, glycogen accumulation, hepatic steatosis, progressive hepatic degeneration and liver tumor development.The availability of an animal model is indispensable to compensate for the limitations of testing patients with rare diseases. The LS-−/−G6pc mice has been determined utilizing the same molecular and informatics tools described in the present manuscript and have now been used to identify the specific manifestations of liver Exo-miR contributions, since in our experimental model only the liver is affected. On the other hand, we also identified a cluster of Exo-miRs, involved in kidney disease, unique to the patients, because this organ is normal in LS-−/−G6pc mice.The profiles of blood Exo-miRs levels in control and LS-A serious complication of GSDIa is renal damage and this is comparable to diabetic nephropathy. In fact, hyperlipidemia is considered to be a risk factor for the progression of diabetic nephropathy and correlates directly with the evolution of renal damage in patients with GSDI. In this respect, the control of hyperlipidemia is important to both prevent severe kidney damage and improve the efficacy of the current treatment. The identification of a set of microRNAs involved in kidney disease may thus be helpful for identifying new drugs to treat renal pathology in GSDIa patients.In this study we have identified the Exo-miR signatures’ target genes and organized them into molecular pathways to gain information on the events potentially controlled by the exosome cargo. Thus, our data provide evidence that the Exo-miR profiles identified may relate to the specific affected organ gene expression and that the long-term consequences of GSDIa can be monitored through Exo-miRs assessment.In conclusion, our results may evolve into protocols to counteract both the progression of liver degeneration leading to HCA and HCC onset as well as kidney disease and failure using circulating microRNAs as biomarkers.