Pediatric patients, especially those with rare diseases, represent a population that has a high tendency towards off-label drug use (OLDU) and needs a more careful practice of pharmacotherapy than in adults. We aimed to investigate biotechnological drug use in children with rare diseases requiring OLDU. This retrospective study examined all single-diagnosed OLDU applications (n = 5792) for 4992 children (<18-year) in Turkey. Applications of rare diseases were selected, and their descriptive characteristics were examined, including demographic features of patients, biotechnological drug utilization status, and disease categories. The off-label statuses of the drugs at the end of 2020 were also examined. In total, 77.7% (n = 4501) of OLDU applications were made for rare diseases. Biotechnological drug use was higher in rare disease applications than in nonrare diseases . Canakinumab was the top applied biotechnological drug (73.2%). Compared to that in small-molecule drugs, the mean age of patients was higher in biotechnological drug-containing applications . Biotechnological drug use was higher in nonneoplastic rare diseases (40.3%) than in neoplastic rare diseases (26.4%), (p < 0.0001). At the end of 2020, the approval status of the off-label indications covered in 2016 was significantly higher for rare (24.4%) vs. nonrare  diseases and for biotechnological (32.3%) vs. small-molecule  drugs. In total, 87.7% of the drugs would have to be still used in the off-label setting at the end of 2020. It was seen that more than three-quarters of the pediatric OLDU applications are for rare diseases, and the need for biotechnological OLDU in this group is almost 2-fold of small-molecule drug use. While further projected findings imply a higher approval tendency for rare diseases and biotechnological drugs, there seems to be more room for improvement for pediatric drug use. Despite differences in its definition, rare diseases are generally considered to cover diseases affecting no more than 5/10.000 people in the community [1]. Studies show that rare diseases in childhood may cause sequelae in children and pose a serious psychological and economic burden on both families and all stakeholders of the health system . A major attempt to alleviate this burden is obviously the novel drug development studies for treating rare diseases. However, apart from the conductibility of a limited number of studies in children due to ethical reasons, it is known that clinical studies in rare diseases have additional methodological challenges and the pharmaceutical industry does not always find it attractive to direct research and development incentives to this group . Although granting extra privileges to clinical trials for rare diseases has been on the agenda of health authorities for a long time, off-label drug use (OLDU) remains prevalent in diagnoses within the scope of rare diseases [3–5]. Moreover, it is estimated that the high OLDU burden already existing in children may escalate even more in the case of rare diseases .Data on pediatric usage of biotechnological drugs is very limited [9]. A biotechnological drug is defined as “a human medicinal product whose active ingredients or substances were produced in or purified from a biological source, where the quality, manufacturing process, and audits are demonstrated through physicochemical and biological tests” [10]. Biotechnological drugs are among the treatment options in a wide range of conditions, including cancers, rheumatological/immunological diseases, and nephrological diseases, and they can also be used in the treatment of many rare diseases in these areas . Available literature has not revealed any study about the management of rare diseases via off-label drugs with a particular focus on biotechnological products . In fact, biotechnological drug utilization data is needed in rare diseases to overcome therapeutic challenges in these diseases, especially in the pediatric population. In this study, we aimed to evaluate the use of biotechnological drugs for rare diseases in children who needed off-label drugs.In this cross-sectional study, we examined OLDU applications conducted between 1 January and 31 December 2016 in Turkey. Applications for OLDU are submitted to the Turkish Medicines and Medical Devices Agency (TMMDA) of the Ministry of Health to be evaluated and approved [13]. We reviewed single-diagnosed applications for the pediatric population (<18 years) submitted by pediatricians, subspecialists of pediatrics, child psychiatry, and pediatric surgeons in 2016 (n = 8272). In this dataset, those applications belonging to a patient for the same drug during the study period were determined as a repeated application and excluded (n = 2480).The applications were grouped according to their indicated diagnoses as rare and nonrare diseases. Whether the diseases in the applications were rare or not were decided according to the Orphanet database supported by the European Commission [14]. The biotechnological or small-molecule statuses of the applied drugs were determined, and their distributions were compared according to the groups. In addition, single or multiple drug application statuses were compared in OLDU applications for rare or nonrare diseases. The age and sex of the patients and the distribution of the physician’s specialties were compared according to the diagnosis groups. OLDU applications for rare diseases were further analyzed in detail. The drugs in these applications were grouped according to the “Anatomical Therapeutic Chemical” (ATC) coding system. The first five most common biotechnological and small-molecule active substances were identified. The distribution of the drugs at ATC-1 level was evaluated, and the percentage of biotechnological or small-molecule drugs in each sublevel was determined. In addition, ATC-1 level status within biotechnological and small-molecule drugs was evaluated. The most frequently applied biotechnological and small-molecule drug in an off-label setting was determined for each ATC-1 group. The mean ages of patients for whom OLDU was applied and their biotechnological and small-molecule drug statuses were compared for each ATC-1 group.The distributions of the main diagnosis groups according to “International Statistical Classification of Diseases (ICD)” in rare disease applications were examined according to their status as biotechnological or small-molecule drugs. The most frequently applied biotechnological or small-molecule drugs were examined in each main diagnosis group. The diagnoses were also classified as neoplastic and nonneoplastic and were compared in terms of off-label biotechnological drug use. Single/multiple OLDU applications were compared to determine whether these were made for biotechnological drugs or not. These applications were also evaluated in terms of off-label drugs’ pharmaceutical form and route of administration.As the regulatory approval statuses of the drugs may alter by time due to the completion of trials, we also examined the off-label statuses of the drugs at the end of 2020 to reflect the progression of the safe and effective use of drugs for children. For this purpose, the approval statuses of all drugs applied for all off-label indications in 2016 were analyzed as if they were applied at the end of 2020. This projection was detailed for rare vs. nonrare diseases and for biotechnological vs. small-molecule drugs.Statistical analyses were made through SPSS 24.0 software. Categorical variables were expressed as number/percentage, and continuous variables were mentioned as mean/standard deviation. The comparisons between categorical and continuous variables were analyzed via chi-square and t-test, respectively. An overall 5% type-I error level was used to infer statistical significance.The data were collected after the study was approved by Ethics Committee of Institute of Health Sciences of Marmara University .A total of 5792 OLDU applications were detected for 4992 children in the study period. It was determined that the applications with rare diseases constituted 77.7% (n = 4501) and that these applications were made for 3894 (78.1%) patients. There were no significant differences between children with rare diseases and nonrare diseases in their applications (p > 0.05) in terms of age  and sex . The need for multiple distinct off-label drugs for one patient during the year was similar for rare and non-rare diseases (p > 0.05). The percentage of biotechnological drug use for rare diseases was significantly higher than that for nonrare diseases , (p < 0.0001), (Table 1). OLDU applications were made most frequently by pediatric rheumatologists/nephrologists (28.1%) and child neurologists (39.0%) in rare and nonrare diseases, respectively.When the drugs in the applications were examined at the ATC-1 level, the most commonly encountered groups were “L-Antineoplastic and immunomodulating agents” (48.2%), “A-Alimentary tract and metabolism” (12.4%), and “B-Blood and blood-forming organs” (7.4%). When biotechnological drug ratio was examined in each subgroup at ATC-1 level, the highest rate was “H-Systemic hormonal preparations” (63.2%), followed by “L-Antineoplastic and immunomodulating agents” (57.5%) and “B-Blood and blood-forming organs” (27.5%) .When the distribution of biotechnological and small-molecule drugs in the applications was analyzed at the ATC-1 level, it was found that both biotechnological and small-molecule drugs more commonly belong to the “L-Antineoplastic and immunomodulating agents” group . Canakinumab and mycophenolate were the most common biotechnological (20.8%) and small-molecule (29.6%) drugs in this group. The mean age of the patients using biotechnological drugs (9.7 ± 4.9 years) was significantly higher than that of the patients using small-molecule drugs . This age difference between biotechnological and small-molecule drugs was preserved in patients using “A-Alimentary tract and metabolism” (p < 0.0001), “J-Antiinfectives for systemic use” (p = 0.008), and “M-Musculoskeletal system” drugs at ATC-1 level (p = 0.025), (Table 2).The most common five biotechnological drugs used off-label were canakinumab (15.3%), rituximab (13.3%), eculizumab (11.2%), somatropin (10.0%), and anakinra (7.9%). In small-molecule drugs, this ranking included mycophenolate (9.8%), sapropterin (6.9%), iloprost (5.1%), sirolimus (4.0%), and tacrolimus (3.5%). “Endocrine, nutritional and metabolic diseases” main ICD group were the most common indications for both biotechnological (33.9%) and small-molecule drugs (27.2%). The most commonly encountered biotechnological and small-molecule drug in this diagnosis group was canakinumab (31.7%) and sapropterin (25.1%), respectively (Table 3).It was detected that 17.5% of the rare diseases were neoplastic in nature. The use of biotechnological drugs was significantly lower in neoplastic diseases (26.4%) than that in nonneoplastic ones (40.3%), (p < 0.0001). In rare diseases requiring multiple OLDU applications, biotechnological drug utilization (31.1%) was significantly lower compared to that in the diseases which needed only a single off-label drug (40.0%), (p < 0.0001), .Analysis of off-label drug administration routes showed that the drugs were administered with injectable forms (49.1%), followed by enteral (47.1%) and other administration routes (3.8%). In almost all biotechnological drug applications, the route of administration was injection (99.6%), whereas the injectable forms constitute 18.2% of small-molecule drugs.We identified that 20.1% (n = 1163) of the off-label indications covered in 2016 would have been approved as of 1January 2021. This projection was significantly more pronounced for rare diseases (24.4%) than nonrare diseases  and for biotechnological drugs (32.3%) than small-molecule drugs . In terms of all drugs applied for off-label use in 2016 (n = 353), twelve drugs (3.4%) had been approved for those particular indications until 2021, whereas 33 drugs (9.3%) were partially approved for certain indications. The rest (87.3%) was observed to be used in the off-label setting until the end of 2020.The present study was the first to focus on off-label biotechnological drug use in children at the national level, where important findings that may help to manage rare diseases were obtained. The pediatric population requires additional care in drug use and frequently needs OLDU [3]. The management of rare diseases in this group, which already has limited drug use data, is a special area with unique balances. Despite the incentives for clinical trials in rare diseases, the number of drugs approved for use in these diseases remains low . Considering that 95% of rare diseases do not have approved treatment, it is often expected to refer to OLDU in rare diseases in children . This was supported by our finding that more than three-quarters of OLDU applications in children were rare diseases. Although rare diseases are thought to affect a restricted population, 8% of the society was reported be faced with a congenital or acquired rare disease . Around 8000 rare diseases have been described worldwide, 70% of which had a pediatric onset . Considering the population of children in Turkey [19], rare diseases (n = 3894) that need to be referred to the health authority to be managed by off-label drugs can affect 17 out of every 100,000 children. Similar to this study, another study conducted in Turkey in 2015 on the use of OLDU in children reported that applications were made more frequently for male patients [8]. Although this may suggest a country-specific difference at first glance, studies in other countries based on drug use in children also reported similar findings, especially in younger children . Biotechnological drug use is on an increasing trend across the globe. Similarly, studies focusing on new drug development show that the number of biotechnological drugs increases with a rising momentum . It is known that many new drugs have been developed in recent years, and biotechnological methods have been used in this process, especially in rare diseases, which are a common area of need for alternative treatment . In our study, the use of off-label biotechnological drugs in rare diseases is approximately twice that of nonrare diseases, indicating the need for biotechnological drugs to manage rare diseases in children. Although there is not enough data on the overall biotechnological drug utilization in children, it has been reported that they are frequently used in immunocompromised conditions and childhood cancers are one of the areas where biotechnological drug use is most needed [9]. Considering the area covered by these conditions in rare diseases, it can be said that the findings obtained in our study are partially compatible with the literature. On the other hand, it can be stated that when off-label drugs are required in rare diseases, it is important that more than one of the three drugs applied is biotechnological in terms of quantitative representation of the need in this area. This high utilization rate and further pharmacoepidemiological study findings are expected to provide the basis for the on-label use of biotechnological drugs in rare diseases. Moreover, despite this increasing trend of biotechnological drug use in rare diseases, information on the pharmacokinetics, pharmacodynamics, and pharmaceutical application of these drugs is still limited in children, especially in those with rare diseases, compared to small-molecule drugs [9]. Besides, the use of biotechnological drugs other than the indications for which they are licensed takes place through extrapolation [24]. Considering their use in rare diseases, it may be suggested that the existing risk management plans of these drugs may be more likely to accompany various uncertainties in rare disease-oriented practice and that additional challenges may arise, indicating the need for additional measures.Consistent with the results of the previously reported OLDU-focused study in Turkey, we determined that nearly half of the drugs applied for rare diseases belonged to the antineoplastic and immunomodulating agents [7]. This main group was ranked first in both biotechnological and small-molecule drugs for rare disease applications. This may have been due to the procedure related to the application of OLDU in Turkey, rather than the fact that biotechnological drug use in rare diseases brought this main group to the top rank. In fact, the literature findings outside Turkey showed that the most commonly encountered off-label used drugs in children indicated other drug groups . This can be attributed to the diversity of different countries in OLDU-related practices. For instance, drugs in antineoplastic and immunomodulating agents group can be used after meeting more rigorous clinical requirements than other drugs due to their difficulty in safety, cost, and compliance [9]. The fact that the OLDUs included in our study did not cover “routine use of off-label drugs that do not require additional procedures” but consisted of OLDUs that are subject to the application to the health authority may have led to this risky drug group being the main group most commonly encountered.Although the definition of orphan drugs covers drugs from different groups for different reasons, it is a commonly used concept for many drugs used to treat rare diseases due to its small market share [16]. It is noteworthy that in our study, the most common biotechnological drug was canakinumab and the most common small-molecule drug was mycophenolate. In addition, canakinumab was observed to be the most common biotechnological drug in applications containing endocrine, nutritional and metabolic diseases, the most common diagnostic group. In fact, both these drugs were reported to be at the top in other studies on OLDU in Turkey . Canakinumab, a recombinant human monoclonal antibody, is an interleukin-1 beta inhibiting orphan drug. Systemic juvenile idiopathic arthritis and extremely rare cryopyrin-associated periodic syndromes (CAPS) constitute the main indications of this drug [27]. Another monoclonal antibody, eculizumab, which was reported to be the most frequently encountered drug in pediatric OLDU practice in Turkey [8], ranked third in our study.The mean age of the patients requiring off-label biotechnological drugs was higher than that of those requiring small-molecule drugs. This might be attributed to several reasons. The first may be that the diagnosis of diseases requiring biotechnological drugs, such as amyloidosis, which is common in OLDU applications, can be diagnosed in older age groups . Another important reason is that access, application, awareness, cost-effectiveness, reimbursement condition, etc. aspects of the small-molecule drugs can bring up their use before biotechnological drugs . Therefore, this age-related condition can be explained by the fact that physicians are likely to apply for biotechnological drugs later for children with rare diseases requiring OLDU.The status of neoplastic cases in rare diseases is another debatable point of the research. It was reported that 11.1% of rare diseases identified in Orphanet were neoplastic [18]. We found that around one-sixth (17.5%) of rare diseases have neoplastic origin. We further observed that biotechnological drugs were required less frequently for rare neoplastic diseases than nonneoplastic ones. Various studies reported that neoplastic diseases constituted the main indication group with the highest number of drug development studies and orphan drug approval among rare diseases . This difference seems to suggest that there may be more alternative drugs for the treatment of rare neoplastic diseases.The high share of nearly 80% covered by rare diseases in OLDU raises the question of whether the management of rare diseases with OLDU is routine or a mandatory practice arising from seeking treatment. While the method of the study did not allow for a direct answer to this, we observed that one off-label drug was sufficient for at least 1 year in three out of every four applications in both rare and nonrare diagnoses. This shows the tendency of the situation in the diseases that need OLDU to be partially controlled with a single off-label drug. Accordingly, it can be inferred that management of these diseases could be maintained routinely with OLDU, rather than seeking treatment, at least during the study period, but this was not unique to rare diseases. On the other hand, it was determined that biotechnological drug use in rare diseases requiring multiple applications of OLDU was lower than those requiring application for a single drug. This difference may be related to the tendency of physicians not to replace biotechnological drugs or not to add new biotechnological drugs in the same year in a sensitive therapeutic area like rare diseases. In other words, it can be said that although biotechnological drugs are often used in rare diseases, physicians tend to insist more on their off-label drug choices. These findings highlight the importance of investigating the use of off-label biotechnological drugs in rare diseases.The projected approval status of the OLDU showed that one-fifth of this special drug use turned into routine clinical practice between 2016 and 2021. We observed that this possibility was near 5-fold higher in rare diseases and 2.3-fold higher for biotechnological drugs, which seems to be consistent with ongoing efforts in these therapeutic areas . On the other hand, the fact that only 12.7% were approved or partially approved during 4 years may suggest that there is further room for improvement for pediatric OLDU.Various research reported that the proportion of parenteral drugs prescribed by routine prescribing procedure in Turkey was between 4% and 6% [30]. However, in OLDU, which is a nonroutine application, this rate was reported to escalate up to 38% [7], with no data on the pediatric population. In our study, the distribution of injectable forms found in rare diseases in children increased approximately 10 times the routine practice, and the share of enteral and injectable pharmaceutical forms was almost equal. This situation can be partly explained by the fact that more than 1/3 of the drugs used to manage rare diseases with OLDU were biotechnological drugs in our study. We further noticed that nearly all biotechnological drugs were administered to the patient in injectable forms. In fact, although various studies are underway to deliver biotechnological drugs in different pharmaceutical forms, intravenous and subcutaneous routes are still the most common ways of administration in biotechnological drugs [31]. On the other hand, we observed that the proportion of injectable drugs in off-label use of small-molecule drugs in children was approximately five times higher than that of routine use. This suggests that injectable pharmaceutical forms are more common in OLDU settings than in routine practice, even considering only small-molecule drugs.The main limitation of this research was that all of the data accessed were OLDU applications with no other medical records of patients. Such lack of data may underestimate the actual reasons that may have led the physician to apply for OLDU, including prior drug use experiences for this indication, other drug use-related ineffectiveness/adverse effects experience or comorbid conditions. In addition, the use of the Orphanet database in the classification of rare diseases can be considered a relative limitation because the rare diseases that do not exist in this database could not be included. In conclusion, the details of particularly biotechnological product-oriented OLDU in pediatric rare diseases were described for the first time at a country-level. It is observed that the majority of OLDU applications made for children consist of rare diseases and that physicians tend to prefer biotechnological drugs more often in these diseases. While projected findings imply a higher approval tendency towards rare diseases and biotechnological drugs, the need for improvement still seems to remain for routine safe and effective pediatric drug use. Important findings obtained in the study, especially those specific to biotechnological drugs, are expected to help manage the pharmacotherapy process in this fragile population in a way that would be less likely to require off-label drugs.This work has been supported by Marmara University Scientific Research Projects Coordination Unit under grant number SAG-C-DRP-150218-0034.The study design does not require obtaining informed consent.


To estimate occupation risk from COVID-19 among teachers and others working in schools using publicly available data on mortality in England and Wales.Analysis of national death registration data from the Office for National Statistics.England and Wales, 8 March–28 December 2020, during the COVID-19 pandemic.The total working age population in England and Wales plus those still working aged over 65 years.Death with COVID-19 as a primary outcome and death from all causes as a secondary outcome.Across occupational groups, there was a strong correlation between COVID-19 mortality and both non-COVID-19 and all-cause mortality. The absolute mortality rates for deaths with COVID-19 were low among those working in schools  relative to many other occupations . There was weak evidence that secondary school teachers had slightly higher risks of dying with COVID-19 compared with the average for all working-aged people, but stronger evidence of a higher risk compared with the average for all professionals; primary school teachers had a lower risk. All-cause mortality was also higher among all teachers compared with all professionals. Teaching and lunchtime assistants were not at higher risk of death from COVID-19 compared with all working-aged people.There was weak evidence that COVID-19 mortality risk for secondary school teachers was above expectation, but in general school staff had COVID-19 mortality risks which were proportionate to their non-COVID-19 mortality risk. We used routinely collected data on all deaths in England and Wales, which are near complete and not susceptible to serious ascertainment biases.We were able to compare mortality data for teachers and other school workers with all other occupational groups and with the working-aged population.The number of deaths due to COVID-19 was small and thus differences between the specific occupational groups were imprecisely estimated.We did not have access to individual-level mortality data so were not able to account for potential confounders such as comorbidities or household size.For those working in schools who were aged over 65 years, we had neither mortality rates per 100 000 nor total numbers within the group; we only had number of deaths and a 5-year average, we do not know whether the denominators have changed for this group over the last 5 years.School closures have been implemented in many countries in an effort to slow the spread of SARS-CoV-2 responsible for the COVID-19 pandemic. This has affected hundreds of millions of children globally and has been contentious due to harms that have arisen to children as a result of school closures.Whether or not teachers and others working in schools are at higher risk of COVID-19 as a result of schools being open is central to decisions on school closures, but until recently there has not been good data on this. Record linkage studies carried out in ScotlandAnalyses of more robust outcomes such as hospitalisation from COVID-19 are less susceptible to detection bias and may be more reliable. The Scottish studyThe Office for National Statistics (ONS) routinely collects mortality data for those living in England and Wales. They released data on deaths with COVID-19 and all-cause mortality by occupation as a publicly available dataset.10The ONS mortality statistics used for this analysis are based on mandatory registration of all deaths in England and Wales. So far, three datasets on mortality with COVID-19 by occupation have been released; the first covered the period from 8 March to 20 April which included the peak of wave 1 deaths, the second covered the period between 8 March and 25 May, and the final dataset  covered the period from 8 March to 28 December.We used these data to: (1) describe the mortality rates among all educational professionals combined and present these alongside rates for all professionals and all working-aged people for the different time periods covered by these data ; (2) com10.1136/bmjopen-2021-050656.supp1Supplementary dataOccupation was reported on the death certificate at the time of death registration by the informant and was coded according to the ONS standard occupational classification 2010.The group ‘teaching and educational professionals’, as defined by the ONS, includes: higher education teaching professionals, further education teaching professionals, secondary education teaching professionals, primary and nursery education teaching professionals, special needs education teaching professionals, senior professionals of educational establishments, education advisers and school inspectors, and teaching and other educational professionals not elsewhere defined.The group ‘teaching and educational professionals’ does not include: teaching assistants, school secretaries, lunchtime assistants or educational support assistants, but these occupations were considered along with teachers working in schools where sufficient data were available.The group defined by ONS as midday and crossing patrol occupations includes some individuals who work as road crossing patrols, however not all schools have these and if they do usually only have one, so the group will mainly comprise of those who supervise children in school during their lunch break and who prepare school lunch. Therefore, midday and crossing patrol occupations will be shorted and referred to in this manuscript as lunchtime assistants.The comparison groups we used were:All working-aged people—for comparisons of mortality rates and risk presented in All occupations—for comparisons of number of deaths presented in All professional occupations—for comparisons of mortality risk presented in Professional occupations are those which are classified by ONS as major occupational group 2, these are occupations which require a degree or equivalent period of relevant work experience and include, but are not limited to: scientists, engineers, architects, doctors, nurses, radiographers, physiotherapists, social workers and solicitors .ONS defines deaths with COVID-19 as those where COVID-19 or suspected COVID-19 was mentioned anywhere on the death certificate, including in combination with other health conditions. If a death certificate mentions COVID-19, it will not always be the main cause of death but may be a contributory factor.We calculated the relative risk (RR) of death for educational professionals, lunchtime and teaching assistants compared with all working-aged (20–64 years old) people and compared teachers with all professionals in England and Wales stratified by sex, for this we used published ONS data on total deaths and age-adjusted mortality rates per 100 000 for each group. We derived the denominator  for each group of interest as:Risk=mortality rate per 100 000 taken from ONS data/100 000.We calculated 95% CIs and p values using the formula:CI=exp(ln(RR)±1.96×√(1/deaths+1/control deaths−1/population−1/control population)2P=exp((−0.717×(RR/SE))–(0.416×(RR/SE)))The ONS data included deaths for eight subgroups of educational professionals; we included those with a sufficient number of COVID-19 deaths (≥10) in our analysis. In addition, sufficient data were available to include female teaching and lunchtime assistants in this analysis. We also performed a fixed-effect meta-analysis across the four occupational groups working in schools among women.We estimated mortality rates for all other causes (other than COVID-19) by calculating the difference between all-cause mortality and mortality with COVID-19 for each minor occupational group as defined by ONS.9 11’ mortality by occupational group for men and women separately and calculated Pearson’s correlation coefficient for these plots.We produced scatterplots of ‘all-cause’ versus ‘with COVID-19’ mortality and ‘all other causes’ versus ‘with COVID-19We also calculated proportionate mortality ratios as mortality rates from COVID-19 divided by all-cause mortality for occupations working in schools.Finally, we used our estimates from above for the SE and the denominators for each occupational group to estimate the variance for COVID-19 mortality. We then performed a weighted least squares regression analysis (weighted by 1/variance) of COVID-19 mortality against other cause to determine the increase in COVID-19 for one death increase in other cause mortality across occupational groups.The public were not involved in the design or conduct of this study.Death rates from COVID-19 and all causes among all educational professionals are presented alongside death rates for all professionals and all working-aged people by time period in Between 8 March 2020 and 28 December 2020, there were 68 757 deaths from all causes among the working-aged (20–64 years) population in England and Wales, 7961 (12%) of these deaths involved COVID-19, of which, 2494 had occurred prior to 20 April, 2267 occurred between 20 April and 25 May, and 3200 occurred between 25 May and 28 December 2020.There were 1326 deaths from all causes among educational professionals during this period; 139 of those deaths were thought to involve COVID-19.COVID-19 was involved in one in eight male deaths and one in nine female deaths among working-aged people; among education professionals, these figures were approximately 1 in 8 deaths among men and 1 in 11 among women. COVID-19 was involved in a higher proportion of deaths early on in the pandemic; data covering the 6 weeks of the first peak in the UK showed that COVID-19 was involved in one in four deaths in male and one in six deaths in female educational professionals during this time. During the 10 months covered by the ONS data, all-cause and COVID-19 mortality rates among educational professionals appeared to be lower than for all working-aged adults and similar to those for all professionals.We examined number of deaths by occupation among those working in schools aged 20–64 years  and amonAmong the older age group, there were much higher numbers of deaths among those working in schools compared with their 5-year average (74% more deaths among men and 37% among women). However, only 33% of excess deaths in men and 37% in women were thought to involve COVID-19. The group of all professionals and of all occupations had fewer excess deaths as a proportion of their 5-year average but a higher proportion of the deaths involved COVID-19 compared with those working in schools.males 0.59, 95% CI 0.46 to 0.75, RRfemales 0.58, 95% CI 0.46 to 0.73). There was also some evidence of a lower risk of death with COVID-19 among female primary and nursery educational professionals . For both male and female secondary school teachers, the RR estimate shows a 25% higher risk of death from COVID-19, although there was a great deal of uncertainty around these estimates . There was little evidence of a difference in risk between teaching assistants or lunchtime assistants compared with working-aged people. A fixed-effect meta-analysis across the four occupational groups working in schools in women showed that risk was similar to all working-aged people , with weak evidence of heterogeneity across the groups .We also calculated RRs for mortality with COVID-19 and all-cause mortality in educational professionals versus all professionals . We foun−16) deaths from COVID-19, with no meaningful difference between men  and women .We also plotted mortality with COVID-19 versus all-cause mortality on separate scatterplots  and founAmong men, the ratio of deaths with COVID-19 to other causes of death, ranged from 1:3 among male nurses and midwifes to 1:13 among male elementary construction occupations . The ratWe used routinely collected data on mortality for the ONS in the UK to study deaths with COVID-19 among teachers and other school staff, during the pandemic in 2020. We found that all educational professionals combined had fewer deaths per 100 000 occurring with COVID-19 compared with the general population. This is to be expected since the total working-aged population of England and Wales will include those who are unable to work due to ill health. The group ‘educational professionals’ includes those working in universities and school inspectors, many of whom have been working from home throughout the pandemic. We therefore investigated excess deaths specifically among school teachers and other staff working in schools and compared them with all those currently working and all professionals. There were fewer deaths than the 5-year average among female primary and secondary school teachers and the number of deaths among male teachers was similar to the 5-year average. There were more deaths among teaching assistants compared with the 5-year average, but only around half of the excess deaths were thought to involve COVID-19. For all women working in schools combined, there was a small increase in the number of deaths (5%) compared with the 5-year average, but the number of deaths occurring with COVID-19 was greater than the excess, suggesting that some of those dying with COVID-19 may have died over the course of a normal year from other causes. For those working in schools who were aged 65 years and over, there were large excesses in deaths compared with the average for the previous 5 years (74% for men and 37% for women), but only around one-third of the excess deaths were thought to involve COVID-19. It is possible that some excess deaths were due to undiagnosed COVID-19 among those who did not have classic symptoms,We found that secondary school teachers had around a 25% higher risk (in both men and women) of dying with COVID-19 compared with the general population, although the evidence was weak. There was stronger evidence of an increased risk to secondary school teachers compared with all professionals. However, all-cause mortality was also increased among secondary school teachers compared with all professionals.Proportionate mortality ratios  are routinely used in occupational epidemiology in situations where calculation and adequate standardisation of mortality rate ratios is not possible.The data we examined from ONS are consistent with the Public Health Scotland study18A study of data on teacher absence due to COVID-19 in England during the autumn term (September–December 2020) found that the proportion of teachers absent due to infection appeared to be similar in both primary and secondary schools.Very few studies have examined risk among teaching assistants and lunchtime assistants as separate groups; we found that despite more deaths occurring in this group in 2020 compared with the previous 5 years, they were not at increased risk of death due to COVID-19 or any cause compared with working-aged people. A study carried out in California used modelling to compare deaths from all causes during the pandemic and found an OR for teaching assistants of 1.28, but no CIs were provided for that study.21We used routinely collected data on mortality which include all deaths in England and Wales, and results are therefore unlikely to be due to ascertainment bias and will be representative of the working-aged population of these countries. However, we did not have access to individual-level mortality data so were not able to account for potential confounders such as comorbidities or household size. For our RR calculations, our comparison group also included school staff  because we did not have age-adjusted mortality rates excluding teachers. The age-adjusted mortality rates calculated by ONS were based on denominators taken from a survey conducted in 2019. If the school workforce has changed as a result of the pandemic, these rates will be inaccurate; if any changes in the school workforce are systematically different to other professions, the results will be biased. While the size of the school workforce in England remained fairly stable between 2015 and 2019, there was a marked decrease in the number of teachers retiring (from 17 853 teachers retiring in 2015 to 12 062 teachers in 2019),In the period covered by these data, many school staff worked remotely for large periods of time during 2020. A survey carried out by the global education company Tes found that just 22% of teachers were engaged in face-to-face teaching during the first lockdown.Teachers, teaching and lunchtime assistants aged 20–64 years were not at high risk of death relative to the working-aged population in England and Wales during the COVID-19 pandemic. For occupations working in schools, COVID-19 mortality was generally proportionate to all-cause mortality. Female primary school teachers and female secondary school teachers seemed to be outliers in opposite directions; however, evidence for heterogeneity in COVID-19 mortality risk among school workers was weak. For policymakers who are considering the impact of schools being open in future SARs-CoV2 pandemics, it will be important to note that staff were not at high risk of death compared with other occupations. Although we did not address these in this manuscript, other outcomes such as hospitalisation due to COVID-19 and long COVID-19 are also important considerations. In addition, for COVID-19, the emergence of more infectious strains since the data for this study were collected plus vaccination of school staff are likely to have changed the situation in schools.


Islet transplantation to the liver is a potential curative treatment for patients with type 1 diabetes. Muscle and the greater omentum are two alternative implantation sites, which can provide excellent engraftment and hold potential as future sites for stem-cell-derived beta-cell replacement. We evaluated the functional outcome after islet transplantation to muscle and omentum and found that alloxan-diabetic animals were cured with a low number of islets (200) at both sites. The cured animals had a normal area under the curve blood glucose response to intravenous glucose, albeit animals with intramuscular islet grafts had increased 120-min blood glucose levels. They also demonstrated an exaggerated counter regulatory response to hypoglycemia. The expression of genes important for beta-cell function was, at both implantation sites, comparable to that in native pancreatic islets. The gene expression of insulin (INS1 and INS2) and glucose transporter-2 was even increased, and the expression of lactate dehydrogenase decreased, at both sites when compared to native islets. We conclude that muscle and omentum provide excellent conditions for engraftment of transplanted islets. When compared to control, 200 islets implanted to the omentum displayed a restored glucose tolerance, whereas animals with intramuscular islet grafts of similar size displayed mild glucose intolerance. However, there is need for improvement since still only around 50% of the transplanted patients remain insulin-independent after 5 years despite substantial immunosuppressive treatment and in most cases repeated infusions of islets from multiple donors. Many of the challenges in islet transplantation are linked to the usage of the liver as implantation site. In the acute setting there is the instant blood mediated inflammatory response due to the direct contact between islets and blood. In fact, in a clinical case report with islets prelabeled with the positron emission tomography tracer  F-fluorodeoxyglucose, the islets could be detected within the liver but close to 50% of the signal was immediately lost indicating massive cell destruction in the acute phase. In experimental studies it has been shown that islets transplanted to the liver have a poor oxygen tension and insufficient revascularization. Furthermore, the islets may suffer toxic effects from the high concentrations of immunosuppressive drugs in the portal blood due to the high uptake and metabolism of drugs in the liver. It has also been shown that there is extensive formation of amyloid in islets transplanted intraportally into the liver. Based on the challenges in intraportal islet transplantation and the need of monitoring or even retrieval possibilities of future stem-cell-derived beta-cell replacement, there is an increasing interest in alternative islet implantation sites. Muscle and bone marrow have even been tested for implantation of islets in the clinical setting and there is currently an ongoing phase 1/2a clinical trial with allogeneic islet transplantation into the omentum . We have previously shown that the hypoxic phase in islets transplanted to muscle is rapidly reversed and that the islet revascularization is excellent. Also, in experimental studies the glucose control in diabetic mice receiving intramuscular islet transplants is superior to that of animals with intraportally transplanted islets. Islets transplanted to the omentum have previously been shown to engraft and cure animals in both small and large animal models, but there have also been reports on a delayed time to normoglycemia when compared to intraportal transplantation. Findings in our laboratory show that islets experimentally transplanted to the omentum have a similar time to normoglycemia as intraportally transplanted islets and a superior function for glucose control 1 month post-transplantation.Islet transplantation is a potential curative treatment for patients with type 1 diabetes and a life-saving treatment for those with severe hypoglycemic events. After the introduction of the Edmonton protocol with a steroid-free immunosuppressive regime and an increased number of transplanted islets, the outcome of islet transplantation dramatically improvedIn the current study we compared the function of islets transplanted to muscle and the greater omentum. We also investigated potential site-dependent changes in the gene expression of the islet grafts, which could be of detrimental importance for future trials using insulin-producing cells derived from stem cells.This study was approved by the animal ethical board of Uppsala County, and all experimental procedures were conducted in accordance with the Institutional Animal Care Guidelines. Adult male C57BL/6 mice weighing ∼25 g were purchased from Taconic . All animals were housed under standardized conditions with ad libitum access to food and water.. Islets were handpicked and cultured free-floating overnight in 5 ml RPMI 1640 medium  supplemented with l-glutamine , benzylpenicillin , and 10% (vol/vol) fetal bovine serum . On the day of transplantation, the islets were again handpicked and manually counted. Islets with central necrosis were discarded.Pancreatic islets from nondiabetic C57BL/6 mice were isolated using collagenase digestion and density gradient purification as previously described. In order to transplant islets to the greater omentum, a midline incision in the skin and abdominal muscle was made and the stomach was exposed. The greater omentum was localized and carefully extended by holding the omental fat and a ligature was placed, but not fixed, around the omentum. An opening between the sheets of the omentum was made with a cannula, a braking pipette filled with the islets was inserted, the islets were infused into the pouch, and the ligature was thereafter closed. The abdominal muscle was carefully sutured followed by suturing of the skin.Two hundred mouse islets were transplanted under aseptic conditions into the abdominal muscle or omentum of recipient nondiabetic or alloxan-diabetic C57BL/6 mice. The animals were anesthetized with an intraperitoneal injection of Avertin (0.02 ml/g), a 2.5% solution of 10 g 97% 2.2.2-tribromoethanol  in 10 ml 2-methyl-2-buthanol . Islets were transplanted to the abdominal muscle as previously describedDiabetes was induced in C57BL/6 mice by a single intravenous injection of alloxan  3–5 days prior to transplantation. Diabetes in mice was defined as repeated blood glucose concentrations exceeding 15 mmol/l, as measured by blood glucose reagent strips  on two consecutive days. Blood glucose concentrations were monitored daily for the first week post-transplantation and then every fifth day. Thirty days post-transplantation an intravenous glucose tolerance test  was performed in cured non-fasted animals (cured was defined as a non-fasting blood glucose concentration <12 mmol/l for two consecutive measurements on separate days). Blood glucose concentrations were monitored for 2 h following glucose injection. On the following day an intravenous insulin tolerance test  was performed during which blood glucose concentrations were monitored for 2 h. Nondiabetic C57BL/6 mice were used as controls for both the IVGTT and the ITT. When the animals thereafter were killed, the pancreas was surgically removed, placed in acid ethanol [0.18 mol/l HCl in 95% (vol/vol) ethanol], sonicated in order to disrupt the cells and thereafter frozen. The remaining insulin content in the pancreas was measured with an insulin enzyme-linked immunosorbent assay  in order to exclude endogenous beta-cell regeneration. An insulin content >10% of that of the pancreas of control nondiabetic animals was used as an exclusion criterion for the study.6 µm2 of islet tissue was collected for each sample.For the gene expression studies nondiabetic recipient animals were used. The animals were anesthetized with an intraperitoneal injection of pentobarbital  30 days post-transplantation, and the muscle or omentum containing transplanted islets as well as the pancreas were surgically excised under sterile conditions and quickly embedded in frozen section medium  and snap frozen in liquid nitrogen. Frame slides  were exposed to UV light overnight for cross-linking of the membrane in order to improve excision of islet tissue on the membrane. Sections were simultaneously stained with hematoxylin to confirm the presence of islets and when confirmed, then consecutive sections (10 µm) were mounted on frame slides, fixed in ice cold acetone for 2 min, and thereafter dried with cold air before storage in RNase free 50 ml tubes  in −80°C. The frame slide with sections was thawed for 30 s before hydration in nuclease free water . Staining was performed using RNase free hematoxylin . Laser microdissection of the islets and islet grafts was performed with a Leica LMD6000 microscope using the Leica Microdissection System software version 7.5.1.5250 . Approximately (1.5–3.0) × 10Total RNA was isolated from the laser microdissected samples according to the manufacturer’s instructions . This kit contains a gDNA Eliminator spin column, which will remove the genomic DNA. The amount and purity [optical density (OD) 260/280] of the total RNA was determined using a NanoDrop 2000C spectrophotometer . Yields of the LMD samples were in the range of 5–15 ng RNA. All RNA samples had OD 260/280 between 1.9 and 2.1, which is in the range for pure RNA. The extracted total RNA was dissolved in nuclease free water and stored at −80°C until cDNA synthesis. The RNA was transcribed to cDNA by Superscript First-Strand Synthesis Super Mix for quantitative-real time polymerase chain reaction  according to the manufacturer’s instructions. Two independent reverse transcriptase reactions were carried out for each RNA sample. The cDNA was amplified using RealTime ready cDNA Pre-Amp Master kit  for the genes of interest (Table 1).INS1, INS2, and HPRT, were purchased from Sigma-Aldrich . Primers for INS1, INS2, and HPRT were purchased from Tebu-Bio ; Catalog no. INS1; MQP027447, INS2; MQP027448, and HPRT; MQP030898.For primer sequences used in this study, see Supplemental Table 1. All primers, except for The qPCR assay was performed using a Light Cycler 480t  and Light Cycler FastStart DNA Master PLUS SYBR Green I kit  for detection. All qPCR samples were run in duplicates. Moreover, cDNA was prepared twice from each RNA sample and thereafter each cDNA was amplified twice with the primer pool to ensure inter-run specificity.(−1/slope). The expression stability of reference transcripts GAPDH, RPS7, and HPRT was evaluated using the Normfinder software. The analysis indicated that the best normalization was obtained by using the geometric mean of the expression of the reference genes GAPDH and HPRT. This normalization was therefore used to compare the islet-specific transcripts in all samples. The cycle threshold (Ct) values were used to calculate the amount of PCR product compared to reference genes by subtracting the Ct value for reference genes from the Ct value for the gene studied (ΔCt). Relative mRNA expression was calculated as 2−ΔCt and data are presented as relative gene expression. For each transplantation site, that is, muscle and omentum, five animals were used and the LMD islet graft material was pooled for the respective site in order to get enough material for three LMD samples. Control samples derived from native pancreatic islets were dissected from two separate animals.To determine the PCR efficiency, the primer pairs were analyzed using a dilution curve with 10-fold cDNA template dilutions between 5 and 0.05 ng/µl. The efficiency was calculated using the formula: Efficiency = −1 + 10To confirm amplicon size, qPCR products were analyzed by electrophoresis using a 3% agarose gel . The PCR products were mixed with five times loading buffer  before loading. A 50 bp ladder  was used to determine the size of the PCR products. Electrophoresis was conducted using an electrical field of 5 V/cm for 80 min and the bands were visualized using GelRed  and detected using the Chemi Doc MP Imaging System .Insulin was stained on the cryosections using an anti-insulin primary antibody , which was detected by Alexa Fluor 488  secondary antibody. ProLong Gold Antifade reagent with 4′,6-diamidino-2-phenylindole  was used for mounting and nuclei staining. From separate animals, islet grafts from muscle and omentum and pancreas were fixed in 4% (vol/vol) paraformaldehyde and embedded in paraffin and sectioned at a thickness of 4 µm. The islet architecture was characterized on paraffin sections, which were double stained for insulin  and glucagon . Secondary antibodies used were Alexa Fluor 488 (goat anti-guinea pig) and Alexa Fluor 594 . Nuclei were stained with Hoechst.Light microscopy images were acquired with a Leica LMD6000 laser microdissection microscope . Fluorescent immunohistochemistry images were acquired with Zeiss LSM780  confocal. The percentage of alpha- and beta-cell area was calculated as the percentage of the respective glucagon and insulin positive area divided by the combined area of insulin and glucagon for that islet/islet graft. The respective areas of insulin and glucagon were calculated by using the image software Imaris  with a fixed intensity cut-off value for each staining. The percentage of each image was calculated as an average for each animal and considered as one experiment.n = 3 for each site) and control animals (n = 4). Glycogen Assay Kit II  was used according to the manufacturer’s instructions. Reading of the plate was performed using Spark Microplate Reader .Glycogen concentrations were measured in 10 mg homogenized muscle and liver tissue from cured transplant recipients  was used for statistical analysis. For comparison of relative gene expression data between the three groups, a nonparametric one-way analysis of variance (ANOVA) was applied with Dunn’s post hoc test using native pancreatic islets as control. For comparison of blood glucose measurements between the three groups, a one-way ANOVA with Tukey’s post hoc test was applied. Comparison of time to normoglycemia was performed as a survival curve using Log-rank test (Mantel-Cox). For all statistical analysis, Syngeneic transplantation of 200 islets reversed diabetes in 9 out of 11 (82%) animals after intramuscular implantation and in 9 out of 10 (90%) after implantation into the omentum. The time to normoglycemia did not differ between the implantation sites; median time to reversal of diabetes (<12 mmol/l) was 10 days for both groups . The feen = 5 for each implantation site) an ITT was performed, which showed similar nadir values of blood glucose in both groups of transplanted animals and in control animals  and liver to audit if there were any differences in glycogen synthesis, depending on portal or systemic insulin release from islet graft transplanted to either omentum or muscle, of the animals. There was no difference in glycogen content in either liver or striated muscle between animals transplanted with islets to the omentum or muscle . SimilarThe present study shows that insulin-deficient diabetes in mice can be efficiently reversed by transplantation of only 200 islets to striated muscle or the greater omentum. For both implantation sites at least 80% of the animals were cured after receiving 200 islets. However, animals with intramuscular islet grafts displayed elevated blood glucose levels 120 min after a glucose challenge, which could reflect either a marginal surviving islet mass, dysfunctional islets, or the systemic instead of portal drainage of these islets., we and others have previously reported of substantial fibrosis formation in intramuscular islet grafts indicative of early cell death. Indeed, our recent studies also show that intramuscularly transplanted islets suffer from prevailing hypoxia in the first week after transplantation with concomitantly increased cellular death. However, site-dependent gene expression changes causing islet graft dysfunction are also known to occur in the liver , the pancreas, and beneath the renal capsule, although this has not been characterized for the presently used implantation sited. The expression of several genes important for maintaining the beta-cell phenotype, such as PDX1, and for function, such as GCK, was decreased in both intraportally and intrapancreatically transplanted islets, and INS1 as well as INS2 was decreased in renal subcapsular grafts. Interestingly, no such detrimental changes were observed for either islets in the muscle or greater omentum in the present study. This could potentially be a very important feature in favor of these two implantation sites when moving forward with clinical trials using stem-cell-derived insulin-producing cells. For both implantation sites there was an upregulated expression of GLUT2, PCX, PDX1, INS1 as well as INS2 when compared to native islets. This could reflect an upregulation due to the increased demands of a marginal islet mass. However, we cannot rule out that the samples obtained from endogenous pancreas could contain small amounts of contaminating exocrine tissue. Moreover, in contrast to previous reports of islets grafted to the pancreas or beneath the renal capsule, the gene expression of LDHA was even decreased at both the intramuscular and omental sites when compared to native islets. Particularly for intramuscular islet grafts this latter findings may be important, since an increased expression of LDHA would likely have cause the transplanted beta cells to secrete insulin not only in response to ambient glucose levels but to generated lactate. Indeed, exercise have previously been shown to induce hypoglycemia in animals with islets transplanted beneath the renal capsule. All in all, there were no signs of beta-cell dysfunction in the grafts and it seems most likely that the glucose intolerance reflects a marginal islet mass at the intramuscular site.Albeit islets implanted to the intramuscular site as into the omentum are rapidly revascularized with a restoration of the vascular network. The C-peptide levels were, however, not increased in the animals with islet grafts with systemic drainage, which indicates that the secretion of insulin was not altered but rather the clearance of insulin. Since insulin or C-peptide levels were not measured in the current study, it cannot be excluded that the reduced clearance of glucose during the IVGTT in the animals with intramuscular islet grafts in part reflected an insulin-resistant state. However, during the ITT the initial glucose lowering response was identical to control in both groups of transplanted animals, which at least is suggestive of a normal response to insulin in peripheral tissue. This is also supported by the finding of similar glycogen levels in liver and muscle irrespective of transplant site.Although both muscle and the greater omentum provide excellent conditions for revascularization, their venous drainage is different. Muscle has a systemic drainage, whereas the omentum as native islets has a portal drainage. Since the hepatic cells are a major target for insulin, the importance of portal drainage has been one of the main arguments for using the liver as an islet implantation site. It has also been shown that a systemic venous drainage of islet grafts increases the systemic levels of circulating insulin despite similar glucose levels, which is suggestive of an insulin-resistant state. However, previous studies indicate that the glucagon response to hypoglycemia may be site dependent. Animals with intraportal islet transplants subjected to hypoglycemia had a blunted glucagon response, whereas animals with islets transplanted beneath the kidney capsule or to the intraperitoneal cavity had a normal response. Clinically, the finding at the intraportal site has since then been debated, since early studies showed that the glucagon response to insulin-induced hypoglycemia is blunted and that a rise in glucagon secretion only occurs after arginine stimulation, whereas a more recent study reported that the glucagon secretion does increase in response to insulin-induced hypoglycemia, although not to the same extent as in healthy controls. Nevertheless, in contrast to our previous observations of decreased percentages of glucagon-positive cells for islets experimentally transplanted to the liver, we found that the islet composition is unaltered in both islets transplanted to muscle and to the omentum.Noteworthy, the blood glucose levels 2 h after the insulin injection was increased in the animals with intramuscular islet grafts when compared to control, suggestive of an exaggerated counter regulatory response to hypoglycemia. The reason for this is presently obscure, since, for example, no glucagon concentrations were measured. However, these two sites have never been compared head-to-head regarding islet graft function. In the current study we report that alloxan-diabetic mice can be cured with a low number of islets (200) by transplantation to either muscle or the greater omentum. The restoration of normoglycemia is rapid and the animals respond well to an IVGTT and can effectively counteract insulin-induced hypoglycemia regardless of implantation site. Moreover, in contrast to previous experimental studies of islets grafted to the liver, beneath the renal capsule or into the pancreas, we observe no detrimental expression changes in genes vital for beta-cell function. These findings support the use of both muscle and the omentum as implantation site for islet transplantation, although differences in the human omentum composition and the need of a much larger islet mass may impact results in the clinical setting. The fact that the expression of genes important for maintaining a beta-cell phenotype is sustained or even augmented at both sites could be of great importance when considering implantation sites for future clinical transplantation of stem-cell-derived insulin-producing cells.Both muscle and the greater omentum has in experimental studies from our group been shown to be superior to the liver as islet implantation sitesClick here for additional data file.Supplemental Material, sj-pdf-1-cll-10.1177_0963689720960184 for Function and Gene Expression of Islets Experimentally Transplanted to Muscle and Omentum by Daniel Espes, Hanna Liljebäck, Petra Franzén, My Quach, Joey Lau and Per-Ola Carlsson in Cell Transplantation


Recent avian influenza infection outbreaks have resulted in global biosecurity and economic concerns. Mallards are asymptomatic for the disease and can potentially spread AI along migratory bird flyways. In a previous study, trained mice correctly discriminated the health status of individual ducks on the basis of fecal odors when feces from post-infection periods were paired with feces from pre-infection periods. Chemical analyses indicated that avian influenza infection was associated with a marked increase of acetoin (3-hydroxy-2-butanone) in feces. In the current study, domesticated male ferrets (Mustela putorius furo) were trained to display a specific conditioned response  in response to a marked increase of acetoin in a presentation of an acetoin:1-octen-3-ol solution. Ferrets rapidly generalized this learned response to the odor of irradiated feces from avian influenza infected mallards. These results suggest that a trained mammalian biosensor could be employed in an avian influenza surveillance program. Avian influenza virus (AIV) has been identified for its potential to disrupt the economy of the poultry industry through devastating losses of farmed fowl ,2. WaterThere is now growing and extensive evidence that certain diseases can alter human and animal bodily odors. In recent studies, it was shown that bodily secretions, such as urine, contain volatile odorants that undergo quality or intensity changes that are detectable by trained mice following events such as immunization, inflammation, or brain trauma ,8. FurthMycobacterium tuberculosis [Biosensors have been used in studies to discriminate various tissues manifesting cancer  from hearculosis  which carculosis , carcassrculosis , and pesrculosis . HoweverMustela putorius furo) were trained to display a specific conditioned response  in response to a marked increase of acetoin in a presentation of a acetoin:1-octen-3-ol solution. It may not be surprising; these same trained ferrets were able to discriminate infected from non-infected samples when confronted with irradiated feces from mallards experimentally infected with a low pathogenic avian influenza (LPAI) on the first attempt.We hypothesized that the success of the mice in detecting AIV in mallard fecal samples could be repeated in a species that had a more malleable behavioral repertoire . Although a study examining the aptitude of dogs in disease detection was desired, the cost and logistical factors proved to be prohibitive without the support of further evidence being provided. Ferrets were chosen as a “bridge” species between the mouse study and a potential canine AI biosensor program to further demonstrate detection of AIV-infected individuals. While ferrets were expected to work well in odor discrimination and detection tasks in the laboratory, it was anticipated that a natural setting would prove too great a challenge to a ferret’s ability to focus. Domestic ferrets were chosen for collecting further evidence in the laboratory because of their learning discrimination capabilities and dog-like social-cognitive skills in human interactions ,16. A prAll biosafety precautions and experimental protocols were approved by the Monell Chemical Senses Center Institutional Animal Care and Use Committee (protocol number 1161).ad libitum access to tap water. Totally Ferret Complete diet  was available ad libitum with the exception of food restriction periods during training and testing.Eight, male, castrated ferrets  born 8/19-21/2013 arrived at Monell as juveniles at 15 weeks of age. Ferrets were housed in pairs in two level, 2.5 cm spaced 12 g wire cages  and maintained at 23°C on a 12-h light (12-h dark cycle). During food restriction feeding periods or in case of illness, the ramp that connected the upper and lower levels of the cage could be locked in a raised and closed position, allowing for each of the ferrets to be isolated. Environmental enrichment was provided both in the cages  and during 60-minute free exercise periods daily on weekdays. Ferrets were given ad libitum access to tap water during training or testing sessions. On days consisting of training or testing, food was provided after the session for 1 h while the ferrets were separated on different levels of the cage. Food bowls were weighed before and after the feeding session and the difference  was recorded.During food restriction periods, ferret weights were recorded every weekday prior to training, and their health was assessed every weekday  before, during, and after training. Training or testing sessions lasted approximately 3.5 hours. Ferrets were also given trans-cinnamic aldehyde , vanillin (CAS: 121-33-5), acetoin (CAS: 513-86-0) and 1-octen-3-ol,  were technical grade . Odorant concentrations were diluted from stock with propylene glycol  and prepared weekly. Ratios of acetoin:1-octen-3-ol were initially chosen based on a previous study of fecal odorants [2O in the glass training vial and the rehydrated fecal samples were stored a 4˚ C.Odorant compounds: Feces collected from eight farm-raised mallards of mixed gender were used from a prior study . BrieflyFor an overall view of ferret training, please see An aluminum “scratch” box  was used to shape the odor alert response and for odor discrimination training and testing. The scratch box (14.6 cm x 5.7 cm x 3.8 cm) was equipped with a sliding, self-locking cover and over 100 drilled holes (2-mm) in the cover and ends of the base compartment to allow for the escape of volatile odors. Initially, tape was used to cover the drilled holes in order to prevent the small nails of the then still young ferrets from being caught and held in the hole. The tape was then punctured with an 18 G needle to conform to the drilled holes. This tape was removed and replaced during cleaning of the boxes. Use of the tape was discontinued following acetoin:octenol ratio training and testing as the nails of the ferrets had grown large enough that they could not catch in the drilled holes. A magnet was attached to the bottom of the base compartment allowing for attachment to a metal surface . Both the cover and base compartment were individually numbered with a permanent marker. The stimulus type or sample number was also written with a permanent marker on one end of the scratch box. In blind trials, this end was pointed away from the ferret handler so they were only visible to the box handler. The base compartment was fitted with a piece of egg crate lighting panel (cut to fit tightly) and customized to allow for the retention of a small 1 ml glass vial . Vial caps (plastic septum-type screw caps with a 9 mm diameter opening) were fitted with 10 mm, Whatman qualitative filter paper, grade 1  that allowed for the escape of volatiles, but prevented escape of samples placed in the vial. The scratch box was placed on the testing room floor during exercise and the ferrets were encouraged to investigate it thoroughly .The odor alert response was defined as the ferret scratching with both fore paws at the box. This response was shaped in steps. The first step  was shaped by responding with a click and a reward  each time they approached the box. The box was allowed to stand freely on the linoleum floor so the ferrets would not be frightened by the sound of metal and the movement of the box in case these events were to occur once the boxes were fixed to a metal panel. Once each ferret behaved consistently (~100%) when the box was present, the reward sequence was withheld until the approaching ferret placed a forepaw on the box. Once the ferrets were each performing this behavior consistently (~100%), the reward was once again withheld until each ferret would scratch with both fore paws at the box. When a consistent response (~100%) to the presence of the box was achieved, a single scratch box was attached by its magnet to a recycled, metal panel (94.6 cm L x 27.3 cm W with a 1.3 cm 45° lip bend on one of the long sides).Five scratch boxes were attached to the metal panel approximately 15.9 cm apart and in the center of the board . One ranNext, ferrets were asked to alert to the scratch box holding 1M cinnamaldehyde (CS+) alone while the remaining 4 boxes remained empty. Upon making a correct choice the ferrets received a reward delivered by the handler using a syringe. Again, daily sessions consisted of 5 trials for each of the eight ferrets with the position of each box being pseudo-randomized for each trial. Six individual ferrets were able to perform this task consistently at 80% or better after four sessions  while two continued to struggle with the task as evidenced by their inability to choose the correct box. To see if contrast was needed for all ferrets to perform consistently, the remaining four boxes held 1 M vanillin for the next 5 sessions. By the third session, all ferrets were performing at accuracy rates of 80–100%. Finally, ferrets were asked to discriminate the box holding a lower concentration of cinnamaldehyde (0.1 M) from boxes holding 1 M vanillin over three sessions to determine if the ferrets would generalize their alert to a different concentration of the same learned odorant. Ferrets as a group performed at 90% or better from the first session for all three sessions.In a previous study from our laboratory, chemical analyses demonstrated that LPAI infection was associated with a marked increase of 3-hydroxy-2-butanone (acetoin) in relation to 1-octen-3-ol (octenol) in feces. The ratio of these compounds was significantly greater in feces of infected ducks (10–50:1) in comparison to ducks pre-infection (1–5:1). Having replicated that ferrets could perform single compound detection , the abiNext, the ferrets were asked to alert to a single, high acetoin:octenol ratio  reflective of feces from LPAI infected ducks (CS+) in the presence of low acetoin:octenol ratios (1:1 or 2:1) reflective of feces from non-infected ducks (CS-). Each panel presented to the ferrets consisted of one scratch box with a randomly selected high acetoin:octenol ratio solution vial (CS+), two 1:1 acetoin:octenol ratio solution vials, and two 2:1 acetoin:octenol ratio solution vials . The position of each box was pseudo-randomized for each trial. After 5 sessions , each ferret had performed at 60% or better (as high as 90%) accuracy and were consistently performing at 60% or better as a group. Again, it is important to note that 20% was the level chance as only 1 of 5 boxes contained the CS+ ratio.To avoid the possibility of the handler inadvertently communicating the position of the CS+ to the ferret, a double-blind procedure was incorporated into the training regimen employing a separate “box handler” and “ferret handler”. The box handler designed the daily schedule, consisting of the position of the CS+ scratch boxes, the order  of CS+ ratio presentation, and the order the ferrets were to perform their individual sessions. The box handler positioned the CS+ and CS- scratch boxes on the board, placed the board on the ground to signal the start of a trial, confirmed or rejected the ferret handler’s call (described in the next sentence) and positioned the boxes on the board for the next trial. The ferret handler controlled when the ferrets were to start a trial and called out “Hit” when the ferret made a decision and alerted to one of the boxes. The box handler responded “correct” or “incorrect” (based on the box handler’s knowledge of CS+ location) so that the ferret handler could rapidly react appropriately to the ferret’s decision. For a correct response, the ferret handler clicked the clicker and provided a small amount of FerretVite with a modified syringe. When the box handler rejected the ferret handler’s call of the alert, the ferret handler picked up the ferret and faced away from the box handler as the box handler picked up the board and prepared it for the next trial. This method was used for all remaining sessions, including additional shaping of behavior, training, and experimental testing.Blind discrimination was performed as described above. After 14 sessions , each ferret had performed at 80% or better in at least one session (as high as 100%) accuracy and were consistently performing at 60% or better (as high as 73%) as a group, but this seemed to be the maximum accuracy they would attain. To improve individual accuracy, extinction trials were introduced into each session. We define an extinction trial as a trial consisting of CS+ and CS- samples that are familiar to the ferret  but is not rewarded or acknowledged in any way. The board was simply picked up immediately after a box selection, regardless if it was a correct or incorrect choice. We incorporated 3 random extinction trials during each session with the following conditions: extinction trials could not occur during the first or last trials and could not occur in two consecutive trials. Each ferret performed at 80% or better (as high as 100%) accuracy and as a group were consistently performing at 80% by the fourth session and 87% by the fifth session. We then added an additional extinction trial for a total of four per session out of 10 trials for three sessions with similar accuracy results.Up to this point, we employed one set of CS- scratch boxes and a different scratch box for each of the CS+ stimuli. To control for the possibility that the ferrets were alerting to the box itself, we began to use three sets of scratch boxes for the CS- stimuli and continued using different scratch boxes for each CS+ stimulus. At this point we also increased the number of trials from 10 per session to 12 per session in order to better accommodate the four extinction trials in each session. There was an immediate drop in accuracy to 53% as a group. While this was still above chance, it did suggest that the identity of the box or the increased number of trials was, in some way, contributing to the choice being made by the ferrets. Scratch boxes were not dedicated to a particular valence and the valence of a particular box was changed frequently . Thus, it was unlikely for CS+ boxes to “acquire a definitive signal” over time because these same boxes had the same chance of being a CS- over the sessions. By the 9th session, the ferrets were performing at 81% accuracy as a group and between 58–92% accuracy as individuals. To ensure that scratch box identity was no longer being used  as a component of the odor discrimination, we began using a new set of boxes for every trial in a session. The results are shown in To determine if the ferrets would be able to generalize to ratios they had not experienced previously, we introduced non-rewarded generalization trials. We define non-rewarded generalization trials here as trials conducted with CS+ stimuli unique in quality, intensity, or donor identity but having the same consequences as an extinction trial . Because the ferrets experienced a neutral response from the handler immediately following a generalization trial, it was assumed that little or no learning occurred during a non-rewarded generalization trial. Two novel ratios (25:1 and 35:1 acetoin:octenol) were randomly presented during the four non-rewarded generalization trials. Results are shown in Next, the ferrets’ responses to 3:1 and 5:1 acetoin:octenol ratio solutions (twice each) were randomly presented in four non-rewarded generalization trials. These acetoin concentrations represent borderline ratios very near the critical ratio delineating LPAI infected and non-infected conditions . Results are shown in To determine if trained ferrets (n = 6) would generalize the training they received with acetoin:octenol ratio solutions to actual fecal samples collected from LPAI infected and non-infected mallards, we presented the ferrets with irradiated duck fecal samples from a previous study. The CS- samples consisted of randomly chosen fecal samples collected pre-infection from all eight mallard donors for the Kimball et al., study (2013). Because of the low number of available samples, post-infection feces had to be presented in both rewarded and unrewarded  trials. The first two days of sessions included only rewarded trials followed by three days of double blind test sessions with unrewarded generalization trials. Daily testing session included four generalization trials randomly presented among twelve trials per session resulting in 72 overall unrewarded generalization trials.Training session results (correct or incorrect selection of the CS+ sample) were first subjected to Cochran-Mantel-Haenszel (CHM) test of general association to determine if correct rates of CS+ identification differed between sessions or among ferrets. Similarly, results of the rewarded trials during the three testing sessions were subject to CHM test. Cumulative responses of all ferrets across all appropriate sessions were calculated for: all training trials, rewarded trials during testing sessions, and generalization trials. Success rates  were subjected to statistical tests of binomial proportion and Wilson confidence intervals were determined (proc FREQ in SAS). Null hypotheses differed for each cumulative score. For training trials, the hypothesis that cumulative score was greater than 80% was tested. For testing trials, the hypotheses that cumulative scores were greater than 75% for rewarded trials and 20% for generalization trials were tested. Cumulative scores for testing were anticipated to show a slight reduction in comparison to training scores as the stimulus being tested was novel.Incorrect selections were further examined to determine if design components influenced box selection. Chi-square tests of association were conducted to determine if any particular CS+ donor , CS+ box position (1 thru 5 from left to right in the box array), or CS- donor  may have resulted in an incorrect selection.In two days of rewarded training trials, identification of the CS+ sample did not differ between sessions or among the six ferrets (p = 0.993). Collectively, ferrets correctly identified the location of the single fecal sample derived from an LPAI infected donor with 86% accuracy, which was greater than 80%  and the ferrets collectively identified the location of the CS+ samples with 81% accuracy, which was statistically similar to 75% .Fecal matter from waterfowl contain viable avian influenza virus. Thus, waterfowl and waterfowl habitats are the central source of avian influenza virus in all other species, including farm raised fowl ,28. CurrThe convenience of training ferrets with acetoin:octenol solutions made conversion to training with duck feces very easy and suggests that a surveillance program consisting of trained biosensors can be developed without need for training with hazardous biological samples. However, in forcing the ferrets to focus on changes in acetoin intensity, did the training and experimental design force them to focus on the acetoin and ignore a potentially superior cue that was not revealed by chemical analysis? This study clearly demonstrates the feasibility of deploying trained biosensors for avian influenza virus surveillance in waterfowl.This experiment provides further evidence that a signature odor results from LPAI. However, there are still important questions to be answered. For example, was fecal irradiation necessary to produce the signature odor of AI infection? Since ferrets are susceptible to influenza A virus infections, what is the likelihood that a ferret become infected with influenza A virus after being used as biosensors to detect acetoin and exposed to IAV in feces when sniffing/inhaling the fecal odors? Can a biosensor differentiate between samples that result from infection with different types of respiratory viruses producing gastrointestinal effects? Most importantly, can a biosensor trained to identify LPAI in mallard fecal samples identify the odor identity of avian influenza infection in another species?The likelihood remains very high that detection and affirmation of avian influenza infection will be as successful in canine biosensors. Domestic ferrets show skill in learning discrimination tasks and dog-like social-cognitive skills in interacting with humans ,16 and t


During the COVID-19 pandemic, journalists reporting on the crisis in the UK were classed as keyworkers. Their role was integral to keeping the public informed of adverse and often fatal consequences related to the spread of COVID-19 as well as government-mandated regulations. Tyson and Wild  raised cWe first wish to thank Van Overmeire for his provocative comment. We welcome the opportunity for scientific debate and agree that clinical researchers should assess for criterion A trauma when investigating PTSD. Assessment of criterion A trauma ensures validity and leads to more accurately reported rates of the disorder. For example, we found in our recent study of healthcare workers that PTSD was most frequently associated with trauma that predated the current pandemic . This isWith reference to Tyson and Wild  and the Contrary to Van Overmeire’s assertion that the journalists in our study failed to experience criterion A trauma, we confirm that all participants had experienced a criterion A trauma as identified by the Life Events Checklist (LEC) . JournalParticipants’ index events were as follows: sudden or unexcepted death of someone close (19%), fire or explosion (14.3%), sudden or violent death (13.1%), severe human suffering (11.9%), combat or exposure to a warzone (10.7%), sexual assault (8.3%), life-threatening illness or injury (7.1%), physical assault (4.8%), transportation accident (3.6%), unwanted sexual experience (3.6%), assault with a weapon (2.4%), or childhood neglect (1.2%). Every journalist had experienced a criterion A event as defined by the DSM-5 and completed the PCL-5 in relation to this event. In our sample, the majority (62%) of journalists experienced occupational trauma through work on the scene (47.6%) or repeated exposure from the office (14.3%). Personal trauma was reported by 38% of journalists. There was no significant difference between the two groups on how the trauma was experienced.Our study advances the work of Osmann et al.  by ensurn = 25) identified occupational trauma as their index trauma with a minority (n = 6) having reported trauma experienced in their personal lives. Van Overmeire expressed concerns over how journalists in different roles (such as editors versus reporters) may have experienced traumatic events. The DSM-5 includes repeated exposure to occupational trauma as a criterion A event. As such, repeatedly reporting on or editing news related to human suffering associated with COVID-19 or the perceived personal threat of catching the virus associated with keywork would fit the definition of a criterion A event in the DSM-5. The majority of editors in our sample  values to examine multicollinearity between variables in the regression analysis. There were no VIF values above 1.8, which is well below the value of 10 . All tolVan Overmeire queried the administration of the COVID-19 impact questionnaire, which assessed repeated reporting of COVID-19 and the effect the pandemic may have had on journalists . This questionnaire was used to identify which category journalists fell into: repeatedly covering COVID-19 news or covering news unrelated to COVID-19. Therefore, those journalists in the ‘not repeatedly covering COVID-19’ group may have reported on COVID-19 once, and subsequently interviewed an individual with COVID-19 once. This accounts for the eight participants in the ‘not covering COVID-19 repeatedly’ group who indicated they interviewed someone with COVID-19. We refute the claim that this questionnaire of COVID-19 impact was biased towards one group. The questions were designed to apply to all journalists working during the pandemic, regardless of whether or not they repeatedly covered COVID-19 stories.Our study adopted a cross-sectional design and as such no causal statements can be or were made. The regression analysis was conducted to investigate the relationship of common strategies for dealing with unwanted memories, a normal feature in the aftermath of trauma, to PTSD symptom severity. We do not see rumination as a symptom of PTSD. It is a thinking style often triggered by an unwanted memory and has previously been shown to predict PTSD in high-risk occupational groups . We would like to thank Van Overmeire for bringing to our attention the typo in the manuscript, which makes the GHQ scores seem very high. The items were scored 1–4, instead of 0–3. The range of scores with the 0–3 scoring is 12–48, and as such, the mean scores are in the mid-range. The 1–4 scoring did not affect the subsequent analysis. The 0–3 scoring was published with the original manuscript as a Correction. “It's almost like you're sort of being attacked by the news because you know, it's our job to be across everything… so I find myself being really on edge, quite a lot. When you finish, go home for the day, you're still kind of on a state of high alert. The offshoot of that is that it's quite hard to switch off, the end of the day. It can affect your sleep, your mood, your mental health.”In efforts to conduct the most rigorous analysis of published research, there is risk that one loses sight of shared intentions: to advance science in a field capable of reducing human suffering. Nothing underscores the value of this work more clearly than the voices of journalists who worked tirelessly to report on COVID-19 at the peak of the pandemic when there was little understanding and immense fear. A BBC journalist tells us: Our research reports rates of PTSD symptoms among journalists working during the pandemic that are consistent with published studies and which advance prior research: we assess for exposure to DSM-5-defined criterion A events and subsequent PTSD symptom severity. Our data demonstrate that exposure to trauma among journalists is high with PTSD symptoms being highest among journalists repeatedly covering COVID-19. We suggest the high rates of PTSD symptoms may relate to exposure to COVID-19 trauma, may be related to prior trauma, which could increase vulnerability to PTSD symptoms during pandemic working, and reflect the persistence of PTSD symptoms associated with prior trauma or the cumulative effect of trauma exposure. Drawing on our data, we offer evidence-based recommendations for future interventions.


Ki-67 is highly expressed in proliferating cells, a characteristic that made the protein a very important proliferation marker widely used in the clinic. However, the molecular functions and properties of Ki-67 remained quite obscure for a long time. Only recently important discoveries have shed some light on its function and shown that Ki-67 has a major role in the formation of mitotic chromosome periphery compartment, it is associated with protein phosphatase one (PP1) and regulates chromatin function in interphase and mitosis. In this review, we discuss the role of Ki-67 during cell division. Specifically, we focus on the importance of Ki-67 in chromosome individualisation at mitotic entry (prometaphase) and its contribution to chromosome clustering and nuclear remodelling during mitotic exit. Multicellular organisms are constituted by cells with different functions resulting from the specific gene expression programs characteristic for each cell type that need to be maintained from one generation to the next. A challenge to the faithful transmission of this information is represented by mitosis. In mitosis, the genome of proliferating cells undergoes remarkable series of structural changes at mitotic entry that lead to chromosome condensation and the formation of mitotic chromosomes ,2. MitosBeing so critical for cell survival, this process is highly controlled not only biochemically but also topologically. The position of chromosomes, spindle and cleavage furrow are important cues for an error-free mitosis. Microscopy analysis of chromosome behaviour during cell division has fascinated many scholars starting from Boveri . As micrWhile chromosomes condense in mitosis, major changes in their mobility occur; these are driven by the very dynamic microtubules and spindle forces. The chromosomes will need to form a tight metaphase plate whilst maintaining their individuality, thus allowing for a swift separation of sister chromatids at the onset of anaphase without causing entanglements. When the migration of sister chromatids is completed, the chromosomes will reach their maximum compaction ,11 and mThe molecular effectors that are in place to maintain the chromosomes separated in the first part of mitosis (a) and to bring them together at the end (b) are starting to emerge. Surprisingly (or not), recent evidence seems to suggest that the same molecule is involved in both aspects: Ki-67.In this review, we are going to provide the reader with the key aspects of this process, the major findings and we will highlight the main open questions.To understand the molecular mechanisms that allow chromosomes to maintain their individuality (inhibit clustering) in early mitosis but then to be able to coalesce (favour clustering) at the end, we need to understand the outermost part of the mitotic chromosomes: the perichromosomal layer. The mitotic chromosome is composed of a chromosome scaffold fraction, the centromere/kinetochore, telomeres and a sheath of proteins surrounding each chromosome defined as the chromosome periphery or perichromosomal layer . During Ki-67 for many years has been used as a prognostic marker for multiple types of cancer , as it iThe mitotic chromosome periphery consists of numerous proteins with diverse functions during the cell cycle. The number of components is still growing as more proteins associated with the chromosome periphery are being identified by proteomic screens of isolated chromosomes ,28. It iAmong the components of the chromosome periphery there are several ribonucleoproteins and RNAs ,13,28,292+ [The perichromosomal layer appears in prophase and it is dismantled at the end of cell division when the nuclear envelope is fully reformed. For identifying the possible functions of Ki-67 through the cells cycle, a human Ki-67-mAID-mClover cell line (HCT116) that allows endogenous Ki-67 to be both visualised (via mClover) and targeted for rapid proteasomal degradation (via the mAID degron) was generated . The rap2+ , no clea2+ .Nevertheless, all the studies are in agreement with the fact that mitotic chromosomes in cells lacking Ki-67 are abnormally clumped together ,18,38, aKi-67 depleted chromosomes are more prone to stick to each other and behave like a single contiguous and relatively immobile mass , thus reThese results raised the hypothesis that Ki-67 acts as a steric and electrostatic coating on chromosomes by forming a sort of biological surfactant important to maintain the individuality of each mitotic chromosome . Indeed,As mentioned before, the maximum chromosome compaction is achieved at the end of mitosis when the chromosomes are tightly clustered before they will start decondensing for re-entering a new cell cycle. A recent study has also implicated Ki-67 at this stage with an apparently opposite effect to the one exerted in early mitosis . At mitot1/2 recovery time of 11.82 ± 3.85 s in metaphase) while the recovery rate for GFP-Ki-67 was considerably decreased after the onset of anaphase (with a t1/2 recovery time were 45.30 ± 14.55 s in anaphase and 48.79 ± 10.97 s in telophase) [The central region of Ki-67 consists of 16 tandem repeats that are encoded by a single large exon 6845 bp)  A 42,43,4342,43.lophase) . At anaplophase) ,24,44 anlophase) . Interes bp  A 42Recently, published data have shed some light on the importance of chromosome clustering at the end of mitosis. In fact, cells lacking Ki-67 displayed nuclei contaminated with cytoplasmic components, including mature ribosomes , thus deThis also opens another interesting possibility regarding the function of the perichromosomal layer. In fact, this compartment could represent a vehicle to mediate a quick start re-assembly of some nuclear sub-compartments. This idea is also supported by the observation that B23 and nucleolin (both nucleolar proteins in interphase and associated with the chromosome periphery in mitosis) were unevenly distributed in Ki-67-depleted daughter cells at cytokinesis . Moreovein vitro  or −194.978 (for the phosphorylation of the repeats only) . This man vitro ( and our n vitro (,49 but in vitro (; indicatWe still do not know exactly what the biological function is of maintaining chromosomes separated from each other in early mitosis. A possible hypothesis could be that if chromosomes cluster prematurely, the spindle could not easily capture the kinetochores thus leading to spindle structural defects or mitotic delay as some studies seem to suggest ,38,52,53The evidence that cells and organisms can survive without a protein that seems to have such important functions in mitosis it is quite interesting and puzzling at the same time but clearly suggests that future work is needed to clarify if adaptation mechanisms are in place to bypass Ki-67 absence or if safe-valve mechanisms exist that take over when one is failing.Highlight the importance of the field. Faithful chromosome segregation requires precise and coordinated chromosome movements during the different stages of mitosis; chromosomes must congress to the metaphase plate but still maintain separation in early mitosis while their clustering at the later stages of mitosis allows for a correct reformation of the nuclear envelope and the establishment of a nuclear/cytoplasmic compartmentalisation.A summary of the current thinking. Recent studies have highlighted the important role of the chromosome periphery in this process and, in particular the pivotal function of Ki-67, a well-known proliferation marker.A comment on future directions. The identification of the molecular mechanisms for the perichromosomal layer structure and function and how Ki-67 can direct almost opposite roles at different stages of mitosis will provide further important details on how cell division is regulated.