Men and younger age groups (18–44) were less informed than women and older age groups (44 +). A significant proportion (21%) of the population would likely travel back to the UK whilst displaying COVID-19 symptoms with many expressing that they would not fully comply with self-isolation guidelines. Overall, males and younger age groups had a reduced perceived personal risk from contracting COVID-19, posing a higher risk of transporting SARS-CoV-2 back to the UK. Poor passenger knowledge and behaviour undermines government guidelines and policies aimed at preventing SARS-CoV-2 entry into the UK. This supports the need for stricter, clearer and more targeted guidelines with point-of-departure viral testing and stricter quarantining upon arrival.Air travel mediates transboundary movement of SARS-CoV-2. To prepare for future pandemics, we sought to understand air passenger behaviour and perceived risk during the COVID-19 pandemic. This study of UK adults ( For example, it is now well established that UK citizens returning from mainland Europe , rather than China, were primarily responsible for initially introducing SARS-CoV-2 into the UK7. Based on sequencing it has been estimated that at least 1300 independently-introduced transmission lineages of the virus were introduced to the UK in early 2020, leading to the first wave of COVID-197. Based on this, we estimate that infected passengers entering the UK represented ca. 0.02% of the 8 million passengers arriving during this period. This was, however, notably lower than the estimated 1.3% of infected international passengers that left Wuhan at the start of the pandemic and which fuelled the global spread of the disease8. At the beginning of the second COVID-19 wave in Europe, in-flight transmission of SARS-CoV-2 between passengers was also documented in a flight from Greece to Ireland, where the attack rate was 10–17%9. This transmission occurred despite the use of face coverings and the implementation of social distancing measures. A range of modelling and epidemiological case studies has also confirmed that one infected person on a flight could transmit the disease to other passengers throughout the plane10. This is not helped by the close proximity of other passengers, intrinsic air circulation patterns and closely confined and high frequency use toilet facilities. The potential for importing and transmitting SARS-CoV-2 on aircraft mirrors the findings for other respiratory viral diseases . In addition, the potential for transmission within crowded airport terminals has also been demonstrated for other respiratory pathogens  suggesting that a single individual carrying the disease may infect multiple people all travelling to different destinations.The importance of air travel in facilitating the long-distance spread of COVID-19 is undisputed16. For example, COVID-19 restrictions on air travel has led to major economic and job losses in many countries18. There is therefore an urgent drive from the travel industry to re-open international air routes post-pandemic. However, this must be done in a socially responsible, practical and economic way that will ensure protection of public health. Key to this is effective disease control and surveillance. This clearly requires the development of practical, cost-effective and socially acceptable surveillance technologies but also necessitates a good knowledge of individual attitudes to COVID-19 and their behaviour before, during and after air travel. From one survey of the public acceptance of quarantining measures it was concluded that public support is vital for any program involving quarantine and isolation19. Further addressing challenges and barrier to enlist the support of the public is essential to optimize compliance21.Although closure of international flights has inevitably helped contain the spread of the disease, it has come at a substantial cost22 and often ineffectual23. Although a range of strategies are now in place for national disease surveillance , we still lack ways to reliably estimate rates of disease entry from overseas travellers. Based on the known trans-national importation of new variants of SARS-CoV-2 into the UK , it is clear that current surveillance strategies remain inadequate both at the point of departure and the point of entry. This is either because (i) current technologies lack scientific credibility , (ii) are not cost-effective for mass deployment, (iii) are subject to error , (iv) fail to capture recently acquired infections (e.g. those acquired within hours of departure), (v) are not available at the point of departure, (vi) cannot capture infections acquired during travel (e.g. in transit lounges or on the flight), or (vii) solely rely on self-reporting which fails to capture asymptomatic, pre-symptomatic, mildly symptomatic individuals and those knowingly concealing symptoms25. This is supported by an ECDC study which estimated that ca. 75% of infected individuals from China arrived at their destination undetected26. To help mitigate this, many countries have implemented policies of quarantining passengers for 10–14 days upon arrival.Appraisal of airport entry screening measures have shown that it is highly resource demanding27. In contrast, disobedience is suggested to be a behaviour where an individual takes a conscious stance against formalized laws or implied social norms27. Therefore, if individuals refuse to comply with quarantining policies, due to obedience being a learned behaviour from childhood, then these individuals may also be less likely to follow other health advice put forward to mitigate the spread of COVID-19 (e.g. mask wearing), posing further risk to themselves and others.Clearly, self-quarantining relies on individual compliance if it is to be effective. In particular this includes obedience and/or agreement to follow quarantining polices. It has been suggested that obedience involves respect of implicit and explicit rules and that is a socially learned behaviourWith a focus on UK air travellers and those that have flown during the pandemic, the primary aims of this study were to evaluate how human behaviour associated with travel can increase the risk of spreading COVID-19. We aimed to (i) evaluate passenger knowledge of COVID-19 symptoms, (ii) their attitudes to catching COVID-19, (iii) evaluate their likelihood of returning back to the UK if they, or a member of their family, were ill, (iv) evaluate their perceived safety during recent air travel, and (v) the likelihood that they would self-quarantine for the full period on return to the UK.28) to carry out this cross-sectional survey, between the 22nd to 23rd October, 2020. Following a full national lockdown introduced in March 2020, at the time of this study the UK remained under tight COVID-19 restrictions that limited social mixing29 although, specific guidance varied between England, Wales, Scotland and Northern Ireland. Participants (n = 2103) were recruited from YouGov's online research panel  and were eligible if they were aged 18 years or older and living in the UK and had undertaken foreign air travel. Comparisons of opt‐in internet panels with traditional stratified random sample interview and random digit dial techniques conclude that the biases introduced by this methodology are small, and in general are more than offset by the much larger sample sizes the internet‐based methodology permits30. The random error on a sample of 2,000 individuals is estimated to be up to 2%. Quota sampling was used, based on age, gender and Government Office Region, to ensure that the sample was broadly representative of the UK general population. All participants provided sociodemographic variables and none were excluded from the subsequent analysis. Participants were invited to participate in the survey by an email with the subsequent survey conducted on-line via the YouGov data portal. Active sampling restrictions were put in place to ensure that only people contacted and registered with YouGov were allowed to participate. Participants were provided with a summary of the surveys purpose and were informed that data collection, storage and analysis would follow the Data Protection Act 2018. Individuals were then asked to consent prior to survey participation.We commissioned the ESOMAR accredited market research company YouGov . The survey questions and responses are available as an open-access data archive on the Zenodo repository, We asked participants to report their age, gender, social grade, employment status, highest educational or professional qualification and marital status. We also asked whether there was a child in their household, what social media/messaging platforms they had used in the last month , whether they had travelled abroad by plane since the start of the COVID-19 pandemic and whether they had ever tested positive for COVID-19. Participants were asked for their postcode to determine indices of multiple deprivation (IMD) and their social grade. The number of respondents for the key demographic variables are summarised in Table This study was performed in accordance with relevant guidelines and regulations, where ethical approval for this study was granted by the Bangor University College of Environmental Sciences and Engineering Ethics Committee .P < 0.05 as the cut-off for statistical significance.Any percentages calculated on bases fewer than 50 respondents  were included with the caveat that they may not represent a true cross-section of the target population and should be used as indicative only. Comparisons between groups was made using chi-squared tests using n > 250), female respondents were better able to recognize the actual COVID-19 symptoms by 25 ± 9% in comparison to the male cohort (P < 0.001). In addition, for the same top 10 ranked symptoms, 9 showed an increased recognition of symptoms with age . When comparing the cohort with least risk to developing severe COVID-19 symptoms (ages 18–24) to those in the older, more susceptible cohort (age 55 +), the older generation were on average 66 ± 29% better at recognizing the symptoms (P < 0.001). No significant effect of social class . The responses of adults with and without children was also very similar, although those with children (< 18 years in age) were on average 9 ± 3% better at recognizing the symptoms. The variation in COVID-19 symptom recognition was not associated with media platforms used by the respondents in the last month .Overall, there was good knowledge of the main symptoms of COVID-19  among the respondents , 17% of respondents were not worried about catching COVID-19, 56% expressed some concern and 27% exhibited strong concern about catching the disease  and older people relative to those in the youngest age group (34 vs. 13%) and also by parents with children relative to those without (31 vs. 22%). Of those surveyed, 1.3% (n = 27) had previously tested positive for COVID-19, while a further 10.3% believed they had contracted COVID-19 but had never been formally tested, with the remainder not knowingly having contracted the disease. The number of confirmed or suspected positive cases was not associated with differences in gender, age, social class or the presence of children in the household.Of those who answered n = 210, 17% of n = 833) had travelled while ill in comparison to those in the older age groups . Travelling while ill was also more frequent in households without children (29 vs 18%).When asked about their previous experience of returning back to the UK on an international flight, 23% of respondents indicated that they had previously boarded a flight while feeling ill . A return to the UK while expressing COVID-19 symptoms was also higher in the younger age groups (ages 18–44) relative to those in the older age groups  while social grade and the presence of children in households proved not to be significant.In the hypothetical situation that an individual started to express potential symptoms of COVID-19, we asked their likelihood of returning on their scheduled flight. Overall, 21% said they would, 52% said they would not and 27% of individuals indicated that they were unsure , the responses were generally similar to responses if they had symptoms, with 27% saying they would still travel home, while 45% would not and 28% of individuals remained unsure . The younger generation were also more likely to break the self-isolate guidance (17%) relative to the older population (6%).To better understand the likelihood that an individual would self-quarantine following return from a country on the UK government’s quarantine list, 83% of individuals reported that they would probably quarantine for the full 10-day period, while 10.2% said they would not Fig. . These rn = 305) of them indicated they had flown since the start of the COVID-19 pandemic. The greatest numbers of flights were taken by the youngest age group (age 18–24) and the more affluent social grades . Of these, 47% expressed that they felt safe from potentially catching COVID-19 during the flight while 13% indicated that they did not  and in some cases in isolation of the more common symptoms35. It is therefore vital to improve the public’s knowledge on symptoms through media reports and education as this has been shown to positively alter behaviour during a disease outbreak36. In this instance, it will allow people to assess their symptoms effectively and subsequently foster more responsible travel behaviour during a pandemic. However, this is particularly challenging as distinguishing COVID-19 symptoms from other common food poisoning agents , viral illnesses , pharmaceutical use and excessive alcohol consumption can be difficult37. There is evidence to show that free testing facilities can effectively alter behaviours to reduce community transmission38; we therefore highly recommend that people are given easy access to testing facilities to allow individuals to distinguish between COVID-19 and other common illnesses when there is uncertainty around the symptoms being experienced.Overall, we found that an individual’s knowledge of the range of symptoms associated with COVID-19 was mixed. Respondents had a good knowledge of the main symptoms of COVID-19  but did not recognise other symptoms . This lack of awareness increases the risk of spreading COVID-19 as individuals are unable to effectively self-diagnose a potential COVID-19 infection and act appropriately to mitigate the spread of COVID-19 when travelling. This poses a particular risk early on within an infection where some point-of-departure detection technologies  are not reliableoms Fig. ;34 and i39, financial worries , social and cultural pressures and mental wellbeing etc.41.A major risk for COVID-19 entry into the UK is a lack of compliance with government mitigation strategies aimed at reducing the spread of COVID-19. Here, we found that 21% of respondents would not follow travel advice where the public are asked to not travel when expressing COVID-19 symptoms. Furthermore, 10% of respondents would not comply with the government self-isolation guidelines upon entry into the UK. Understanding these behaviours is integral in providing ways in which health planners and policy makers can encourage compliance. Although this study does not explore these factors, it is likely that these behaviours are driven by similar factors identified by studies that focus on general compliance behaviours during a pandemic, which are not specific to travel. These include a lack of perceived risk to the virus42. Mitigation strategies have been implemented internationally to reduce the risk of spreading COVID-19 by air travel , however, several risks that are more closely aligned to one’s perception and/or behaviour to flying during a pandemic has the potential to further increase risk despite implemented mitigation strategies. Evaluating the risk perception of air passengers that travelled during the COVID-19 pandemic highlighted that a high proportion of individuals felt safe from contracting COVID-19 during air travel. This suggests that non-pharmaceutical interventions to reduce disease transmission were being correctly practiced during their flights.Multiple factors can contribute to the cumulative risk of contracting COVID-19 from air travel43, handwashing with soap is the most effective way to mitigate the spread of infectious diseases. Despite this, globally, the benefits of hand hygiene are poorly practiced44. This is particularly problematic in cramped aircraft bathrooms where there is a high risk of cross contamination due to the large number of touch surfaces45. Additionally, Suen et al.46 found that many individuals overlook the actual importance of washing their hands (hand hygiene) when taking part in activities where they would be required to wash their hands. In support of this, our research found that only 84% of participants would wash their hands following the recommended guidelines for COVID-19. Furthermore, they proposed that female respondents generally have better knowledge regarding hand hygiene which supports the findings from this study where it was mainly the women and the older participants (44 +) that stated they would follow COVID-19 recommended guidelines with regards to washing hands. It is recommended that when promoting hygiene as a guideline, that more gender specific material be used to ensure that the importance of hygiene, whether it be hand hygiene or hygiene in general is understood and adopted by all individuals to sustain improvement in hygiene practices47.According to Anderson et al.48, we found that women were generally better at identifying COVID-19 symptoms compared to the male participants. We believe that previously reported differences between males and females such as, men having a lower risk perception49, a disbelief that COVID-19 is contagious50 and a sense of COVID-19 immunity51 are likely to also contribute towards the observed differences between age groups and adults with or without children.In agreement with Kamenidou et al.19 that women would be more willing to adhere to various levels of quarantine compared to men. Furthermore, our research agrees with Galasso et al.52 that women were more likely to perceive COVID-19 as a very serious health problem making them more likely to agree and comply with government guidelines. Additionally, Bass et al.19 found that respondents who were older than 65 were more willing than younger respondents to stay at home when government guidelines were in place to do so which also agrees with our findings. Overall, our findings suggest that males and younger ages groups have a reduced perceived personal risk from contracting COVID-19 and our results suggest that they pose a higher risk of transporting SARS-CoV-2 back to the UK and around the UK due to lack of willingness to co-operate with isolation guidelines.Our research supports previous findings by Bass et al.53.We found a higher proportion of older generations (aged 44 +) and adults with children were able to recognise COVID-19 symptoms compared to younger age groups (18–44) and adults without children, respectively. Our findings suggest that males, young adults, and adults without children pose a higher risk in spreading COVID-19 infections and that information needs to be created to target each segmentation in a more unique way. Similar findings have also been reported for the non-air travellers in Africa48. The aforementioned study suggests that the younger population are more interested in solving the issues that COVID-19 presents rather than focusing on how they can prevent the spread. This is compounded by negative media representation of the role of younger generations in spreading COVID-19 and their over-reliance on social media for information54. Again, this suggests that information needs to be targeted to segmented age groups to ensure the correct information is retained and processed.Our findings agreed with previous research that younger participants (under the age of 25) were unable to identify many symptoms connected to COVID-19 and that they would be less likely to pay the required attention when symptoms appeared55 found no age-related differences in compliance with the regulation. They did, however, identify that the 20% who did not follow the strict guidelines, were the same participants that had less confidence in the public health information being provided. These findings strengthen the importance of not only the delivery of information, but also the reliability of the information being presented.With regards to following guidelines set by the government, our findings suggest that the younger generation were more likely to break the self-isolation guidance (17%) relative to the older population (6%). In contrast, research conducted in Italy by Ceccato et al.Test and Trace and Welsh Test, Trace, Protect programmes which still show high rates of SARS-CoV-2 carriage among incoming passengers  and also non-compliance with self-quarantining rules which have subsequently led to major outbreaks . Our recommendation supports the imposition of stricter guidelines to ensure complete compliance with point-of-departure PCR-based COVID-19 testing and stricter quarantining on arrival for UK citizens returning from overseas57 A limitation of this study was that it was only confined to UK citizens. It would therefore be desirable to repeat the study to evaluate the perceptions of non-UK citizens where additional socioeconomic and cultural barriers may prevent non-compliance and enhance the risk of disease entry into the UK. Typically, pre-pandemic these account for ca. 59% of all air travellers entering the UK58, although it should be noted that these have been subject to compulsory quarantining in government facilities, rather than self-quarantining at home like UK citizens. Despite the well understood risks associated with air travel and disease transmission (e.g. confined space and poor airflow) and that mitigations have been implemented to reduce these risks (e.g. compulsory face coverings), we highlight additional risks that should be considered that are associated with an individual’s perception and related behaviours towards COVID-19 that can jeopardise existing strategies to reduce the spread of SARS-CoV-2.Based on this national survey of air passenger behavioural patterns and individual perceptions of COVID-19, alongside known importation of new SARS-CoV-2 variants into the UK, we conclude that past and current government guidelines and policies are insufficient to prevent the frequent entry of SARS-CoV-2 into the UK. This is supported by information provided by the English In terms of future pandemics, our work suggests that despite repeated messaging from national agencies, greater emphasis is needed to ensure that all individuals understand the importance of following public health guidelines and how self-imposed quarantining and extended hygiene can help to reduce infection spread. Additionally, the presentation of disease symptoms (both main symptoms and onset) needs to be addressed in ways in which they are understood and adopted by all individuals to reduce the chance of spreading infection. Different ways of communication are also needed particularly to reach younger generations travelling by air.Supplementary Information.


Future climate scenarios suggest that crop plants will experience environmental changes capable of affecting their productivity. Among the most harmful environmental stresses is drought, defined as a total or partial lack of water availability. It is essential to study and understand both the damage caused by drought on crop plants and the mechanisms implemented to tolerate the stress. In this study, we focused on four cultivars of tomato, an economically important crop in the Mediterranean basin. We investigated the biochemical mechanisms of plant defense against drought by focusing on proteins specifically involved in this stress, such as osmotin, dehydrin, and aquaporin, and on proteins involved in the general stress response, such as HSP70 and cyclophilins. Since sugars are also known to act as osmoprotectants in plant cells, proteins involved in sugar metabolism (such as RuBisCO and sucrose synthase) were also analyzed. The results show crucial differences in biochemical behavior among the selected cultivars and highlight that the most tolerant tomato cultivars adopt quite specific biochemical strategies such as different accumulations of aquaporins and osmotins. The data set also suggests that RuBisCO isoforms and aquaporins can be used as markers of tolerance/susceptibility to drought stress and be used to select tomato cultivars within breeding programs. Global warming, primarily due to the intense emission of carbon dioxide caused by human activities, is constantly increasing the average temperature; this is responsible for the reduction of rainfalls in highly vulnerable areas such as the Mediterranean, which is considered a “hot spot” in the 21st century [At the level of individual plant organisms, water scarcity is perceived as an abiotic stress (drought stress). Drought stress leads to various damages in plants, including incorrect protein folding, alterations in enzymatic functions and higher production of reactive oxygen species (ROS) . The firIn general, abiotic stresses are associated with enzyme and protein dysfunction and it is important for the plant to keep the cells functional during stress. For this purpose, plants increase the expression of various genes under drought stress leading to the production of corresponding proteins. Most importantly, it is necessary to prevent the aggregation of non-native proteins and to maintain the proteins in their functional conformation. Therefore, some of the most important proteins induced by drought stress include chaperones, such as heat shock proteins (HSPs), and abundant proteins of late embryogenesis (LEA) .Among the proteins induced by ABA are Heat Shock Proteins (HSPs), a class of chaperones involved in protein folding and thus relevant in the defense against abiotic stresses ,10. HSPsCyclophilins (CYPs) are another ubiquitous chaperone proteins, involved in a wide range of cellular processes ,16,17. CThe synthesis of Late Embryogenesis Abundant (LEA) proteins is also induced by ABA . DehydriAbiotic stresses activate many intracellular signals which lead to the accumulation of osmoprotectants and production of Pathogenesis-related (PR) proteins . Osmotin2 transporters [2 and H2O transport. Indeed, some work has shown that overexpression of PIPs in Arabidopsis, rice, or tobacco results in enhanced CO2 assimilation in leaves [Proteins important for the efficiency of photosynthesis are also the aquaporins. These are known as water and COsporters . Accordin leaves ,36,37. Fn leaves . For exan leaves . In toman leaves ,41.2O and CO2. In the dark phase , the leading enzyme is Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO) which catalyzes the carboxylation reaction that initiates CO2 fixation. The catalytic activity of RuBisCo progressively decreases with increasing duration and severity of drought conditions [Other biochemical adaptations of plants consist of the regulation of the photosynthetic process. Photosynthesis might be limited due to the scarce availability of substrates such as Hnditions . This canditions . The degnditions . FurtherAt the level of sink tissues, sucrose can enter cells in two ways. It can be split by the activity of cell wall invertases into glucose and fructose, which are transported into cells by monosaccharide transporters . SucroseIn this manuscript, we analyzed the biochemical response of tomato cultivars subjected to drought stress. Tomato is a widely cultivated plant that can suffer dramatically from water stress conditions . AlthougThe purpose of this work was to test the hypothesis that drought susceptibility or tolerance of tomato cultivars are based on the different production of metabolic proteins, such as RuBisCO and SuSy that regulate the level of osmoprotective sugars, and on the change in the content of proteins specifically involved in the stress response, such as HSP70, cyclophilins, osmotin, dehydrin, and aquaporins.In this manuscript, we focused on four tomato cultivars characteristic of the Tuscany Region . This manuscript is based on previous works  in whichWe initially examined the general response to drought stress by evaluating the relative content of HSP70 A. ExpresComparing the content of HSP70 in DS samples of the four cultivars, the increase in protein levels from the most tolerant to the most susceptible cultivar is evident. The cultivar Perina showed lower content than the others, especially compared to Pisanello. Even in the CTRL samples, the content of HSP70 increased from the most tolerant to the most susceptible cultivar. Furthermore, in the control samples, basal levels of HSP70 in Perina are lower than in Quarantino or Pisanello. Therefore, results cannot be interpreted only in terms of quantity, but it is necessary to compare the increase of HSP70 from CTRL to DS. Both the cultivars Perina and Fragola increase the content of HSP70 more than 200% in DS plants compared to those in the CTRL group, whereas in Quarantino, HSP70 levels increase about 50%. In contrast, Pisanello, while showing an abundant basal content of HSP70, only reveals a smaller increase than the other cultivars. Therefore, it is inferred that the increase in content of HSP70 in Pisanello under drought stress is the lowest among all cultivars.Consistent with morpho-physiological analysis , the FraAs a further general analysis of the stress response, we subsequently investigated cyclophylins (CYP). The cultivars Perina and Fragola were selected in previous studies based on their tolerance to drought stress; however, the cultivar Quarantino showed both traits of tolerance and susceptibility ,54. The In rice, drought and salt stress induce considerable expression of a specific cyclophilin; furthermore, overexpression of this protein in transgenic rice and Arabidopsis plants increased drought tolerance . TherefoAfter depicting a preliminary picture of the overall response to drought stress, we moved on to the analysis of proteins more specifically involved in the stress response. Furthermore, the increase in dehydrin content in stressed samples is inversely proportional to the tolerance of cultivars toward drought stress B. The inTo cope with abiotic stresses, plants have several defense mechanisms including the protein osmotin, which belongs to the PR-5 family of pathogenesis-related (PR) proteins . The blo2O and CO2, two important substrates for photosynthesis [Aquaporins are proteins located in the plasma and intracellular membrane and are well known transporters of Hynthesis . The bloThe role of aquaporins in plants under drought conditions has not yet been fully investigated. Aquaporins have been related to salt stress in tomatoes , as well2 in the mesophyll [2 and H2O transport and more efficient photosynthesis. This hypothesis is also strengthened by work on transgenic tomato plants expressing a drought-inducible aquaporin gene PIP1;3, which derived from Malus domestica Borkh [Forty-seven genes encoding for aquaporins have been identified in tomatoes. As for the family of plasma membrane intrinsic proteins (PIPs), among the five forms recognized by the antibody we used only one is present in mature tomato leaves, namely PIP1;3. The PIP1;1 protein is strongly expressed in roots and fruits, PIP1;2 in the young leaf and root, PIP1;5 only during fruit development, and PIP1;4 is not present in tomatoes . In addiesophyll ,37. Consca Borkh . The tom2 fixation. This enzyme can account for up to 50% of the protein content in a leaf. There is no need to emphasize that it is an extremely critical enzyme in the metabolism and life of plants and that any alteration in the levels or activity of this enzyme has major consequences in the production of plant biomass. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the first enzyme in the Calvin-Benson cycle. It catalyzes the carboxylation reaction that initiates CO2 concentration caused by stomata closure induces an increase in the oxygenase activity of RuBisCO [2, which helps to keep the dark phase of photosynthesis active and protects chloroplasts from excessive ROS production. Under drought stress conditions, decreased transcription of genes encoding for small subunits of RuBisCO may occur, thus leading to the loss of enzyme stability [ RuBisCO . To inittability . In additability ; this maAnalysis by 2D electrophoresis and immunoblotting on RuBisCO revealed a series of spots, whose total number is shown in the virtual master blot of The 8 spots identified are more or less present in each case analyzed, at the level of cultivars as well as in the comparison between control and stressed samples. However, some substantial differences emerge. From a qualitative point of view, the differences between cultivars are minimal. Some cultivars are characterized by spots present only in stressed samples. For example, the cultivar Perina expresses the isoform 9905 only in the stressed sample. Even the cultivar Fragola shows a typical expression with the isoforms 9904 and 9905 being present only in the stressed samples. Others, such as the cultivar Quarantino, lack a specific spot, in this case 9906. Pisanello differs because some spots, such as 9904, are present only in the stressed sample, while 9905 is detected only in the control sample. Apart from the qualitative aspect, it is also noteworthy that the four cultivars differ in terms of the quality (usage) of individual spots. In the cultivar Perina, some isoforms are present correspondingly between control and stressed samples while others are typical of control samples (such as spot 9902) while 9905 is typical of stressed samples. The cultivar Fragola differs substantially from Perina because spots can be classified as almost exclusive to the control sample  and as exclusive to the stressed sample . The cultivar Quarantino has a similar behavior to the Perina cultivar; the Pisanello cultivar is similar to the case of the Fragola cultivar with spots almost exclusive to the control sample and two spots (9904 and 9906) exclusive to the stressed sample. In the case of Pisanello, it is worth noting the absence of spot 9908.In summary, the data indicate that the 9905 RuBisCO isoform is typical of the most tolerant cultivars (Perina and Fragola) and is therefore preferentially used; this isoform is partially expressed in Quarantino and is completely absent in the most susceptible cultivar (Pisanello). Since the large subunit of RuBisCO is encoded by a single plastidial gene, the different isoforms may be the result of post-translational modifications. Indeed, RuBisCO is characterized by several potential co-/post-translational modification sites . It is aSuSy is a key enzyme in sucrose metabolism as it cleaves sucrose producing UDP-glucose and fructose. While fructose can be directed toward respiration, UDP-glucose provides a conservative form of energy that can be redirected toward both intracellular metabolic processes and in the building of cell wall polysaccharides . Thus, aIndeed, the accumulation of Susy can also be related to an increased production of fructose that accumulates in plants under drought stress, like in wheat . HoweverSuSy is just one of the many enzymes regulated by phosphorylation events . In geneCarbohydrates produced by photosynthesis in plant leaves provide energy and building blocks for growth and productivity. In addition to their energetic action, soluble carbohydrates  act as important osmoregulatory substances capable of maintaining cell turgor under conditions of osmotic stress  . TherefoSucrose is the main product of reactions involving 3-carbon sugars generated by photosynthesis and represents a form of energy storage and transport . Figure In addition to being cleaved into glucose and fructose, sucrose can be cleaved into fructose and UDP-glucose by sucrose synthase (SuSy) . In leavBecause the content of sucrose and related sugars in leaves is the result of different metabolic pathways, the data on sugar content do not perfectly match the expression of SuSy . In partThe analysis of soluble sugars in tomato cultivars was also extended to water-soluble pectins D. GeneraThe plants studied in the present work are a subset of the nine tomato cultivars previously analyzed  from a mPerina and Fragola, the most tolerant cultivars,Quarantino, the cultivar with medium tolerance,Pisanello, the most susceptible.The analysis in this manuscript was carried out on the above four cultivars. Biochemical aspects related to proteins involved in the defense mechanisms against drought stress were investigated. All samples were taken at the final stress phase (after 16 days of drought stress). The timing of drought stress was chosen according to ,102,103.g for 10 min at 4 °C and the upper phase was taken to which 3 mL of Extraction Buffer were added; samples were vortexed for 3 min and centrifuged at 5500× g for 10 min at 4 °C. The upper phase was collected and supplemented with four volumes of precipitation solution (0.1 M ammonium acetate in methanol), mixed by inversion, and incubated at −20 °C for at least 4 h or overnight. The mixture was centrifuged at 5500× g for 10 min at 4 °C and the supernatant was removed. The pellet was washed with the precipitation solution, centrifuged at 5500× g for 5 min at 4 °C and the supernatant was removed. This last step was repeated twice. The last pellet was washed with cold acetone, centrifuged at 5500× g for 5 min at 4 °C and the supernatant was removed. Samples were then dried at RT under a fume hood for 10 min. Afterwards, 100 μL of 0.2 M NaOH were added and samples incubated for 2 min for more effective solubilization. A volume of 200 μL of LSB1X for 1-D electrophoresis and 200 μL of Rehydration Buffer (RB) for 2-D electrophoresis were added to the samples. Finally, samples were centrifuged for 15 min at 10,000× g at RT, the supernatants were collected, and the protein concentration was calculated using the 2-D Quant Kit .Protein extraction was performed as described by Faurobert and coworkers . Leaves Citrus L. and pepper plants [The Anti-HSP70 (ADI-SPA-820-D) was a mouse monoclonal type antibody and was diluted 1:5000 (Enzo Life Sciences). This antibody was purified from human HeLa cells. It recognizes protein homologues in plants and its efficiency has been confirmed in r plants  as well r plants .Solanum sogarandinum O. [Pyrus L. pollen [The Anti-Cyclophilin (Anti-CYP) was a rabbit polyclonal antibody and was diluted 1:3000 . This andinum O. . Our wor. pollen .Solanum licopersicom L., as well as in other species such as Pistacia vera L. and Cucumis sativus L. [The Anti-Dehydrin (AS07 206A) was a rabbit polyclonal antibody and was diluted 1:1000 (Agrisera). This antibody binds to the dehydrin family, which are proteins involved in protective reactions against dehydration. Specifically, the antibody binds to the k-segment peptide sequence (TGEKKGIMDKIKEKLPGQH) conserved in a wide range of different plant species. The reactivity of this antibody has also been confirmed in tivus L. ,110,111.Nicotiana tabacum L. protein sequence, ranging from amino acid 22 to 246. The predicted reactivity is also on Solanum lycopersicum L.The Anti-Osmotin (AS19 4336) was a rabbit polyclonal antibody and was diluted 1:1000 (Agrisera). This antibody was derived from the Raphanus sativus L. The peptide is conserved in PIP1;1, PIP1;2, PIP1;3 N-terminus of Raphanus sativus L. and in all 5 isoforms  of Arabidopsis thaliana L. The reactivity in Solanum lycopersicum L. is not confirmed but predicted.The Anti-Aquaporins (AS09 489) was a rabbit polyclonal antibody and was diluted 1:1000 (Agrisera). The immunogen for aquaporin antibody is a KLH-conjugated synthetic peptide derived from N terminus of Solanum lycopersicum L. However, the reactivity against tomato was evaluated in a previous work on the Micro-Tom cultivar [The Anti-RuBisCO was a rabbit polyclonal antibody and was diluted 1:10,000 (Agrisera). The immunogen for the RuBisCO antibody was a synthetic KLH-conjugated peptide preserved in all known plant, algal and cyanobacterial protein sequences. Reactivity was confirmed and predicted on several plant species but not on cultivar .Zea mays L. on the complete protein [The K4 anti-SuSy was a rabbit polyclonal antibody and was diluted 1:1000 . The ant protein .Electrophoresis was conducted on 10% bis-Tris SDS-PAGE  at pH 6.Subsequently, membranes were washed twice with 1X TBS and then incubated with peroxidase-conjugated secondary antibodies for 1 h. The Anti-rabbit IgG  and Anti-mouse IgG  were polyclonal antibodies and were diluted 1:3000. After rinsing the membranes with 1X TBS, the immunological reactions were visualized with ClarityTM Western ECL Substrate . Images of blots were acquired using a Fluor-S apparatus  and analyzed with the Quantity One software . Finally, densitometric analysis was performed with the same software for a relative quantitative evaluation of band intensity (expressed as Integrated density).2D electrophoresis and immunoblotting on RuBisCO were performed as described in ,85. Brie2O. Samples were homogenized by Ultra-Turrax® T-25 basic , centrifuged at 3000 RCF for 5 min; the supernatants were transferred to 2 mL Eppendorf® tubes and then centrifuged again at 12,000 RCF for 5 min . Samples were filtered (0.45 µm) and 20 µL of each extract was injected into a Waters Sugar-Pak I ion exchange column (6.5 × 300 mm) at a temperature of 90 °C. The mobile phase consisted of MilliQ H2O (pH 7) with a flow of 0.3 mL min−1. The overall duration of the separation was 30 min. Identification of components was done using a Waters 2410 refractive index detector by comparing the retention times with those of reference standards. The experiment was conducted in three technical replicates for each sample. Finally, the mean and standard deviation were calculated. To verify the significance of the data obtained, the t-test  were carried out.High Pressure Liquid Chromatography (HPLC) was used for the analysis of sugars  ,114. Bri® Diamond Blot Reagent & Buffer (Thermo Fisher) was used to highlight the phosphoprotein patterns according to the protocol provided by the company. Proteins were separated by electrophoresis and then transferred to PVDF membranes (pre-moistened in methanol). After electroblot, membranes were allowed to dry completely. Proteins were fixed on membranes by dipping it face down in 25 mL of Fix Solution  for 10 min. Membranes were washed by immersion in 25 mL of dH2O for 5 min (three times). Proteins were stained by immersing the membrane in 25 mL of the diluted Pro-Q® Diamond Phosphoprotein Blot solution for 15 min. Membranes were destained by washing in 30 mL of Destain solution  for 5 min (three times). Fluorescent phosphoproteins could be visualized by the Fluor-S apparatus  by illuminating membranes with UV light using a 615 nm bandpass filter; exposure times were 10–30 s. The resulting electrophoretic lanes were scanned by Quantity One software (Bio-Rad).Pro-QIn this work, four Tuscan tomato cultivars were characterized using a panel of biochemical analyses. These revealed critical differences among cultivars in the response to drought. Some mechanisms, such as increased levels of HSP70 and cyclophilins, are common and implemented by all cultivars, albeit with some differences among cultivars . These data confirm the important protective role of HSP70 and cyclophilins in the correct folding of proteins. In fact, previous works on the morpho-physiological aspects of the above cultivars already indicated Perina as the most tolerant, while Pisanello was the most susceptible.Dehydrin and osmotin contents are higher when plants are severely affected by drought stress. While dehydrins are substantially expressed by all cultivars under stress, osmotins are found only in Pisanello. This can be explained by the fact that the susceptible cultivar Pisanello shows more damage to the photosynthetic system. Therefore, it is the only cultivar that requires expression of osmotin, which plays a role in chlorophyll protection. Analysis on RuBisCO corroborated this hypothesis. In fact, a drastic decrease in RuBisCO content is shown in the cultivar Pisanello under drought stress. Moreover, the 9905 isoform of RuBisCO is apparently typical of the most tolerant cultivars (Perina and Fragola) but not of the Pisanello cultivar. RuBisCO is an enzyme with several co/post-translational modification sites; therefore, under stress these modifications may generate isoforms that are more suitable to counteract a demanding situation such as drought. This concept is further supported by the evidence that Pisanello, compared to other cultivars, shows a pronounced generic dephosphorylation pattern. Phosphorylation/dephosphorylation may contribute to an increase or decrease in sucrose synthase (SuSy) activity. The content of this enzyme increases in all stressed cultivars, especially in Pisanello. SuSy allows the cleavage of sucrose into fructose and UDP-glucose, thus feeding the biosynthesis pathway of osmoprotective sugars. However, the Pisanello cultivar still produces sucrose in large quantities without breaking it down into glucose and fructose, which could instead be more useful during stress.2 and H2O.In conclusion, this study confirmed at the biochemical level the results previously obtained by morpho-physiological analyses on the tolerance or susceptibility of tomato cultivars to drought stress. More specifically, we have examined the biochemical mechanisms that are activated by drought and that increase stress tolerance. From this and previous studies, the Perina cultivar is confirmed as the most tolerant, because it can activate all the mechanisms necessary for tolerance. Most importantly, it can keep the photosynthetic system active by selecting the best RuBisCO isoforms and increasing the content of aquaporins, beneficial for the transport of CO


Previous study showed that both melatonin supplement and physical exercise intervention could improve sleep quality in children with autism spectrum disorders (ASD) with the increase in endogenous melatonin level. However, none of the studies have directly compared the effectiveness between the two interventions on treating sleep disturbance in children with ASD. Without direct comparison, we do not know which intervention is better. Thus, we designed a study to compare which intervention is more effective to treat sleep disturbance in children with ASD and to examine whether the combination of the two could be the most efficacious. We present a protocol for conducting a randomized controlled trial to compare the effectiveness of physical exercise and melatonin supplement on treating sleep disturbance in children with ASD.The proposed study will be a four-group randomised control trial (RCT) design, with equal allocation of participants to the three intervention groups and one control group.p < .05). The results of this trial will be submitted for publication in peer-reviewed journal.All eligible participants will be randomly allocated to a morning jogging group, a melatonin supplement group, a combination group and a control group. Changes in sleep quality will be monitored through actigraphic assessment and parental sleep logs. Melatonin levels represented by 6-sulfoxymelatonin will be measured from the participants’ 24-h and the first morning void urinary samples. All the assessments will be carried out before the intervention (T1), in the mid of the study (5 weeks after the commencement of the study) (T2) and after the 10-week intervention (T3). Level of statistical significance will be set at 5% (i.e. The findings will provide evidence to determine whether physical exercise or melatonin supplement or the combination of interventions is the most effective to treat sleep disturbance in children with ASD. Autism spectrum disorder (ASD) is a neurodevelopmental disorder. In Hong Kong, the latest prevalence rate is 1.5% and the rate is predicted to increase  for reviTo achieve so, we need to understand the causes of sleep disturbance in the population. Over decades, a growing body of literature has shown that physiological differences in children with ASD may be the major cause of sleep problems in children with ASD  to determine which intervention is more effective in improving sleep quality in children with ASD and 2) to explain how these interventions impact on sleep via melatonin-mediated mechanism model. We hypothesise that the combination intervention (i.e. jogging and melatonin supplement) is the most effective because it may provide sufficient melatonin level to enhance sleep quality through both physical exercise and melatonin supplement.The proposed study will be a single blinded four-group randomised control trial (RCT).Participants will be screened using the following inclusion criteria: (1) clinical diagnosis of ASD by a physician based on the Diagnostic and Statistical Manual of Mental Disorders, 5th edition, (DSM-5) ; (2) ageExclusion criteria are: (1) with one or co-morbid psychiatric disorders; (2) with other medical conditions that limit their physical exercise participation and sleep ; and (3) with a complex neurologic disorder  and (4) participants who are currently meeting physical exercise guidelines .st August, 2022 and the estimated ending date will be 30th June, 2023.Each participant will attend 3 one-week-long assessments in their respective schools, where we will assess their habitual sleep patterns and endogenous melatonin level before the intervention T1), in the mid of the study (5 weeks after the commencement of the study) T2) and after the 10-week intervention (T3). The mid-assessment is valuable to know how much variance in sleep behaviors is accounted by variance in melatonin, as well as to assess the adherence of the interventions. SPIRIT schedule of the study and a flow diagram of the study are shown in Figs , in the  and afteThe training protocol, assessment and instructional technique will be referenced on our previous protocol in children with ASD . HoweverParticipants in this intervention group will undergo a 10-week melatonin supplement intervention period, where melatonin supplement  will be provided 30 minutes before bedtime . The preParticipants will receive the jogging program and supplemental melatonin dose with identical format as that in the intervention A and B . Meanwhile, they will also be required to wear an actigraph to control for their physical activity level at the assessment points . They will be expected for following their daily routine without participating in any additional formal physical exercise training program throughout the whole study period (T1-T3). After T3, they will be assisted with jogging program to recognize their contribution as controls.To ensure all the interventions are delivered as intended, regular meetings among the research team will be hosted by the PA to monitor the progress and to discuss any difficulty encountered. Meanwhile, each participating family will be provided with a financial incentive, which is a good strategy evidenced by a high compliance rate in our ongoing study.The demography and developmental history of each participant will be obtained from their parents or schools. And the following measurements will be carried out at T1, T2 and T3.Sleep. Four sleep parameters including sleep onset latency ; sleep efficiency ; wake after sleep onset  and sleep duration  will be objectively measured by a GT3X accelerometer [erometer . Particierometer , 20. Theerometer , which ierometer . MoreoveEndogenous melatonin level. The endogenous melatonin level of the participants, reflected by the 6-sulfatoxymelatonin , will be measured from their 24-h and first morning urinary samples during weekend and Monday respectively [ectively , and thePhysical activity level. The physical activity level of the participants will also be measured as secondary data to examine its relationship to cognitive functions as suggested by previous studies [ studies , 23. It  studies . The dayd of about 0.9) on improving sleep onset latency, sleep duration and sleep duration. We assumed that melatonin supplements have similar effects on sleep. A sample of 20 participants per group is computed by G*power software to achieve a power of 80% and a level of significance of 5% and 95% confidence level. Assuming 25% attrition rate, 25 participants per group will be recruited .Previous study , 14 showParticipants will be randomly assigned to three intervention groups or a placebo-control group by a trained research staff using MS Excel randomization function. Block randomization with block size of 5 will be used for equal allocation ratios.Patients and public will not be involved in the study.The person analysing the sleep parameters and melatonin level will be blinded for the group assignment.https://clinicaltrials.gov/.Prior to the study, information about the study were provided to all participants and their parents. Written consent and verbal assent were obtained from them. All data were encrypted with passwords and only the authors could access to the data. Ethical approval was obtained from the first author’s institution (Reference no: 2019-2020-0470). The results of the study will be published in a peer-reviewed journal. Findings of the study will also be shared with other university and non-governmental organisations in Hong Kong that specialise in autism by means of a formal dissemination webinar. The trial registration number is NCT04878198 from All statistical analyses will be conducted using SPSS (version 26.0) for Windows . All the data will be entered into SPSS by the research assistant. One-way  analysis of variances (ANOVA) with repeated measures will be performed to compare changes in actigraphy data and endogenous melatonin level between and within groups over different time points (i.e. T1-T3). Post-hoc analyses will be performed when any significant difference both between- and within-group is found in any of the outcome variables. Bonferroni correction will be used to adjust the alpha levels. Depending on the proportion of missing data and the assumption of normality, multilevel regression or a generalized estimating equation (GEE) will also be used as a sensitive analysis to assess the effects of the physical exercise intervention, melatonin supplement, time effect, and their interaction with sleep outcomes and melatonin level outcomes if there is any missing data. Average time spent in both daily sedentary activity and MVPA at baseline will be used as covariates. Linear regression will be used to analyse the association between participant’s adherence to the intervention and the outcome variables as a secondary analysis. Mediation analysis will be conducted to examine the mediating effect of endogenous melatonin level at T2 on the relation between physical exercise and sleep at T3. Preacher and Hayes’ bootstrapping and conditional process modeling will be used to determine the significance of the mediating effect. A p-value of <0.05 will be considered statistically significant. Both proposal analysis and intention-to-treat analysis will be carried out. Demographic variables  will be adjusted if they demonstrate clinically significant prognostic effects on the outcomes (with absolute correlation or Cohen’s d effect size of >0.2) for both main analysis and mediation analysis.This study protocol is designed largely based on our previous protocol and study , 17 thatS1 Checklist(DOC)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Methods: We compare the effect of these two different therapies on oxygen saturation measured by pulse oximetry (SpO2) over time. Result: In both patients SpO2 increased significantly from 65–70% to 95%. Above 80% this increase was slower in AP-Pat compared with O2-Pat. Therapy started immediately in AP-Pat but was delayed in O2-Pat because of organizational and logistic reasons. Conclusions: The well-established therapies of HAPE are always the option of choice, if available, and should be started as soon as possible. The advantage of Auto-PEEP is its all-time availability. It improves SpO2 nearly as well as 3 L/min oxygen and furthermore has a positive effect on oxygenation lasting for approximately 120 min after stopping. Auto-PEEP treatment does not appear inferior to oxygen treatment, at least in this cross-case comparison. Its immediate application after diagnosis probably plays an important role here.Background: Improvement of oxygenation is the aim in the therapy of high-altitude pulmonary edema (HAPE). However, descent is often difficult and hyperbaric chambers, as well as bottled oxygen, are often not available. We compare Auto-PEEP (AP-Pat), a special kind of pursed lips breathing, against the application of bottled oxygen (O High-altitude pulmonary edema (HAPE) is a life-threatening illness that may affect hitherto healthy persons after fast ascent to high altitudes . It has As the primary underlying mechanism of HAPE is hypoxia the therapy of choice is to improve oxygenation. The general recommendation is to descend to the last asymptomatic sleeping altitude or at least 500 m of altitude. This is often difficult to realize because of organizational reasons  or the patient’s condition. Another option to improve oxygenation is the use of a hyperbaric chamber or the use of bottled oxygen as the recommended gold standard ,8,9. Bot2). Auto-PEEP is a special breathing pattern we described earlier [2O . As the vocal cords are open during Auto-PEEP we conclude that intrathoracic pressure is in the range of approximately 55 cm H2O.We compare two patients with HAPE to demonstrate the effect of supplemental oxygen and Auto-PEEP on peripheral tissue oxygenation measured by pulse oximetry (SpO earlier . Control earlier  and coul® with a reusable flexible finger sensor (Adult Flexi-Form 9000A) placed on the index finger to visualize SpO2. This device logs the data for SpO2 and pulse every 4 s and has a capacity of 72 h [In both cases we used a Nonin PalmSat 2500 of 72 h . Data we2-Pat), male 55 years of age, a very experienced ultra-marathon runner, suffered from HAPE during a trek to Mera Peak (6461 m) located in the region of Mount Everest in Nepal. During the ascent to the high camp of Mera Peak on day 9 of the trek, at an altitude of about 5000 m and above, he lagged significantly behind the rest of the group. Up to this point he had been ascending without any problems and had always been in the upper performance level of the group. He complained of a sudden loss of performance and hallucinations. Since the terrain was easy, the wayfinding clear, and there was light for several more hours, he turned around shortly after the Mera La Pass at about 5450 m and descended alone to the last camp with permanent shelters at Karre (4870 m). The rest of the group climbed Mera Peak and returned to Karre the next morning. The health of the patient had subjectively improved somewhat in the meantime, with increasing appetite, but he felt very weak.The first patient  [2 steadily from <70% to >95%   was 10 ( to >95% .2 was 62% (59–65%), which is low compared with the findings of Roach et al. who reported an average SpO2 of 81.5 ± 4.4% in 102 healthy mountaineers in the same location [2 continuously on the display of the pulse oximeter. Nifedipine, Dexamethasone, and Acetazolamide were available but not used.The second patient (AP-Pat), male 33 years old, an experienced climber in the European Alps, tried to ascend Denali (6198 m) in Alaska. After his flight from Talkeetna (350 m) to the glacier (2100 m) he ascended to 4380 m within 4 days  carrying heavy loads. There he suffered from initial HAPE with significantly impaired physical performance and dyspnea and an LLS of 11 . His SpOlocation . At this2 increased rapidly from ~64% to 97% within 2 min in O2-Pat (2), he was evacuated to Lukla by helicopter for a first checkup. Several hours later the patient was transferred to a hospital in Kathmandu by helicopter. A physical examination delivered no pathological findings. In the chest X-Ray bilateral prominent bronchovascular markings were diagnosed, with blood and urine samples showing no abnormalities. The patient’s health improved rapidly and by the evening of the same day the patient felt subjectively almost healthy. He was discharged 3 days later in good health.After the administration of bottled oxygen SpOn O2-Pat . A solid2 increased immediately after starting Auto-PEEP from <65% to ~80% with a further slow increase up to 95% at the end of the Auto-PEEP period . Four days after Auto-PEEP he climbed with his wife to the summit of Denali (6190 m), starting from ABC (4330 m), and returned to it within one day without setting up high camp at 5200 m.2 immediately after the start of therapy. The initial increase up to SpO2-levels of ~80% is similar in both patients within a few minutes with a constant fast increase in O2-Pat but subsequently slower increase in AP-Pat up to values close to 95% in both patients. What is remarkable is the slow decrease in SpO2 in AP-Pat after finishing Auto-PEEP.Both patients showed a distinct increase in SpO2 in both patients is distinctive but both patients differ in several aspects.The similar increase in SpO2-Pat (5450 m vs. 4330 m). In general, one would expect a higher risk/severity of HAPE at a higher altitude. However, the impact of a delta of 1120 m relativizes for several reasons. First the impact of 5450 m in O2-Pat was only for a short time as he solely crossed Mera La Pass and descended down to Karre at 4870 m to his accommodation.The altitude at the onset of HAPE is higher in OSecond, there is a different density of the atmosphere, the “physiological” altitude near the arctic circle (63° north latitude) compared to Denali which is higher than in the Everest region (28° north latitude) ,17,18. A2-Pat to reach the altitude where he developed HAPE versus 4 days for AP-Pat. It is well-known that the risk of developing HAPE increases if the rate of ascent is fast resulting in an impaired acclimatization process [2-Pat should have been better and more protective with regard to the risk of HAPE. In addition, AP-Pat had to carry heavy loads as at Denali a lot of equipment including fuel and nutrition for the whole expedition must be carried by the mountaineers themselves. AP-Pat had to carry more than 50 kg on a slide and in his rucksack. Heavy exercise is also a known risk factor for developing HAPE [Third, the altitude profiles of both patients are different. It took 9 days for O process . With reing HAPE ,22,23.Comparing the two patients and their overall risk profiles, including their fitness we can state that they are similar.Considerable difference exists regarding their age. There is a difference of 22 years that may result in different recovery abilities and even though younger persons are more prone to develop HAPE, there is evidence that systolic pulmonary arterial pressure increases more with age ,25,26. F2-Pat is in line with the general recommendations for the therapy of high-altitude illness and a descent of nearly 600 m of altitude is within these recommendations [2-Pat recovered initially but symptoms worsened again. Probably the 600 m of descent were not enough [2-levels of 50% and below, even if it is questionable if these values are valid due to the complexity of SpO2 measurement at high altitude [2 of 62%, which is substantially higher than that measured by the patient. This value is similar to that of AP-Pat (58–64%). The treatment with Dexamethasone and Nifedipine is effective for HAPE [2-curve . The striking difference was that their SpO2 decreased to initial levels after having finished Auto-PEEP making it likely that the high Auto-PEEP pressure probably leads to a structural improvement of oxygen transport into the bloodstream, most likely a reduction in the intra-alveolar fluids of the pulmonary edema and a persistent recruitment of atelectasis and thereby reduced hypoxic pulmonary vasoconstriction. We discussed this in more detail in our previous publication [There are some anecdotal reports of PLB being effective in the therapy of HAPE even at the same location at Denali ,34 but nlication . Other slication ,36. This2 [2 as well as heart rate increased under PEEP breathing. The authors conclude that HAPE patients benefit disproportionately well from improved oxygenation with PEEP breathing, but that this positive effect is offset in healthy subjects by the increased effort of breathing. Thus, healthy climbers are more likely to benefit from hyperventilation than from PEEP breathing [Although Auto-PEEP breathing is strenuous, AP-Pat obviously benefited from the improved oxygenation because his pulse decreased during Auto-PEEP breathing and remained lower than the baseline values see . This is2 . By contreathing .2 and breathing difficulties is recommended. The pulse rate displayed simultaneously by the pulse oximeter can be used as an indicator of possible overexertion and thus a limited benefit of Auto-PEEP therapy [Since Auto-PEEP breathing is easy to apply, based on our results it is recommended to have patients suffering from AMS or HAPE perform Auto-PEEP breathing every 2 h for 30 min as soon as severe AMS or HAPE is suspected. Accurate temporal monitoring of respiratory rhythm  is useful to avoid hyperventilation. Pulse-oximetric monitoring of these sick individuals as well as control of the therapy success on the basis of SpO therapy . If it iThe most important limitation of this study is the comparison between two individuals and the fact that the respective situations are similar but not identical. Therefore, no general conclusions can be drawn from this case report.2 can often be observed but none of them is 100% evidential for HAPE. Both patients showed these symptoms and the typical anamnesis, the altitude profile, and the course of the disease make it most likely that both suffered from HAPE. However, even if they suffered (only) from severe AMS, which is documented by their LLS-Score, the treatment (except Nifedipine in the O2-Pat) would have been identical.In remote areas the diagnosis of HAPE is based on the clinical finding with the cardinal symptom of a sudden significant loss in physical performance. In addition, dyspnea, a high LLS and low SpO2  or logistic reasons (availability of hyperbaric chambers and bottled oxygen) and therefore leads to considerable delays in the start of the therapy. Auto-PEEP ensures an immediate start of therapy with an immediate increase in SpO


Background: SARS-CoV-2 is a coronavirus that has rapidly spread across the world with a detrimental effect on the global population. Several reports have highlighted an increased mortality rate and a higher severity of COVID-19 infection in chronic kidney disease (CKD) individuals. Upon the development of various SARS-CoV-2 vaccines, mRNA vaccines including BNT162b2 and mRNA-1273 were deemed safe, with a high efficacy in preventing COVID-19 in the general population. This review investigates whether  SARS-CoV-2 mRNA vaccines are as effective in triggering an immune response in Dialysis Patients (DPs) and Kidney Transplant Recipients (KTRs) and if a third dose is required in this  population.Methods: A systematic search employing the PRISMA criteria was conducted in several major databases, with the data being extracted from publications for the period January 2021 to May 2022 .Results: 80 studies were included in this analysis with a total cohort number of 15,059 participants. Overall, 85.29%  and 41.06%  of DPs and KTRs included in this review showed positive seroconversion after two doses of either mRNA vaccine, respectively. A total 76%  of the cohort given a third dose of an mRNA vaccine demonstrated positive seroconversion, with 61.86%  of the cohort that was assessed for a cellular response displaying a positive response.Conclusions: This data emphasises a reduced incidence of a positive immune response in DPs and KTRs compared to healthy controls, albeit a better response in DPs than when compared to KTRs alone was observed. A third dose  appears to increase the occurrence of an immune response in the overall DP/KTR cohort. WHO Coronavirus (COVID-19) Dashboard).In December 2019, an outbreak of atypical respiratory disease was reported in Wuhan, Hubei Province, China . ShortlySARS-CoV-2 is a β-coronavirus; coronaviruses consist of enveloped, positive-sense, single-stranded RNA, and non-structural, structural and accessory proteins . This prin vivo study highlighted that chronic pharmacologic inhibition of ACE2 could worsen glomerular injury  2.52), dialysis patients (aHR 3.69) and transplant patients (aHR 3.53), constituting some of the top risk categories in this study  Dashboard). Despite several studies investigating the safety and efficacy of vaccinations, there is no conclusive evidence as to whether the SARS-CoV-2 vaccines provide the same level of immunity in CKD patients. Overall, it has been deemed safe to use the mRNA vaccines BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) in immunocompromised patients, including CKD patients  guidelines, published in 2020. Seedelines  for the 2 test using RevMan (version 5.4) by two independent reviewers .Studies were grouped together as described above with two independent reviewers assessing whether they met the inclusion criteria . Studies were assessed and data was collected via Microsoft Excel 2016 by the same reviewers. The information extracted included sample size and sample groups, treatment regimen, number of doses, whether there is a history of COVID-19 and how long after vaccination any assessments took place. The effects and responses to the vaccination including humoral and cellular immunity responses after the second and third doses were also reviewed (the PRISMA flow diagram is given asRevMan (version 5.4). Odds ratios (OR) and 95% confidence intervals (CI) were calculated to evaluate the seroconversion and cellular response in DPs and KTRs after two and/or three doses of the BNT162b2 and mRNA-1273 vaccinations. The seroconversion is a measure of antibody response to SARS-CoV-2-specific antibodies including IgG, IgA, and IgM antibodies. The cellular response incorporated the number of patients that developed a T-cell response across the studies included in this analysis, including CD4+ and CD8+ T cells. The population was divided into subgroups i.e., dialysis patients and transplant recipients; two dose and three dose groups; seroconversion and cellular response group. A pooled 95% CI not including 1 for the OR was considered as statistically significant. Risk of bias/heterogeneity was assessed using RevMan (version 5.4) with the Cochrane’s Q test and I2 test and presented in funnel and forest plots. If a P value of < 0.1 and/or I2 was > 50%, heterogeneity was determined as significant.Two independent reviewers assessed studies for eligibility for each synthesis using Microsoft Excel 2016. Studies were grouped in accordance with data availability before being included in the synthesis, with those with missing data being excluded from analysis. Funnel plots, forest plots and statistical analysis were performed usingA total of 80 studies  were included in this work, with an overall cohort number of 15,059 participants. Of these, 11,509 participants were recipients of the BNT162b2 vaccine, with 4,263 being KTRs and 7,246 participants being DPs. Separately, a total of 3,550 participants were vaccinated with the mRNA-1273 vaccine. Of these, 826 participants were KTR, and 2,724 participants were DP. Finally, 292 participants had been given the ChAdOx-1 nCoV-19 vaccination, with 11 participants being KTR and 281 participants being DP. Due to the lack of studies for ChAdOx-1 nCoV-19 vaccination in this population, this data was excluded from analysis and one study was ruled out  , neverthIt is important to note that it is not possible to deem immunity levels or identify protective thresholds from antibody testing alone. Further to this, we can confirm there is no specific antibody titre for protection against severe COVID-19. Upon performing an extensive review of the studies in this meta-analysis, antibody titres and thresholds varied significantly between studies due to the use of different immunoassays.It has also been reported that IgAN or other kidney diseases could be triggered as a response to the COVID-19 mRNA vaccinations in certain populations . IgA nepThis study set out to establish the efficacy of the COVID-19 mRNA vaccines in DPs and KTRs. The seroconversion and cellular response were assessed in 15,072 participants after either two or three doses of an mRNA vaccine. The vaccines that were included in this analysis include BNT162b2 and mRNA-1273, with ChAdOx-1 nCoV-19 being excluded due to lack of available data.Of the 9,913 DPs that were assessed for seroconversion after two doses of an mRNA vaccination, 85.29% showed positive seroconversion, with 14.71% of participants displaying no response. Comparatively, in the 4,822 KTRs that were assessed, the response was deemed ineffective when compared to DPs. 41.06% of KTRs displayed positive seroconversion, with the remaining 58.94% of participants showing no significant response to the vaccinations after two doses.We also assessed the seroconversion response after three doses of an mRNA vaccine. Of 1,513 participants, 76% of patients showed positive seroconversion with the remaining 24% of patients showing no response. It was not confirmed in the studies whether seroconversion after a third dose was compared to unvaccinated individuals or individuals who had received a second dose. Thus, in our study, we were unable to conclude whether there was an increase in antibody production after the third dose in those who did not show positive seroconversion after the second dose. When assessing cellular response in DPs and KTRs after two doses, 978 participants were included in the analysis, with a positive cellular response being observed in 61.86% of patients. The remaining 38.14% of participants showed no or little cellular response.ClinicalTrials.gov number: NCT04470427;ClinicalTrials.gov number: NCT04368728) -specific antibodies  individuals’.O/63PJX .Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).Data are available under the terms of the Authors have responded adequately in their revised manuscript.Are the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?YesAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:NephrologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I thank the authors for their time to address my comments appropriately. I also understand their response on the limited number of studies available to enable them to perform statistical analysis for the detection of positive seroconversion for DPs and KTRs after a third dose of an mRNA vaccine separately.  The paper is well-presented with sound evidence from their results to support their paper.Are the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?YesAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:Cancer genetics, immunological studiesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors present a thorough systematic review on the effectiveness of mRNA vaccines for COVID-19 infection in DPs and KTRs patients. Overall, the review is well-presented with a detailed description of methods used for the analysis and good presentation of Figures. The review is highly cited with up-to-date references. The statistics used for the analysis are appropriate for systematic reviews and with sufficient details. The results are presented nicely albeit some additional information would be beneficial for this review. The authors investigated the efficacy of the 2 COVID-19 mRNA vaccines in DPs and KTRs by analysis of the seroconversion and cellular response in patients after either 2 or 3 doses of an mRNA vaccine. The authors found that a third dose of mRNA vaccine increased seroconversion by 76% in both DPs and KTRs together compared to an average of 63.18% after two doses. It would be helpful if the authors could do a separate analysis per set of patients  after administration of a third dose of mRNA vaccine rather than grouping them together and then compared each set with the ones after a second vaccine dose. I believe the results of this analysis would help to understand better the requirement or not of a third dose of vaccine in these groups of patients. The discussion and conclusions support the results of this review.Are the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?YesAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:Cancer genetics, immunological studiesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We thank the reviewer for their comments. We have taken their points on board and edits to the initial manuscript can be seen as tracked changes. Please note that unfortunately, there are a limited number of studies available  and thus, to perform statistical analysis, the percentage of patients displaying positive seroconversion for both DPs and KTRs after a third dose were grouped together. Despite this, our data shows that separately, 89.13% and 61.29% of DPs and KTRs displayed positive seroconversion after a third dose of an mRNA vaccine, respectively. In this systematic review the authors assessed the response rate and overall effectiveness of vaccination against Covid-19 with mRNA vaccines in patients on dialysis or patients after kidney transplantation.nd dose, had a substantial increase in antibody production after the 3rd dose. The work overall is clearly presented and the most contemporary literature is cited. The design meets the standards of systematic reviews and analysis details are provided clearly in methods. The statistical analysis is appropriately performed, nevertheless some results need clarification. Firstly, it is stated that 17 studies with 1,513 participants were included to determine the effectiveness of 3 doses of the mRNA vaccines (either BNT162b2 or mRNA-1273) in both DPs and KTRs. In my opinion the seroconversion results after a third mRNA vaccine dose should be presented separately for DPs and KTRs. Moreover, it should be noted whether those patients, either DPs or KTRs, who did not show seroconversion with the 2 In general, although the authors describe in detail the seroconversion ratio of DPs and KTRs after Covid-19 vaccination with an mRNA vaccine, they do not discuss about the actual protective threshold of antibodies that is reached in these groups of patients. Thus, I would propose to mention apart from seroconversion ratio, the ratio of patients that have a titer that is actually protective against severe Covid-19 infection as well. Finally, the source data are reported to be available and the final conclusions of the study are sound and are supported from the original results.Are the rationale for, and objectives of, the Systematic Review clearly stated?YesIs the statistical analysis and its interpretation appropriate?YesAre sufficient details of the methods and analysis provided to allow replication by others?YesAre the conclusions drawn adequately supported by the results presented in the review?YesReviewer Expertise:NephrologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. We thank the reviewer for their comments. In response to them, we have submitted a revised version of the article, with any amendments shown as tracked changes. Please note: 1) There were a limited number of studies available and thus, to perform statistical analysis, the percentage of patients displaying positive seroconversion for both DPs and KTRs after a third dose were grouped together. Despite this, our data shows that separately, 89.13% and 61.29% of DPs and KTRs displayed positive seroconversion after a third dose of an mRNA vaccine, respectively. 2) It was not confirmed in the studies whether antibody production/seroconversion after a third dose was compared to unvaccinated individuals or individuals who had received a second dose. Thus, in this study, we were unable to conclude whether there was an increase in antibody production after the third dose in those who did not show positive seroconversion after the second dose. 3)Not all studies published the antibody titre levels, therefore it was not possible to conclude what the exact protective threshold was in DPs and KTRs vaccinated against severe COVID-19. Nonetheless, we have provided in Table 1 the threshold over which an individual's test is scored as seroconverted. It is important to note that it is not possible to deem immunity levels or identify protective thresholds from antibody testing alone. Further to this, we can confirm there is no specific antibody titre for protection against severe COVID-19. Accordingly, the U.S. Food and Drug Administration (FDA) states that “antibody testing is not currently recommended to assess immunity after COVID-19 vaccination” because a result from currently authorized SARS-CoV-2 antibody tests is not an indication of a specific level of immunity or protection from SARS-CoV-2 infection after the person has received a COVID-19 vaccination.