This study aims to design a set of the visual artificial intelligence system based on medical information mining for hospital emergency care management. A visual artificial intelligence emergency first aid nursing management system is designed by analyzing the needs of the emergency first aid nursing management system. The results show that system personnel allocation, comparative management, record management, query management analysis, basic setup analysis, nursing management basis, and nonfunctional requirements all need to be optimized for the emergency first aid management system. In this study, the comparative management module, log management module, and the query management module are designed, and the emergency first aid management system of different APP terminal functions in different modules is described in detail. The nursing document query business is tested, and the corresponding time of query of nursing assessment sheet, nurse shift record, nurse record, and physical sign observation sheet is 375.50 ms, 351.48 ms, 336.36 ms, and 245.57 ms, respectively. It shows that the visual artificial intelligence emergency nursing management system based on medical information mining can provide convenience for clinical work to a large extent and has potential application value in hospital emergency nursing work. Medical information (MI) is a combination of medicine and computer science that aimed to facilitate the acquisition, storage, retrieval, and utilization of health and biomedical information . As an eThe medical treatment of the emergency department is the key of the key in the hospital. The emergency department mainly serves to patients with acute and severe diseases, and the diagnosis and treatment level of this department is directly related to the quality of life of patients in the later period and the degree of satisfaction of patients with the hospital. However, the large flow of emergency patients and the high management cost and the difficulty of emergency care require the rational allocation of resources of the emergency department. Buscema M used automatic compressed mapping (Auto-CM) to find out the subtle associations between various variables . This meThen, a visual artificial intelligence nursing system is developed. It has functions of comparative management, query management, record management, nursing document management, basic settings, and basic nursing management, which are mainly realized through an APP end , 13. TheAbove all, this study focuses on the design of the medical information visualization emergency first aid system. With first aid measures in hospital emergency departments as the breakthrough point, the work procedures of the nurse, head nurse, and nurse directors are analyzed. According to specific requirements, the APP end of the emergency first aid management system is designed, and the submodules of the visual artificial intelligence emergency first aid management system are described in detail. Finally, the system is tested. The study provides a more practical management system for hospital emergency nursing and convenience for clinical work and improves the work efficiency of medical workers.P(X) represents the probability that transaction X appears in transaction set D.The probability-based interest model algorithm is as follows:P(Y) represents the probability that transaction Y appears in food set D.P(Y/X) represents the probability of simultaneous occurrence of transactions X and Y in transaction set D.X⟶Y)), Supp(Y) is an evaluation standard, which is used to ensure that Int(X⟶Y)<1 is established.The interest model based on differences is as follows.The interest model based on correlation is as follows.X⟶Y) illustrates the relationship between the preceding and subsequent items X and Y in the association rule (X⟶Y).The degree of interest based on relevance Int(The information-based interest model is as follows.X⟶Y), the amount of information, and the similarity of the probability distributions of the preceding item X and the subsequent item Y, and the probability of the preceding item is used as a measure of the conciseness of the preceding item of the association rule.The interest degree model based on the amount of information comprehensively considers the conciseness of the association rule (The influence-based interest model is as follows.Through the comparison and analysis of various interest degree models, as well as the mining of medical information, the improved algorithm is as follows.There are many problems in the hospital emergency nursing system. Nurses are not efficient in the processes of infusion and dispensing, and it is difficult for nurses to quickly query the patient's basic information, doctor's orders, and other medical information in their daily work. There lacks an effective way to manage nursing document information, and basic nursing management also has various shortcomings. Therefore, a visual nursing system for the emergency department based on artificial intelligence is developed in the form of an APP. The system requirements are shown in In the APP end of the medical information-based visual artificial intelligence emergency nursing system, many new services have been added to meet the various needs of nurses, such as management of prescribing, infusions, and doctor orders; in record management module, nurses can view and deal with physical signs and documents; the query management module includes patient information, medical order information, and treatment of patients; and statistical performance, monitoring of nursing links, and application settings are included in the basic settings module. The business flowchart of the visual artificial intelligence emergency nursing management system based on medical information is shown in The visual artificial intelligence emergency nursing management system includes nurses, head nurses, and nursing department. The system APP enables the operator to carry out a series of operations, such as comparison, query, and record management. The roles involved in the of emergency nursing management system are shown in In the functional section of the APP, nurses in the emergency department can enter the comparison management interface through the APP to process fluid dispensing and infusion-related services to achieve rapid emergency nursing. The comparison management module is shown in The record management module reflects a series of nursing-related requirements, and it can collect nursing signs, record nursing sign information, generate nursing documents, process document information, and manage nursing measures. The record management module is shown in The query business concerns patient information, doctor's order information, and diagnosis and treatment expenses. The basic information above can be obtained through query module. The query management module is shown in The basic setting module reflects the relevant needs of statistical nursing work, monitors nursing links, and obtains notifications. In addition, users can also set according to their own habits, such as signature keyboard, automatic data reference, and writing input. The basic setting analysis module is shown in Basic nursing management involves basic management businesses, including nursing document management, human resource management, nursing quality management, adverse event management, and nursing statistical analysis. The basic nursing management module is shown in The visual artificial intelligence emergency nursing management system based on medical information mining puts forward higher requirements for adaptability and safety. Adaptability: the mobile service components of the system should have large memory storage and can adapt to the complex and changeable use environment. Safety: due to irresistible factors such as slow network communication speed, mobile users will encounter poor communication or slow speed when logging in; in order to ensure the security of user logging in, the system should identify users who are offline multiple times in per unit time. Ease of use: the hospital has a large number of object-oriented nursing tasks of complex types. Therefore, the system should be able to provide a personalized setting system, so that nurses can flexibly set up according to their own operating habits.Comparison management window and comparison management business are the basic categories of comparison management modules. The business category includes liquid dispensing management business, infusion management business, specimen collection business, medical order execution business, and liquid preparation entity, infusion management entity, specimen collection entity, and medical order execution entity. The comparison management design is given in The nurse sends an application for processing the preparation business. The system accepts the processing application, sequentially calls the comparison management window and business categories, and returns to the liquid preparation business. The processing of the liquid to be dispensed is shown in The functional category of the record management module includes record management window, record management business, nursing physical sign management business, nursing document management business, patrol management business, and nursing measure management business. The record management design is given in The query management module includes query management window, query management business, patient information management business, medical order management business, diagnosis and treatment expense management business, and special patient management business. The entity category includes patient information entity type, diagnosis and treatment expense entity type, inspection and examination entity type, special patient entity type, and doctor order entity type. The query management design is given in The basic setting module includes basic setting window, basic setting business, performance statistics business, nursing link monitoring business, information push business, application setting business, nursing link entity, information entity, and performance statistics entity. The design of basic setting is given in The monitoring business in the nursing link is shown in System test environment: Windows Server 2012 operating system, 1 TB hard disk, 4G processing memory; the Web server runs Windows 7 and has 500 GG hard disks and 2G processing memory; the browser kernel is IE and Safari; the APP terminal is the Android 5.0 operating system and can connect to the WiFi network. On the basis of formulating test cases, the test process is standardized, striving for the accuracy of system test results. In the study, the requirements on the visual artificial intelligence emergency nursing system are analyzed. Then, the role of the system is analyzed, each functional module is established, and the functional requirements are analyzed. As for the system design, the overall functional architecture of the system is designed.n + 1 item through n items. This method is mainly divided into two steps, which are called connection steps and pruning steps [The development of the APP adopts the MVC design pattern, Spring MVC framework, and other technologies. The MVC design pattern stipulates the responsibilities of the components and reduces the coupling between the components . The useng steps , 23.In the study, the steps of emergency nursing and the needs in the process of emergency nursing are analyzed. It is found that there is a lack of effective means for nurses to compare the information of infusion and liquid dispensing. It is difficult for nurses in daily work to quickly query the patient basic information, such as orders, and the head nurse and nursing managers on lack of systematic methods of management for nursing documents information. Based on these problems, the visualization APP emergency care management system is designed and tested. The results show that the visual artificial intelligence first aid nursing management system APP designed in this study has certain operability, but there are still some limitations in this study. In this study, only APP window is designed, without Web window application. In subsequent studies, Web window design should be added to meet the needs of more functions. In conclusion, this study promotes the application of the artificial intelligence-based visual nursing system for hospital emergency care in clinical work, which provides convenience for clinical work and improves the work efficiency of medical workers.


Dichlorodiphenyldichloroethylene (DDE), the primary persistent metabolite of dichlorodiphenyltrichloroethane (DDT), has toxic effects on cells, but its dose-dependent impact on mitochondrial proteins involved in mitochondrial fusion and fission processes associated with cell viability impairment has not yet been analysed. Mitochondrial fusion and fission processes are critical to maintaining the mitochondrial network and allowing the cell to respond to external stressors such as environmental pollutants. Fusion processes are associated with optimizing mitochondrial function, whereas fission processes are associated with removing damaged mitochondria. We assessed the effects of different DDE doses, ranging between 0.5 and 100 µM, on cell viability and mitochondrial fusion/fission proteins in an in vitro hepatic cell model ; the DDE induced a decrease in cell viability in a dose-dependent manner, and its effect was enhanced in conditions of coincubation with dietary fatty acids. Fusion protein markers exhibited an inverted U-shape dose-response curve, showing the highest content in the 2.5–25 μM DDE dose range. The fission protein marker was found to increase significantly, leading to an increased fission/fusion ratio with high DDE doses. The low DDE doses elicited cell adaption by stimulating mitochondrial dynamics machinery, whereas high DDE doses induced cell viability loss associated with mitochondrial dynamics to shift toward fission. Present results are helpful to clarify the mechanisms underlying the cell fate towards survival or death in response to increasing doses of environmental pollutants. Mitochondria are vital organelles involved in ATP production and in regulating apoptotic and necrotic cell death ,2,3. SuiMitochondria can use several mechanisms to maintain their physiological homeostasis ,3,45. MiThe main aim of the present study was to analyze the dose-dependent response to DDE exposition in terms of cell viability, mitochondrial protein fusion, and fission content in the human hepatocarcinoma cell line (HepG2). Scientists consider this cellular model suitable for in vitro studies on hepatic toxicology ,60. Firsv/v) fetal bovine serum, 1% (v/v) non-essential amino acids, 0.2 mM L-glutamine, 50 U/mL penicillin and 50 µg/mL streptomycin . Cells were maintained at 37 °C in a 5% CO2, 95% air-humidified atmosphere and passaged twice a week. To perform treatments with DDE, cells were seeded at the density of 4.0 × 104/cm2 and cultured for 24 h before stimulation. A stock solution of DDE was prepared by dissolving the pesticide in dimethyl-sulfoxide (DMSO). In each treatment, the final concentration of DMSO in the culture medium was 0.1%, which represents a non-toxic concentration for hepatocytes [A HepG2 cell line was cultured in Minimum Essential Medium supplemented with 10%  was only treated with DMSO (0.1%). In the coincubation experiments with fatty acids, cells were co-treated for 24 h with the different doses of DDE and 250 µM of palmitate, oleate or eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) 1:1 mixture.v/v, respectively. The resulting 8 mM fatty acid stock solutions were filtered using a syringe equipped to filter (0.2 μm), aliquoted and stored at −20 °C. Concerning EPA and DHA , 40 mM stock solutions were prepared by dissolving fatty acids in methanol as indicated by the manufacturer and mixed 1:1 v/v to obtain a stock solution EPA + DHA. Then, fatty acids were co-incubated with BSA in a culture medium. The final fatty acids concentration used in the study (250 µM) was freshly prepared in a cell culture medium at the time of the treatments.Oleate and Palmitate (Merck) 40 mM stock solutions were prepared by dissolving fatty acids in NaOH 0.1 M at 70 °C for 15 min under stirring. A 10% BSA fatty acid-free  solution was dissolved at 55° in NaCl 0.9% and mixed to fatty acid solutions 4:1 The cytotoxicity was assessed by a 3--2,5-diphenylte-trazolium (MTT) assay. This technique is based on the enzymatic conversion of MTT in mitochondria . To asseWestern blot analysis was performed as previously described . The folOil red stain quantitative (spectrophotometric) and qualitative (microscopic) analyses were performed using a classic protocol to detect lipid droplets in cells . Brieflyn = 3) or interquartile of range (n > 3). Each experiment was performed in biological triplicate (n = 3) or more (n > 3). Technical replicates (1 to 3 replicates) of the same biological samples were also performed. One-way ANOVA test followed by Bonferroni’s post hoc test was used to test significant differences . Differences were considered statistically significant with a p-value < 0.05.All data were expressed as median with range , were observed with high DDE doses  A. Cell vQualitative and quantitative tests were performed using the oil-Red O method to detect total intracellular lipid accumulation in cells treated with DDE alone or associated with fatty acids. In a recent work, we analysed the time- and dose-dependent effect of palmitate or oleate on lipid accumulation and cell viability in HepG2 cells . We showTo evaluate the dose-dependent effect of DDE on mitochondrial dynamic machinery, western blot analysis was performed to measure cellular contents of proteins involved in mitochondrial fusion and fission processes, namely Mfn2 and Opa1, as a marker of outer and inner mitochondrial membrane, respectively, and DRP1 as a fission marker. As for fission marker DRP1, the relative protein content described a progressive-increase pattern in response to DDE concentrations up to the dose of 50 μM, with a significant increase (+45%) in the 5–50 μM range compared to control cells. On the other hand, the increase in DRP1 content was about +18% in cells treated with 100 μM of DDE . TherefoTo study the effects of DDE on the mitochondrial antioxidant system, we analyzed the levels of mitochondrial enzyme SOD2 on total cell lysates. Results showed a progressive dose-dependent reduction in SOD2 protein content with a significant decrease (−25%) with the highest DDE dose (100 µM), compared to DMSO treated cells . AnalyseAdverse effects of DDE, including cellular stress generation ,72, apopOur study confirmed that DDE reduced cell viability in HepG2 cells in line with literature data in other cellular models . Low DDEIt should also be noted that, in the presence of fatty acids, xenobiotics toxicity could be associated with lipo-toxicity with an additive adverse effect on cell survival . Lipo-toNoteworthy, the coincubation of cells with fatty acids and increasing doses of DDE induced a progressive decrease in lipid accumulation compared to cells treated only with fatty acids. The reduction degree was similar for all fatty acids. A 20% decrease with low DDE doses and a 50% decrease with the highest DDE dose could be observed. Further experiments are needed to evaluate whether lower doses of fatty acids could induce a similar impact. However, present finding is in line with our previous results in an animal model of hepatic steatosis, where we showed that simultaneous treatment with DDE and high-fat diet elicited a significant reduction in hepatic lipid content associated with increased beta-oxidation rate and detoxification, compared to high-fat diet treatment in the absence of DDE . It coulPresent findings in cells cotreated with increasing DDE doses and different types of dietary fatty acids suggested that it could be of interest to further investigate the interaction between DDE, as a lipophilic pollutant, and dietary fatty acids, given that they could be the main route of entry of DDE into the body. Further studies could shed light on the complex interactions among environmental pollutants and nutrients and suggest whether different dietary nutrients (such as saturated or unsaturated fatty acids) could potentiate the adverse effects of environmental pollutants or could be protective towards them.A further goal of the present work was to analyze the dose-dependent response of mitochondrial markers to increasing doses of DDE in HepG2 cells. Mitochondria are cellular targets susceptible to chemicals, such as DDE . Given tIn the present study, we also analysed SOD2 content as a marker of mitochondrial function and antioxidant system and GRP75 as a marker of mitochondria/endoplasmic reticulum interaction.SOD2 represents one of the first mitochondrial-associated antioxidant defenses used by cells to quench superoxide anion produced by mitochondria . It has 2O2 and CCl4-induced oxidative damages, ameliorating ATP production and preserving cell viability. In our study, GRP75 protein levels showed a similar inverted U-shape dose-response curve as observed for Mfn2 and Opa1 levels, with the highest significant increase (about +30%) with 5 and 10 µM DDE vs DMSO-treated cells. This result suggested that an additional adaptive phenomenon involving ER-mitochondrial contact point formation/communication could be associated with mitochondrial increases in dynamic protein in response to low DDE doses. This mechanism could be used by cells to regulate cell survival and mitochondrial function in the presence of low DDE doses. On the one hand, the increase in mitochondrial dynamic machinery and GRP75 chaperone in response to low DDE doses could play a role in the mitochondrial network and mitochondria/endoplasmic reticulum interaction maintenance to maintain cell viability. On the other hand, in cells treated with high DDE doses, decreased levels in mitochondrial fusion proteins, GRP75, and SOD2 content associated with the increase in DRP1 levels suggested mitochondrial fragmentation and dysfunction in response to high DDE doses, which could trigger the observed reduction in cell viability.We also analysed the content of intracellular chaperone GRP75, a protein involved in mitochondria/endoplasmic reticulum interaction . This chThe present study added new scientific notions regarding adaptive/toxic phenomena induced by increasing dose of DDE in hepatic cell culture. Cellular adaptation to low doses of the environmental pollutant seemed to include maintaining mitochondrial network and mitochondria-endoplasmic reticulum interaction, which could play a pro-survival role. On the other hand, high doses of DDE induced a toxic effect that reduced cell viability by impairing mitochondrial fusion/fission balance and antioxidant defense. Present results are useful to clarify the mechanisms underlying the cell fate towards survival or death in response to increasing doses of environmental pollutants. Further studies are needed to shed further light on the metabolic events associated with our findings and highlight the different roles of cellular organelles and their adaptation to environmental pollutants.


Lockdown measures due to the COVID-19 pandemic have led to lifestyle changes, which in turn may have an impact on the course of headache disorders. We aimed to assess changes in primary headache characteristics and lifestyle factors during the COVID-19 lockdown in Germany using digital documentation in the mobile application (app) M-sense.We analyzed data of smartphone users, who entered daily data in the app in the 28-day period before lockdown (baseline) and in the first 28 days of lockdown (observation period). This analysis included the change of monthly headache days (MHD) in the observation period compared to baseline. We also assessed changes in monthly migraine days (MMD), the use of acute medication, and pain intensity. In addition, we looked into the changes in sleep duration, sleep quality, energy level, mood, stress, and activity level. Outcomes were compared using paired t-tests.p > 0.999). MMD, headache and migraine intensity neither showed any significant changes. Days with acute medication use were reduced from 4.50 ± 3.88 in the baseline to 4.27 ± 3.81 in the observation period (p < 0.001). The app users reported reduced stress levels, longer sleep duration, reduced activity levels, along with a better mood, and an improved energy level during the first lockdown month (p ≤ 0.001).The analysis included data from 2325 app users. They reported 7.01 ± SD 5.64 MHD during baseline and 6.89 ± 5.47 MHD during lockdown without significant changes (p = 0.688 in the ANOVA). Migraine frequency, headache and migraine intensity, and acute medication use were also not different during the entire observation period.In an extension analysis of users who continued to use M-sense every day for 3 months after initiation of lockdown, we compared the baseline and the subsequent months using repeated-measures ANOVA. In these 539 users, headache frequency did not change significantly neither (6.11 ± 5.10 MHD before lockdown vs. 6.07 ± 5.17 MHD in the third lockdown month, Despite slight changes in factors that contribute to the generation of headache, COVID-19-related lockdown measures did not seem to be associated with primary headache frequency and intensity over the course of 3 months. Patients with primary headache disorders perceive external factors such as stress, sleep, and changes of everyday routine as common trigger of their headache attacks . EspeciaOn March 11, 2020, the World Health Organization (WHO) declared COVID-19 a global pandemic, and on March 22, the German government imposed strict social distancing measures on public life . The resStudies on mental health during the COVID-19 pandemic suggest a high psychosocial impact of such measures with a negative effect on quality of life, beginning in the first lockdown weeks , 7. PatiA digital headache diary is a reliable tool to track headache attacks and potential triggers. Compared to paper-and-pencil diaries, digital documentation is associated with a higher compliance and better data quality . We aimeM-sense is a commercial mobile headache application (app) available via app store for Android and iOS in Germany, Austria, and Switzerland. At the time of the data analysis, M-sense was marketed as a migraine app in a free-of-charge “Basic” version with approximately 85,000 registered users and an additional “Active” version for purchase.Functions of M-sense comprise the documentation of headache attacks in an interactive electronic diary, along with a daily entry of predefined factors, which may influence the course of headaches. Headache features are entered according to a standardized scheme, which includes start and end of the headache, pain intensity, headache localization, headache character, presence of aura, accompanying symptoms, and acute medication use. A validated algorithm classifies single headache episodes as migraine, tension-type headache (TTH) or non-migraine / non-TTH headache . A detaiThe app developers provided us with aggregated data from all M-sense users, who entered headache-related data in this app in the four-week periods before March 22 (baseline) and after March 22 (first month of lockdown) every day. Primary outcome of this retrospective analysis was the change in monthly headache days (MHD) between baseline and the first lockdown month. Our analysis also included changes in monthly migraine days (MMD), monthly days with use of acute headache medication (AMD), mean headache and migraine intensity . A headache day was defined as any calendar day with a registered headache attack, regardless of the individual headache features. A migraine day was defined as each calendar day at which the user experienced a headache attack classified as migraine by M-sense. Acute headache medication consisted of triptans and non-steroidal anti-inflammatory drug (NSAID). We also assessed changes of several predefined factors including sleep duration , self-assessed sleep quality (on a NRS 0–10), energy level (0–10), mood (0–4), stress (0–10), and level of activity (0–10).We then performed an extension analysis of M-sense users who continued to use the app for at least 3 months after lockdown begin. In these users, we analyzed changes in the above mentioned parameters between the baseline phase, the first, second and third lockdown month.p-value ≤0.05 was considered statistically significant. P-values were adjusted for multiple comparisons using the Bonferroni procedure. We calculated effect sizes using Cohen’s d.Statistical analysis was performed using R, version 3.6.2. For data protection, we developed the statistical code on a dummy dataset. The team of Newsenselab ran the finalized code on the real dataset. By doing so, the research team did not access personal data but received only aggregated results for the predefined endpoints. Demographics and monthly headache characteristics were summarized with descriptive statistics, using frequencies and percentages or means ± standard deviation. We compared outcomes between baseline and first month of lockdown using paired, two-tailed t-tests. In the extension analysis, outcomes were compared between all months using repeated-measurement analysis of variance (ANOVA). Post hoc pairwise comparisons were performed only if the ANOVA revealed significant results. A n = 2325 users (mean age 38.69 ± SD 11.09 years) entered data in M-sense every day. The sample consisted of n = 1699 females (73.1%), n = 271 males (11.6%), and n = 2 diverse sex (0.1%), while n = 353 (15.2%) did not provide information about sex.During the primary observation period, p = 0.999). MMD, headache and migraine intensity did also not change significantly over time .Headache frequency did not show any statistical difference, with 7.01 ± 5.64 MHD during baseline and 6.89 ± 5.47 MHD in the first lockdown month . MMD, AMD, migraine or pain intensity did also not show any significant change. Stress levels remained numerically below baseline in every month, but without statistical significance. Table They reported 6.11 ± 5.10 MHD during baseline and 6.07 ± 5.17 MHD in the third lockdown month .Previous research on sleep patterns during the COVID-19 pandemic is scarce: In an Indian cohort, 325 students reported an increased sleep duration during lockdown, while 203 office workers indicated reduced sleep hours . Our finA major concern for patients with primary headache disorders in the COVID-19 pandemic may be the worsening of their condition due to reduced access to medical care . HoweverThe reduction of physical activity due to home confinement in the pandemic has already been reported in large survey-based cohort studies , 19. We This study is an example on how headache data collected by an app can be used for research purposes in specific situations like the COVID-19 pandemic. Digital data collection offers the possibility of a large sample size. Nevertheless, several methodological limitations should be considered when interpreting our results. The M-sense algorithm classifies the single headache attacks, but cannot provide a final headache diagnosis. Due to the analysis of aggregated anonymous data patients could not be assessed for individually diagnosis. Previous assessments have shown that more than 70% of users have headaches compatible with the diagnosis of migraine . TherefoIn conclusion, we did not observe a change in headache frequency and intensity in patients suffering from migraine and/or TTH during the first 3 months of the lockdown in Germany. A small stress reduction in the first lockdown month was not associated with an improvement in these primary headache disorders.


FH) cells. Postpartum memory B cells of MS patients expressed higher levels of CCR6 and preferentially developed into plasma cells under TFH-like in vitro conditions. These findings imply that memory B- cell differentiation contributes to postpartum relapse risk in MS.In MS, pathogenic memory B cells infiltrate the brain and develop into antibody-secreting cells. Chemokine receptors not only define their brain-infiltrating capacity, but also assist in their maturation in germinal centers. How this corresponds to pregnancy, as a naturally occurring modifier of MS, is underexplored. Here, we aimed to study the impact of pregnancy on both ex vivo and in vitro B-cell differentiation in MS. The composition and outgrowth of peripheral B cells were compared between 19 MS pregnant patients and 12 healthy controls during the third trimester of pregnancy (low relapse risk) and postpartum (high relapse risk). Transitional, and not naive mature, B-cell frequencies were found to drop in the third trimester, which was most prominent in patients who experienced a pre-pregnancy relapse. Early after delivery, these frequencies raised again, while memory B -cell frequencies modestly declined. CXCR4 was downregulated and CXCR5, CXCR3 and CCR6 were upregulated on postpartum memory B cells, implying enhanced recruitment into germinal center light zones for interaction with T follicular helper (T In the relapsing phase of MS, naive B cells escape peripheral tolerance checkpoints2 and develop into memory populations that activate CNS-infiltrating, IFN-γ-producing CD4+ T cells3. This is further supported by the reduction in T-cell activation in MS patients after receiving anti-CD20 therapy4. Normally, memory B cells interact with T cells within germinal centers (GCs) of peripheral secondary lymphoid organs. Our group recently demonstrated that B cells utilize IFN-γ to drive their CXCR3-expression, boosting their ability to recruit to MS brain tissue as well5. Hence, in MS, CNS-infiltrating memory B cells are probably reactivated by T cells in perivascular spaces6 and ectopic lymphoid structures in the cerebral meninges7 to further mature into plasmablasts/plasma cells and contribute to focal inflammation6.MS is a chronic inflammatory and demyelinating disease of the CNS, for which a key role for B cells in the pathogenesis has been shown in recent years9. This is further controlled by the expression of other chemokine receptors, such as CXCR4, CXCR5 and CCR6. CXCR4 and CXCR5 orchestrate the GC response by guiding B cells into dark and light zones respectively. Centroblasts undergo several rounds of proliferation and somatic hypermutation in the dark zone, while centrocytes interact with follicular dendritic cells and T follicular helper (TFH) cells to undergo antigen-specific selection in the light zone10. CCR6 has been reported to promote their development and antigen responsiveness in the light zone12. Therefore, specific aberrancies in chemokine receptor profiles may affect the outgrowth of B cells into memory and plasmablasts/plasma cells13. Currently, it remains to be determined whether this is related to relapse risk in MS.IFN-γ-induced CXCR3 expression on B cells is not only associated with increased homing to inflamed tissue and the formation of ectopic lymphoid structures, but also with aberrant plasma cell differentiation within GCs14. Although current diagnostic and treatment strategies probably contribute to an attenuation of this fluctuation15, these specific phases of pregnancy can still be considered periods of relatively low and high relapse risk in MS. Pregnancy is known to cause a shift from TH1 to TH2 responses16 and promote the expansion of circulating TFH cells17, therefore likely affecting the maturation of B cells in GCs.Notably, in pregnant MS patients, relapse risk is reduced by approximately 70% in the third trimester. This increases in the first three months after delivery, with almost 30% of patients having a postpartum relapseFH-like cell culture system to investigate how in vitro memory B-cell differentiation into plasmablasts or plasma cells differs between periods of relatively low (third trimester) and high (early postpartum) relapse risk.In this experimental study, our primary aim was to determine the effects of pregnancy as naturally occurring disease modifier on peripheral B-cell differentiation in MS. We assessed the frequencies and GC-related chemokine receptor profiles on ex vivo B-cell subsets in paired first trimester, third trimester and early postpartum blood samples of MS patients and healthy controls. In addition to this, we used a T18 and retrospectively validated to match the most recent McDonald 2017 criteria19. Patients did not use any immune modulatory medication before, during and early after pregnancy. Two patients gave birth via a cesarean section and all others had vaginal deliveries. No relapses were observed during pregnancy. During the early postpartum period, 6 patients developed a clinically defined relapse20. These patients did not differ from non-relapsing patients in their third trimester expanded disability status scale (EDSS) scores. No MRI evaluations for disease activity were performed. Additionally, 12 age-matched healthy pregnant women were included, who did not have central nervous system or inflammatory disease and were seen at the outpatient obstetric clinic at Erasmus MC. None of the healthy pregnant women used immunomodulatory medication before or during the study, were hypertensive, experienced recurrent abortions or were diagnosed with diabetes mellitus. Clinical characteristics of patients and controls are depicted in Table Nineteen pregnant women with RRMS were included at the MS Center ErasMS as part of our previous study® tubes containing sodium heparin according to manufacturer’s instructions . After isolation, cells were taken up in RPMI 1640  containing 20% fetal calf serum (Lonza) and 10% dimethyl sulfoxide  and stored in liquid nitrogen until further use. Cells were pre-incubated with Fixable Viability Stain 700 (BD Biosciences) for 15 min at 4 °C. The following monoclonal antibodies were used stained for 30 min at 4 °C: CD27 , CD138 , CXCR4 , CXCR5 , IgD , IgG , CCR6 , CD19 , CD38 , CXCR3 , CXCR4  and IgM . Stained cells were measured using an LSRII‐Fortessa flow cytometer. For in vitro culture experiments, memory (CD19+CD3-CD27+) B cells were purified using a FACSAria III sorter. Data were analyzed using FACS Diva software, version 8.0.1 . Since ex vivo B-cell phenotyping of the relapsing MS group was performed in a separate study, it was not possible to compare the MFI of functional markers with the non-relapsing MS or control groups.PBMCs from patients and controls were collected in the first and third trimester of pregnancy as well as 4–8 weeks after delivery (postpartum). For sample collection, we used Vacutainer CPT21. In short, irradiated murine 3T3 fibroblasts expressing human CD40L were co-cultured with sorted memory B cells in the presence of IL-21 . After 6 days of culturing, viable CD19+ cells were analyzed using flow cytometry and supernatants were collected and stored at -80 °C until further use.In vitro B-cell differentiation assays were performed as recently describedIgM and IgG levels were determined in supernatants of memory B cells cultured for 6 days using ELISA. After overnight coating with goat anti-human Ig  at 4 °C, flat-bottom 96-well plates  were washed with PBS/0.05% Tween-20 and subsequently blocked with PBS/5%FCS for 2 h at RT. Samples were added for 1.5 h at room temperature. After washing, peroxidase-conjugated goat anti-human IgG (Thermo Fisher Scientific) or rabbit anti-human IgM  were used to detect bound antibody. 3,3′,5,5′-Tetramethylbenzidine substrate (Thermo Fisher Scientific) was used to reveal peroxidase activity. Reactions were stopped with sulfuric acid and optical densities were measured at 450 nm using a BioTek Synergy 2 reader . Concentrations were calculated using standard curves for IgM and IgG.P values < 0.05 were considered statistically significant.Graphpad Prism software (version 8) was used for statistical analyses. Both percentages and mean fluorescent intensity (MFI) are shown as individual data points together with the corresponding mean. We compared paired data using Wilcoxon signed-rank tests and data between clinical groups using 2-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison tests, unless stated otherwise. 18 to determine their relation to MS. Finally, we verified whether relapses are associated with alterations in B-cell profile and compared B-cell development into antibody-secreting cells in vitro for samples from low and high relapse risk periods .Our overall aim was to explore how pregnancy as naturally occurring disease modifier influences the peripheral B-cell lineage in MS. To properly address the impact of pregnancy without relapses as a potential confounder, we first analyzed ex vivo B-cell differentiation profiles in paired first trimester T1), third trimester (T3) and early postpartum (PP) blood samples from 13 MS patients without a postpartum relapse. These profiles were compared with blood samples from the same periods of 12 healthy controls , naive mature (CD38-/dimCD27-IgM+) as well as IgM+ and IgG+ memory (CD38-/dimCD27+) B cells using flow cytometry  implies an increased potential of these cells to recruit and further develop in the GC light zone.Together with the increased Ig surface levels Fig. E, the diFH cells within the GC light zone. After reaching the CNS, memory B cells are likely reactivated to develop into potent antibody-secreting cells in MS patients. To assess whether B cells are prone for such recall responses in the high-risk postpartum period in MS, memory (CD27+) B cells were purified from paired third trimester and postpartum samples of 11 MS patients and compared for their outgrowth into plasmablasts/plasma cells in vitro. Under IL-21- and CD40L-inducing conditions, which mimic a TFH cell response, more antibody-secreting cells (CD38highCD27high) were formed in cultures with postpartum versus third trimester memory B cells  frequencies, these were mainly increased for postpartum memory B cells from MS patients, which were significantly higher than those from healthy controls  were more induced in both third trimester and postpartum memory B-cell cultures  found for ex vivo postpartum B cells implied a marked propensity to recruit to the TFH cell-containing GC light zone11. The elevated expression of CCR6 on ex vivo postpartum memory B cells of MS compared to healthy females pointed toward increased, T cell-dependent recall responsiveness23. This was supported by their ability to differentiate into plasma cells under TFH-like conditions in vitro.Pregnancy causes a transient period of immune suppression, which is lost early after delivery in MS. Here, we studied the functional impact of pregnancy as a disease modifier on peripheral B-cell subsets (primary aim), whether this is differentially regulated between MS patients and healthy controls (secondary aim) and if this is affected by a pre- or post-pregnancy relapse (tertiary aim). The chemokine receptor expression pattern 25, a glycoprotein which pumps steroids out of cells. We recently found a similar impact of steroid treatment in patients with MS, AQP4-IgG+ neuromyelitis optica spectrum disorder and MOG-IgG-associated disease26. Because of the rise in IgMhigh transitional B cells early after delivery, one could speculate that this results in increased entrance of potentially autoreactive naive mature B cells to GCs, resulting in the development of pathogenic memory subsets that are destined to enter the CNS and contribute to an MS relapse. Although we did not touch upon their CNS-infiltrating ability in this study, we can at least assume that the postpartum increase in CXCR3 expression mediates local B-cell enrichment in MS5. This may be further induced by the observed abundance of CCR6 on postpartum B cells of MS patients.Regarding naive B cells, the proportion of circulating transitional B cells was found to be decreased in third trimester compared to postpartum samples. A similar observation was made in a previous study27. Our group previously reported an upregulation of CXCR4 during MS onset in non-pregnant patients28. The observed reduction in CXCR4 expression during the high relapse risk postpartum period is therefore counterintuitive. A possible explanation for this discrepancy may be that during a primary response, CXCR4high naive mature B cells escape from TFH-mediated selection in the GC light zones of peripheral secondary lymphoid organs13, while CXCR4low memory B cells are more prone to interact with TFH cells and develop into long-lived plasma cells during a recall response in the CNS. CCR6 could facilitate such recall responses23 and, together with CXCR3, has also been associated with the production of high affinity antibodies29.The postpartum upregulation of CXCR5 especially seen in MS patients is likely involved in B-cell organization rather than recruitment in the CNS30. The clinical relevance of local Ig production has become apparent from the increased risk of CIS to MS conversion in patients with CSF oligoclonal bands31, which are present in more than 95% of MS patients and indicates ongoing IgG production in the CNS. Consistently, the absence of B cells in brain lesions of MS patients is associated with a lack of CSF oligoclonal bands, a lower intrathecal IgG production, and a more favorable outcome32. The observation that anti-CD20 treatment reduces CSF B cell numbers while oligoclonal bands persist33 suggests that intrathecal IgG are mainly produced by (CD20-) long-lived plasma cells in the CNS. Recently, it has been shown that MS myelin is bound by IgG and that IgG immune complexes trigger human microglia, resulting in enhanced production of pro-inflammatory cytokines34.Thus far, the potential of functionally distinct memory B cells to develop into plasma cells has been relatively understudied in MS. Despite the current focus on antibody-independent B-cell functions, this is of high relevance as long-lived plasma cells reside within the chronically inflamed CNS of MS patients35. Furthermore, we did not perform in vitro B-cell cultures with pregnancy-related sera or hormones to verify whether the observed GC light zone phenotype is controlled by extrinsic factors. Finally, although beyond the scope of this study, intrinsic factors such as memory B-cell EBV load could contribute to the increased CXCR3 expression and plasma cell formation21 as seen early after delivery.Our study has some limitations. The relatively low numbers of included subjects hampered the analysis of MS risk groups based on disease activity before and after pregnancy. Sequential data collection of such patients is difficult, but the accumulating evidence for the safe continuation during pregnancy of various disease modifying therapies may increase optionsTogether, this work provides new insights into how B-cell development is affected during high and low risk periods associated with pregnancy in MS. We demonstrate a first link between chemokine receptor expression profiles and the capacity of  memory B cells to differentiate into plasma cells, which should be further studied in the near future. This may not only help to decipher underlying mechanisms of local B-cell accumulation and antibody production, but also offer new tools to better predict disease activity in patients with MS.Supplementary Figures.


Background: Multiple sclerosis (MS) patients are protected from relapses during pregnancy and have an increased relapse risk after delivery. It is unknown how pregnancy controls disease-contributing CD4+ T helper (Th) cells and whether this differs in MS patients who experience a postpartum relapse. Here, we studied the effector phenotype of Th cells in relation to pregnancy and postpartum relapse occurrence in MS.Methods: Memory skewing and activation of effector Th subsets were analyzed in paired third trimester and postpartum blood of 19 MS patients with and without a postpartum relapse and 12 healthy controls. Ex vivo results were associated with circulating levels of pregnancy-induced hormones and mirrored in vitro by exposing proliferating Th cells to corresponding serum samples.Results: Based on HSNE-guided analyses, we found that effector memory proportions of Th cells were increased in postpartum vs. third trimester samples from MS patients without a postpartum relapse. This was not seen for relapsing patients or healthy controls. CXCR3 was upregulated on postpartum memory Th cells, except for relapsing patients. These changes were verified by adding sera from the same individuals to proliferating Th cells, but did not associate with third trimester cortisol, estradiol or progesterone levels. For relapsing patients, activated memory Th cells of both third trimester and postpartum samples produced higher levels of pro-inflammatory cytokines.Conclusion: Effector Th cells are differentially regulated during pregnancy in MS patients, likely via serum-related factors beyond the studied hormones. The pro-inflammatory state of memory Th cells during pregnancy may predict a postpartum relapse. Women with multiple sclerosis (MS) have a reduced relapse rate of ~70% in the third trimester of pregnancy , which c+ T helper (Th) and not CD8+ T cell clonotypes are reduced in the blood  containing sodium heparin for cell-based analysis. Serum and peripheral blood mononuclear cells (PBMCs) were isolated according to manufacturer's instructions. PBMCs were taken up in RPMI 1640  containing 20% fetal calf serum  and 10% dimethyl sulfoxide  and stored in liquid nitrogen until further use. Serum samples were stored at −80°C.highCD127low) and defined naive, central memory and effector memory populations based on CCR7 and CD45RA expression. Th1, Th2, Th17, Th17.1 and Th17 “double-positive” (DP) subsets were discriminated based on differential expression of CCR6, CCR4, and CXCR3  using CD4 microbeads and the autoMACS Pro Separator , frozen as described above and stored in liquid nitrogen until further use. Thawed cells were labeled with CFSE  for 10 min at 37°C. After washing twice with RPMI 1640 containing 100 U/ml penicillin and 100 μg/ml Streptomycin  and 5% FCS (Thermo Fisher Scientific), cells were plated at a concentration of 1 × 10E6/ml in RPMI1640 containing Pen/Strep. Anti-CD3/-CD28 dynabeads (Thermo Fisher Scientific) were added (1:5) together with paired third trimester or postpartum serum samples from MS patients or healthy controls till a concentration of 5%. After 72 h of culture, dynabeads were removed and cells were assessed by flow cytometry.CD4+CD4+CD8−CD25dim−/CD45RA−) were isolated using a FACSAria-III machine (BD Biosciences) and plated at a concentration of 0.5 × 106/ml in RPMI 1640 containing 5% inactivated human AB serum (Sanquin) and Pen/Strep. Cells were rested overnight (37°C) and were stimulated with phorbol 12-myristate 13-acetate  and ionomycin  for 5 h. Subsequently, supernatants were harvested and stored at −80°C until further use. Supernatants were diluted 2-fold and analyzed for GM-CSF, IFN-γ, IL-2, IL-6, IL-17A and TNF-α using a custom Luminex multiplex bead immunoassay . Measurements were performed on a Bio-Plex MAGPIX machine and data were analyzed using Bio-Plex Manager MP software .Memory Th cells . Both steroids were calibrated using commercially available calibrators ; article number P-069 (progesterone) and C-106 (cortisol). Trueness was verified for progesterone using reference materials ERM-DA347 and ERM-DA348. For cortisol, trueness was verified using the ERM-DA451 IFCC cortisol reference serum panel (34 levels). Both analytes were measured without bias when compared to the reference materials. Measurement uncertainty was assessed for both analytes by measurement of tri-level matrix matched controls . For progesterone, the coefficients of variation (CVs) over a period of 1 year were 5.2, 6.6, and 10.4% at 1.4, 17.9, and 65.3 nM, respectively. For cortisol, CVs were 3.6, 3.7, and 4.0% at 40.0, 98.6, and 573.0 nM, respectively. Estradiol levels were measured by automated immunoassay on . CVs over 19 runs were 4.4 and 3.9% at 345 and 1,099 pM, respectively.p-value of < 0.05 (*) after multiple testing correction was considered significant. Categorical data were analyzed using Chi-squared, Kruskal-Wallis or Wilcoxon rank-sum tests.Generalized linear mixed models (GLMM) with a gamma distribution were used to assess longitudinal effects and the false discovery rate (FDR) method of Benjamini and Hochberg (BH) was used to correct for multiple testing . Data between clinical groups were compared using Kruskal-Wallis tests incorporating Dunn's multiple comparisons or 2-way analysis of variance (ANOVA) with Bonferroni's multiple comparison tests . Longitudinal results were displayed as connected data points for each individual. For the comparison of data between clinical groups, Box and Whisker plots were used containing individual data points and displaying the median with the interquartile range (IQR). For all tests, a In this exploratory study, pregnant relapsing-remitting MS patients visiting MS center ErasMS were included and retrospectively validated to match the most recent McDonald 2017 criteria , 17. Excex vivo flow cytometry data of CD4+ Th cells from paired third trimester and early postpartum blood of healthy controls  and MS patients without a postpartum relapse  in an unbiased manner using HSNE (lowCD45RAlow) was decreased, while cluster P1 was increased (CCR7highCD45RAhigh) in third trimester vs. postpartum blood (dimCD45RAlow) was proportionally decreased during pregnancy relative to the presence of other clusters. For the MS-NR group, this was confirmed by manual gating, showing significantly reduced effector memory (CCR7lowCD45RAlow) and increased naive (p = 0.002 and p = 0.012) Th cell frequencies. For the HC group, these frequencies did not differ (+ Th cells in vitro (ex vivo (p = 0.026 and p < 0.001). In contrast to our ex vivo results (p < 0.001). Interestingly, these frequencies appeared higher than in the MS-NR and HC group. Overall, these data suggest that effector memory Th cells are kept in check during pregnancy by serum-related factors, which is lost and may contribute to an MS relapse early after delivery.To assess shifts in phenotypic effector profiles, we first analyzed ing HSNE . For botum blood . Clustert differ  and weret differ . To detein vitro . Th cell results , reduceddim/lowCD45RAlow) Th clusters P2 and P3 (p = 0.002) and MS-NR groups (p < 0.001), but not for the MS-R group. In line with our HSNE analysis (−CXCR3+CCR4−), Th17 DP (CCR6+CXCR3+CCR4+) and Th17.1 (CCR6+CXCR3+CCR4dim−/) cells (in vitro proliferating memory Th cells exposed to MS-NR and MS-R third trimester vs. postpartum sera (p < 0.001). This was comparable to the in vitro effects on effector memory phenotypes . As expected, mass spectrometry revealed that cortisol levels were strongly elevated in third trimester sera from each clinical group (p < 0.001). Progesterone and estradiol could not be detected in postpartum samples. Third trimester cortisol, progesterone and estradiol levels did not differ between clinical groups  cells . Interesm) cells  were decm) cells , 3. In senotypes . Althougl groups . These dhighHLA-DRhigh memory Th cells were increased in postpartum compared to third trimester samples (p < 0.001). These proportions were higher than those in the HC group (p = 0.008). To address how this is related to their ability to produce pro-inflammatory cytokines, memory Th cells were purified from paired third trimester and postpartum samples and stimulated with PMA and ionomycin. Although not significant for each cytokine, higher levels of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), IL-2, IL-17A, interferon gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were found in supernatants from postpartum memory Th cells of the MS-R group  cells are selectively enriched in the blood of MS patients who clinically responded to nalalizumab  and Th17.1 (IL-17low) cells  cells .+ effector memory Th subsets during pregnancy corresponds to a postpartum relapse in MS. Due to the limited number of samples available for this study, our results should be validated in a larger cohort of similar well-defined treatment-naive pregnant MS patients, preferably with radiological data available to confirm the absence of immunological activity in cases not experiencing a clinical postpartum relapse. Since the composition of effector Th cells can easily be monitored in the blood, subsequent studies should clarify whether their pro-inflammatory nature could be evaluated as a prognostic cellular marker of disease activity during and after MS pregnancy.Our study provides additional insights into how the suppression of CXCR3The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Medical Ethics Committee Erasmus MC. The patients/participants provided their written informed consent to participate in this study.SK performed experiments, analyzed data, interpreted results, and wrote the manuscript. RN organized the clinical study and critically revised the manuscript. AW-W, M-JM, AR, and RW performed experiments. CM assisted with the statistical analysis. WD, BB, and SB analyzed data and critically revised the manuscript. JS and ML designed the research, obtained funding, interpreted results, and critically revised the manuscript. All authors contributed to the article and approved the submitted version.JS received lecture and/or consultancy fee from Biogen, Merck, Novartis, and Sanofi-Genzyme. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition, for the [68Ga]Ga-DOTATOC examinations, tracer uptake quantitation and liver lesion detectability were assessed.The aim of this prospective study was to evaluate a data-driven gating software’s performance, in terms of identifying the respiratory signal, comparing [68Ga]Ga-DOTATOC PET/CT examinations. Prospective DDG was applied on all bed positions and respiratory motion correction was triggered automatically when the detected respiratory signal exceeded a certain threshold , at which point the scan time for that bed position was doubled. These bed positions were reconstructed with quiescent period gating (QPG), retaining 50% of the total coincidences. A respiratory signal evaluation regarding the software’s efficacy in detecting respiratory motion for [68Ga]Ga-DOTATOC was conducted and compared to [18F]FDG data. Measurements of SUVmax, SUVmean, and tumour volume were performed on [68Ga]Ga-DOTATOC PET and compared between gated and non-gated images.Twenty-four patients with confirmed or suspected neuroendocrine tumours underwent whole-body [R ≥ 15 was exceeded and gating triggered on mean 2.1 bed positions per examination for [68Ga]Ga-DOTATOC as compared to 1.4 for [18F]FDG. In total, 34 tumours were evaluated in a quantitative analysis. An increase of 25.3% and 28.1%, respectively, for SUVmax (P < 0.0001) and SUVmean (P < 0.0001), and decrease of 21.1% in tumour volume (P < 0.0001) was found when DDG was applied.The threshold of 68Ga]Ga-DOTATOC PET/CT. DDG yielded significantly higher SUVmax and SUVmean values and smaller tumour volumes, as compared to non-gated images.High respiratory signal was exclusively detected in bed positions where respiratory motion was expected, indicating reliable performance of the DDG software on [ Respiratory motion has an impact on positron emission tomography (PET) imaging of the thorax and abdomen, usually in the vicinity of the diaphragm. Non-corrected respiratory motion might lead to severe lesion blurring which affects several aspects of the imaging assessment such as delineation, detectability, and quantitation , 2. ApplData-driven gating (DDG) constitutes a device-less alternative by which respiratory motion is derived directly from the PET-acquisition data. A number of different techniques for DDG have been developed during the last decade, and many have shown promising results when applied on retrospective data –13. DDG R. The R value is a measure of how much the signal resembles respiratory motion in each bed position and hence provides information on to what extent this motion might affect the resulting image. An R value threshold can be predefined by the user and during the scanning, when this threshold is exceeded in a bed position, motion correction is triggered automatically. This results in activation of quiescent period gating (QPG), extending the scan time and retaining images from a desired percentage, typically 50%, of the total acquisition. When the level of respiratory signal in a bed position is below the set threshold, it is assumed that there is no need for respiratory gating and the scan time is not extended FDG PET data. However, since this DDG software is based on PCA, which is a signal processing method, its sensitivity is expected to depend on noise levels and tracer distribution [18F]FDG.To the best of our knowledge, all previously published studies evaluating the MotionFree software’s performance regarding identification of the respiratory signal have been conducted retrospectively on [ribution . We hypo68Ga]Ga-DOTATOC is used for PET/CT-imaging of neuroendocrine tumours (NETs) [68Ga]Ga-DOTATOC would most likely benefit from respiratory motion correction.The somatostatin analogue Ga-DOTATOC and [18F]FDG examinations conducted with the same scanning parameters. In addition, for the [68Ga]Ga-DOTATOC examinations, tracer uptake quantitation and liver lesion detectability were assessed.The aim of this prospective study was to evaluate the MotionFree software’s performance, in terms of identifying the respiratory signal, comparing [68Ga]Ga-DOTATOC was produced in-house on an automated synthesis platform  using disposable cassette system (C4-Ga68-PP) and pharmaceutical grade 68Ge/68Ga generator . The product was formulated in sterile saline and sterile-filtered in-line. The quality control regarding radiochemical purity, chemical purity and quantity was conducted using high performance liquid chromatography with UV- and radio-detectors connected in series.The [68Ga]Ga-DOTATOC. The examinations were performed at Uppsala University Hospital during a one-year period, from June 2019 to June 2020, when the clinical workflow allowed for additional acquisition time. The study was approved by the regional ethics board for medical research (Dnr 2019/00092) and the need for written consent was waived.Twenty-four patients  with confirmed or suspected neuroendocrine tumours underwent whole-body PET/CT examinations with [68Ga]Ga-DOTATOC mean ± SD 64 ± 8 min (range 56–79) prior to the examinations conducted on a Discovery MI digital 4-ring PET/CT system  with 198 mm axial field of view. Before each PET-acquisition, a CT was performed in end expiration breath hold for attenuation correction as according to our centre’s standard protocol. The vendor’s integrated software MotionFree  was applied prospectively for DDG on all scans and motion correction was triggered automatically when the measured respiratory signal exceeded a threshold set to R = 15. The threshold was chosen according to the vendor’s recommendation.The patients were injected with mean ± SD 1.4 ± 0.2 MBq/kg (range 1.1–2.0) of FDG PET data from 62 consecutive patients, scanned at Karolinska University Hospital, Huddinge, Stockholm on an identical scanner with the same acquisition time and settings as for the [68Ga]Ga-DOTATOC examinations was utilized. The [18F]FDG patients were injected with 3 MBq/kg using an autoinjector (MEDRAD® Intego), and the scans were started approximately 60 min post-injection. No further analysis was conducted for [18F]FDG PET. The [18F]FDG data was merely used to compare the DDG algorithm’s performance in terms of detecting respiratory motion.For comparison of the number of triggered bed positions, [R values were extracted from the scanner's database retrospectively for each bed position. The centre of each bed position in relation to that of the liver dome was calculated for every patient. Localization of the liver dome utilized the CT performed for attenuation correction.The previously calculated 18F]FDG and [68Ga]Ga-DOTATOC were assessed using a Mann–Whitney test. A P < 0.05 was considered statistically significant.Differences in the number of triggered bed positions for [68Ga]Ga-DOTATOC PET-images, well-defined tumour lesions located in bed positions that were triggered by DDG during live acquisition, were assessed for measurements of SUVmax, SUVmean, and volume using auto contour volume of interest (VOI) delineation with 41% of SUVmax, including up to 5 lesions per patient and a maximum of 3 lesions per organ. The VOIs were transferred to the corresponding gated images. To compare SUVmax, SUVmean and lesion volume, in gated versus non-gated images, an orthogonal regression was performed. In order to quantify the changes in background activity, a spherical VOI of 16 cm3 was placed in normal liver tissue in non-gated images and copied to the corresponding gated images. SUVmean in the spherical liver VOI and lesion-to-liver measures (lesion SUVmax/liver SUVmean) were evaluated. Statistical significance was considered for P values below 0.05 using Wilcoxon’s signed rank test. Quantitative measurements were made using an Advantage Workstation Server . Statistical analyses were performed in Prism 8 (GraphPad Software Inc).In non-gated [68Ga]Ga-DOTATOC PET/CT examinations in which liver metastases were detected. Liver lesion detectability was evaluated by a senior nuclear medicine physician with 21 years of PET/CT experience. The non-gated and gated PET-image sets of each patient were assessed and compared side by side, and the number of detected metastases was registered. Patients with more than 20 liver metastases were excluded from the comparison. The physician was not blinded to gated versus non-gated image sets.In order to achieve some initial data on the clinical performance of DDG in comparison to the non-gated image sets, lesion detectability was analysed, including all [68Ga]Ga-DOTATOC PET/CT scans and 352 bed positions from 62 [18F]FDG PET/CT scans were analysed. There were significantly more triggered bed positions in the [68Ga]Ga-DOTATOC PET/CT examinations than in the [18F]FDG PET/CT scans (P = 0.0074). In the [68Ga]Ga-DOTATOC examinations the average number of beds per patient was 5.3. When using a threshold of R = 15, QPG was triggered in 22 of the 24 patients and 50/128 bed positions (39%), resulting in mean 2.1 gated bed positions per scan. Bed centre positions in which QPG was triggered were found in the range 7.1 cm superior to 29.5 cm inferior of the liver dome. In the [18F]FDG PET/CT examinations, QPG was triggered in 47 of 62 patients and in 87/352 bed positions (25%) using the same threshold (R = 15). The number of triggered beds for [18F]FDG scans was mean 1.4 and the centre position for the bed positions were found in the range 9.9 cm superior to 40.6 cm inferior of the liver dome . The orthogonal regression analysis, comparing gated and non-gated, of SUVmax measurements, resulted in a slope of 1.192  1.055–1.328). SUVmean was also significantly higher in gated than in non-gated images , with an orthogonal regression slope of 1.212 (95% CI 1.058–1.365). There was a mean 21.1% decrease in lesion volume in gated as compared to non-gated images (P < 0.0001), with an orthogonal regression slope of 0.782 (95% CI 0.628–0.937)  in non-gated images and 4.37 (range 2.03–7.65) in gated images (P = 0.4). The ratio between lesion SUVmax and liver SUVmean was increased by 19.6% on average in the gated as compared to non-gated images (P < 0.0001).The mean SUV68Ga]Ga-DOTATOC PET/CT and were included in a subsequent lesion detectability analysis. Two patients harboured more than 20 lesions and were thus excluded from the analysis. In two patients, 8 and 5 metastases were detected in the non-gated and 10 and 8 in the gated images, respectively , however, was not significantly different when comparing gated and non-gated images. The NET-patients´ eligibility for peptide receptor radionuclide therapy (PRRT) with 90Y and 177Lu labelled DOTA-peptides Ga-DOTATOC PET examinations. As illustrated by Fig. 68Ga]Ga-DOTATOC PET/CT imaging routine could potentially increase lesion detection and reader confidence. This, however, has yet to be assessed in a separate trial in a larger number of patients, employing several observers and grading reader confidence scores. Also, an evaluation including different image reconstructions, such as Bayesian penalized likelihood reconstructions, could also be valuable since different reconstruction methods applied together with DDG, are likely to impact image quality and PET quantification FDG data, in terms of evaluating the respiratory signal, quantitative measurements and image quality, and where the performance of DDG was found superior Ga-DOTATOC PET/CT and gated images yielded significantly higher lesion SUV and lower lesion volumes as compared to non-gated images. This supports that DDG might be a useful tool to provide more accurate quantitation of [68Ga]Ga-DOTATOC tumour uptake.High respiratory signal was exclusively detected in bed positions where respiratory motion was expected, indicating reliable performance of the DDG software on [


Fusarium spp., is a major disease of field pea (Pisum sativum). The development of genetic resistance is the most promising approach to manage the disease, but no pea germplasm has been identified that is completely resistant to root rot. The aim of this study was to detect quantitative trait loci (QTL) conferring partial resistance to root rot and wilting, caused by five fungal isolates representing Fusarium solani, F. avenaceum, F. acuminatum, F. proliferatum, and F. graminearum. Evaluation of the root rot-tolerant cultivar “00-2067” and susceptible cultivar “Reward” was carried out with the five species. There was a significant difference (p < 0.001) between the mean root rot values of the two cultivars inoculated with the F. avenaceum (F4A) and F. graminearum (FG2) isolates. Therefore, in the QTL study, the F8 recombinant inbred line (RIL) population derived from “Reward” × “00-2067” was inoculated in the greenhouse (4 ×) with only F4A and FG2. The parents and F8 population were genotyped using 13.2K single nucleotide polymorphisms (SNPs) and 222 simple sequence repeat (SSR) markers. A significant genotypic effect (p < 0.05) and high heritability (79% to 92.1%) were observed for disease severity, vigor, and plant height following inoculation with F4A and FG2. Significant correlation coefficients were detected among and within all traits. This suggested that a high proportion of the genetic variance was transmitted from the parents to the progeny. However, no significant QTL (LOD > 3) were detected for the RILs inoculated with F4A. In the case of the RILs inoculated with FG2, 5 QTL for root rot severity and 3 QTL each for vigor and plant height were detected. The most stable QTL for plant height (Hgt-Ps3.1) was detected on Chrom5/LGIII. The two most stable QTL for partial resistance to FG2, Fg-Ps4.1, and Fg-Ps4.2 were located in a 15.1-cM and 11.2-cM genomic region, respectively, on Chrom4/LGIV. The most stable QTL for vigor (Vig-Ps4.1) was found in the same region. Twenty-five major and moderate effect digenic epistatic interactions were detected. The identified region on chrom4/LGIV could be important for resistance breeding and marker development.Fusarium root rot, caused by a complex of  Fusarium spp., Aphanomyces euteiches, Pythium spp., Phytophthora spp., Rhizoctonia spp., Didymella spp. (formerly Mycosphaerella spp.), and Ascochyta spp.  in the major field pea cultivation regions in Canada and worldwide  includes the major pathogens causing Fusarium head blight (FHB) of wheat, barley, oats, and other small grain cereals , while four minor QTL were found on LGIII, IV, VI, and VII . These QTL explained 44.4–53.4% of the total variance for resistance  in a rough map generated with 14 SSRs. The QTL identified in most of these studies had very large confidence intervals due to the limited number of markers used. The low marker density makes it difficult to apply the identified markers in pea breeding.Genetic resistance offers the most promising way to control FRR and wilt in pea. However, there is no complete resistance to FRR in field pea, and only a few studies have reported QTL associated with partial resistance to this disease , 2019. Csistance . McPhee Fusarium spp. an important research objective.On the Canadian prairies, cereals are grown in tight rotations with canola, while the cultivation of field pea and other pulses is increasing . Boom-anFusarium spp. recovered from surveys for root rot in Alberta, Canada; (2) map the QTL associated with partial resistance to FRR using a segregating recombinant inbred line (RIL) pea population genotyped by Fusarium isolates; and (3) determine the stability of the QTL, accounting for disease severity, vigor, and plant height.Therefore, the objectives of this study were to: (1) evaluate the partially resistant pea cultivar “00-2067” for resistance to different 1 plants, which were then used to develop an F8 RIL population (One-hundred thirty-five RILs used by pulation  by the spulation .F. solani), F4A (F. avenaceum), F037 (F. acuminatum), F039 (F. proliferatum), and FG2 (F. graminearum), representing the Fusarium species most frequently recovered from symptomatic pea plants in root rot surveys in Alberta, were used to screen the parental cultivars “00-2067” and “Reward.” Briefly, to obtain the SSIs, surface-sterilized pieces of root tissue with disease lesions were placed on potato dextrose agar (PDA) and incubated at 25°C for 2–3 days and then transferred to the peptone-pentachloronitrobenzene (PCNB) medium for further selection. Mycelial tips of the fungal isolates were cut from selected colonies under a stereomicroscope , and the water agar (WA) procedure was used to obtain SSI (Five single-spore isolates (SSI), S4C , with the resulting suspension decanted into 250-ml Erlenmeyer flasks, containing a 100-ml autoclaved CMC medium . The flasks were covered in aluminum foil to block light and incubated on a rotary shaker at room temperature for 2 weeks. The suspension was centrifuged to collect conidia. The concentration of conidia was estimated with a hemocytometer and adjusted to a final concentration of 2 × 106 spores ml–1 with sterile-deionized water.Conidial suspensions of the five isolates were generated following Plastic cups (9-cm diameter and 10.5-cm depth) were filled with a sterilized potting mixture . In the greenhouse tests with each SSI , the roots of seven 5-day-old seedlings of the partially resistant parent “00-2067” and the susceptible parent “Reward” were immersed in the conidial suspension for 15 min and transplanted into the soilless mixture in a cup. An aliquot (1 ml) of conidial suspension was pipetted onto the roots before they were covered with the potting mix. The plants were kept in a greenhouse at 20–25°C/15–18°C day/night and a 16-h photoperiod with daily watering to maintain the potting mix at saturation conditions conducive for FRR development. Each experiment was repeated two times. After 3 weeks, disease severity was estimated for the parental cultivars on a scale of 0–4, where: 0 = completely healthy; 1 = brown or black spots on the main root; 2 = lesions covering the main root, but the rootlets still healthy; 3 = lesions spread to the entire root system; and 4 = root totally dead .Fusarium isolates was used as inoculum to screen the 135 RIL population and parents under greenhouse conditions. The inoculation and the maintenance of the plants were as described above. The pots were arranged in a randomized complete block design (RCBD) with four replicates. The greenhouse experiment was repeated four times. After 3 weeks, plant height (cm) was measured from the base of the stem to the top leaf. Plant vigor was evaluated as a measure of the wilting severity on a scale of 0–4  (The most virulent of the ly dead) . The plaP-value of the Shapiro–Wilk test was used to determine the normality for each variable using R  searches of the Pulse Crop Database1 to determine their possible functions.Linkage analysis was carried out using the filtered SNP and SSR markers, following F. graminearum (FG2). This was then repeated for the RILs inoculated with F. avenaceum (F4A). The analysis was conducted using means for the four single greenhouse experiments, LSM, and BLUPs of the total data by Composite Interval Mapping (CIM) using WinQTL Cartographer v2.5  from the RILs inoculated with her v2.5 . The prop < 0.05 . The LODFusarium isolate was indicated, followed by “Ps” = Pisum sativum, the first number = the pea linkage group (Fg-Ps4.1” represents the QTL for disease severity caused by F. graminearum located on linkage group IV of the pea genome. The chromosomes and pseudomolecules were named in accordance with Vig-Ps2.1) and plant height .The QTL names were defined according to the QTL detection studies by ge group , and theR2) was determined for each QTL. Furthermore, QTL with R2 > 10%, 5–10%, and <5% were arbitrarily classified as major-, moderate-, or minor-effect QTL, respectively. The origins of favorable alleles for individual traits were assigned to different parents, following R2 > 15%, 7.5–15%, and <7.5%, respectively. Epistatic-effect QTL were named with the prefix “E,” followed by the QTL name and a serial number .Quantitative trait loci identified in at least two of the four environments were classified as stable. The percentage of variation (p < 0.001) between the mean root rot values of the tolerant parent “00-2067” and the susceptible parent “Reward”, following inoculation with F. graminearum isolate FG2  (p < 0.001)  ,b. This < 0.001) ,b. The G< 0.001) ,b.t-test indicated a significant difference between the parents for disease severity in all four experiments. Frequency distribution  existed among the single experiments, pooled, and BLUPs for vigor  of “00-2067” were 234.5 ± 54.6 cm, 157.3 ± 50.6 cm, 155.5 ± 59.5 cm, 159.7 ± 6.7 cm, 176.7 ± 56.6 cm and 158.9 cm, respectively. For “Reward”, the plant heights were 177.5 ± 36.1 cm, 120.7 ± 31.5 cm, 129.5 ± 26. cm, 178.5 ± 34.6 cm, 151.5 ± 36.9 cm, and 100.8 cm, respectively. The frequency distribution of plant height of the RIL population for all six variables was not normal and slightly skewed  was found for plant height among the single experiments, pooled, and BLUPs data  on the parental cultivar “00-2067” inoculated with FG2 were 1.5 ± 0.7, 1.3 ± 0.6, 2 ± 1.2 and 1.5 ± 0.7 for the four greenhouse experiments, 1.6 ± 0.8 for LSM and 1.2 for the BLUPs. This was comparable with the estimated mean of 1.1 ± 0.4 obtained in the preliminary screening of the parents . On the ribution  indicateribution . High coribution . The difribution . The Sharibution . A signior vigor . The heiy skewed . A high UPs data .r < –0.90, p < 0.001), indicating the adverse effect of FG2 on root and aboveground growth. Plant height showed low to moderate correlation with disease severity  and vigor .Collectively, the correlation analysis among traits indicated that root rot caused by FG2 was negatively correlated with vigor and plant height. High correlation coefficients were detected between disease severity and vigor in all conditions   for “00-2067” were 4.0 ± 0.0, 3.7 ± 0.5, 3.5 ± 0.6, and 4 ± 0 for the four individual greenhouse experiments, 3.8 ± 0.5 for LSM for the pooled data and 4.0 for BULPs of the pooled greenhouse experiments (p < 0.001) (The estimated means (±SE) of disease severity for “00-2067” were 1.0 ± 0.0, 1.0 ± 0.8, 1.3 ± 0.5, 1.0 ± 0.0, 1.1 ± 0.4, and 1.0, while, for “Reward”, they were 3.3 ± 0.5, 3.3 ± 0.5, 3.5 ± 0.6, 3.0 ± 0.0, 3.3 ± 0.4, and 3.0 for DSGH1, DSGH2, DSGH3, DSGH4, LSM of pooled data, and BLUPs, respectively . These vribution , althougribution . The cor< 0.001) . Based oeriments . The esteriments . Additioeriments . The cor< 0.001) .t-test. The estimated plant height for “00-2067” for the individual experiments, LSM, and BLUP was 210.8 ± 128.2 cm, 174.5 ± 104.8 cm, 159.5 ± 13.5 cm, 210.0 ± 53.1 cm, 188.7 ± 82.8 cm, and 208.0 cm, respectively. The estimated plant height for “Reward” was 118.3 ± 100.2 cm, 193.5 ± 104.5 cm, 125. ± 98.9 cm, and 194.0 ± 38.4 cm for the individual experiments, 157.7 ± 88.5 cm for LSM and 133.1 cm for the BLUP. The frequency distribution for the RIL population was continuous and slightly skewed to the right. In addition, HGH2, HGH3, HGH Pooled, and HGH BLUPs followed a normal distribution   .r < –0.95, p < 0.001) and with plant height . Plant height was positively correlated with vigor .The correlation among the traits for plants inoculated with F4A was similar to that of plants inoculated with FG2. Disease severity was highly correlated with vigor  retained markers were as described by F. avenaceum isolate F4A. As such, no QTL likelihood profiles are shown. In the case of RILs inoculated with F. graminearum isolate FG2, a total of 11 QTL were detected for the three parameters and six variables  by the CIM using Win QTL Cartographer v2.5 (R2) values ranging from 4.05 to 36.35%  for disease severity, vigor, and plant height were detected for the RILs inoculated with her v2.5 ; Table 3F. graminearum isolate FG2, Fg-Ps4.1, and Fg-Ps4.2 were located in the middle of Chrom4/LGIV at positions 59.3–74.4 cM and 74.0–85.2 cM, respectively  of Chrom5/LGIII and with flanking markers of AA5 and PsCam036163_21311_1095) and Fg-Ps3.2  explained 9.62–9.88% of the total variance (Fg-Ps5.1 [detected on the top part (0.9–9.2 cM) of Chrom3/LGV and flanked by the SNP markers PsCam059449_39630_321 and PsCam011153_7569_125] explained 14.2% of the total variance in greenhouse Experiment 1 (Fg-Ps5.1) had a negative additive effect, indicating that genomic regions for resistance in Fg-Ps4.1, Fg-Ps4.2, Fg-Ps3.1, and Fg-Ps3.2 originated from “00-2067,” while Fg-ps5.1 derived its resistance from “Reward”  > Vig-Ps3.2  > Vig-Ps3.1  both on Chrom5/LGIII  of the same chromosome or linkage group, was flanked by the SNP marker PsCam013763_9362_423 and the SSR marker AD270 . This QTL was located on the bottom segment of Chrom5/LGIII  of four greenhouse experiments (R2 = 7.04–20.04%). These QTL were located 142.3–168.0 cM on Chrom7/LGVII and were flanked by the SNP markers PsCam002756_2184_427 and PsCam045262_28962_162  (R2 = 13.63–14.06). The additive effect was negative for Hgt-Ps3.1, but positive for Hgt-Ps7.1 and Hgt-Ps7.2  , was detgt-Ps7.2 . This suTwo hundred eight putative digenic epistatic pairs were identified using all variables for disease severity, vigor, and plant height. These comprised 65 (12-24) for disease severity, 57 (10–21) for vigor, and 86 (15–28) for plant height. The 208 putative digenic interactions consisted of one major epistatic effect (PVE ≥ 15%), 13 moderate epistatic effects (7.5% ≤ PVE ≤ 15%), and 194 minor epistatic effects (PVE ≤ 7.5%). BLUPs for disease severity, vigor, and plant height detected 20, 18, and 21 putative digenic interactions, respectively. Within the 59 digenic interactions among BLUPs of all traits, epistatic analysis identified one major QTL pair, three moderate QTL pairs, and 55 minor QTL pairs . In contFg-Ps3.1, Fg-Ps3.2, and Vig-Ps3.1). The remaining 15 epistatic QTL were not related to any of the additive-effect QTL , followed by E.Hgt-Ps7 (R2 = 19.1%) and E.Hgt-Ps4 (R2 = 13.5%). The fourth was E.Hgt-Ps3 (R2 = 13.5%), which was linked to Fg-Ps3.2 and Vig-Ps3.1. Only E.Fg-Ps7 and E.Vig-Ps7 were linked to Fg-Ps3.1, showing moderate epistatic effect .Twenty-five digenic epistatic interactions with major and moderate effects were identified by 33 flanking markers, of which 10 epistatic-effect QTL with 14 flanking markers were linked to three additive-effect QTL  of all the Fusarium species isolated from 1,812 wheat, 71 durum, 596 barley, and 177 oat samples  only for the isolates representing F. avenaceum and F. graminearum. Therefore, the F8 RIL population derived from “Reward” × “00-2067” was screened with F4A (F. avenaceum) and FG2 (F. graminearum) for the detection of partial resistance to the two Fusarium species. The greenhouse experiments were repeated four times to determine the G × E interaction for all traits. In addition, the best linear unbiased predictors (BLUPs) and LSM were applied to minimize environmental effects  was comparable. R2 = 68–80%) identified by F. oxysporum were also located on Chrom4/LGIV, while three minor QTL (R2 = 2.8–5.4%) were located on Chrom5/LGIII. Despite identifying the same chromosomes, the similarity of the location of the QTL cannot be confirmed, given the different markers used in the two studies. However, the coincidence of the QTL is not surprising, since very few partially resistant pea cultivars are used in breeding programs across the world. F. avenaceum were located on Chrom7/LGVII. F. solani were located on Chrom6/LGII, while several minor QTL were located on Chrom5/LGIII, Chrom4/LGIV, Chrom6/II, and Chrom7/LGVII.In this study, 11 QTL accounting for disease severity, vigor, and plant height were identified. The major QTL for disease resistance were located on Chrom4/LGIV, while two minor QTL were detected on Chrom5/LGIII and one QTL on Chrom3/LGV. These QTL were coincident with the QTL detected for resistance to Aphanomyces root rot . The majSignificant QTL × QTL interactions were found between the minor QTL for disease severity and plant height but not for vigor. An interaction of the major QTL for disease severity, vigor, and height was not observed. F. graminearum. The use of high-density SNP markers and SSR anchor markers contributed to the construction of a fine linkage map and the identification of two stable QTL located on Chrom4/LGIV associated with partial resistance to F. graminearum. The identified QTL showed broad resistance to F. graminearum, F. solani, F. avenaceum, F. acuminatum, and F. proliferatum, as well as to A. euteiches. This study, together with our previous report (To the best of our knowledge, no genetic studies have been carried out to determine the genomic regions associated with the partial resistance of field pea to s report , suggestHeritability of each trait, marker information, linkage information, and QTL profiles are available in the main manuscript or as Fusarium spp., greenhouse screening of RIL population and parents for resistance to Fusarium root rot, disease rating, measurement of vigor and plant height, phenotypic data analysis, DNA extraction, PCR, genotyping with SSR markers, and writing of the manuscript. RF-A: supervision of molecular marker work, molecular data analysis, linkage map construction, QTL mapping, and writing of the manuscript. SS: principal investigator, grant application, supervision and provision of technical support to graduate student, and revision of the manuscript. K-FC: grant application, supervision, and provision of technical support for graduate students for RIL population screening in the greenhouse. S-FH: principal investigator, grant application, supervision and provision of technical support for graduate students for RIL population screening in the greenhouse, and revision of the manuscript. All authors contributed to the article and approved the submitted version.LW: inoculum preparation of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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