To investigate the influence of prestroke physical activity (PA) on acute stroke severity.Data from patients with first stroke were retrieved from registries with a cross-sectional design. The variables were PA, age, sex, smoking, diabetes, hypertension and statin treatment, stroke severity, myocardial infarction, new stroke during hospital stay, and duration of inpatient care at stroke unit. PA was assessed with Saltin-Grimby's 4-level Physical Activity Level Scale, and stroke severity was assessed with the National Institutes of Health Stroke Scale (NIHSS). Logistic regression was used to predict stroke severity, and negative binomial regression was used to compare the level of PA and stroke severity.The study included 925 patients with a mean age of 73.1 years, and 45.2% were women. Patients who reported light or moderate PA levels were more likely to present a mild stroke (NIHSS score 0 to 5) compared with physically inactive patients in a model that also included younger age as a predictor (odds ratio = 2.02 for PA and odds ratio = 0.97 for age). The explanatory value was limited at 6.8%. Prestroke PA was associated with less severe stroke, and both light PA such as walking at least 4 h/wk and moderate PA 2–3 h/wk appear to be beneficial. Physical inactivity was associated with increased stroke severity.This study suggests that PA and younger age could result in a less severe stroke. Both light PA such as walking at least 4 h/wk and moderate PA 2–3 h/wk appear to be beneficial. However, although physical activity (PA) is a well-known, self-monitored, cost-efficient method to increase health in individuals of all ages, physical inactivity is a global pandemic.3 Previous research in animals has shown that PA before stroke has a neuroprotective effect leading to less severe stroke with fewer neurologic deficits5 and better recovery.5 Human studies have, however, been less consistent. Some studies have shown that PA before stroke is associated with milder neurologic symptoms after stroke,6–10 whereas another showed no effect.11Stroke is the second leading cause of disability-adjusted life-years lost worldwide after ischemic heart disease.The objective was to investigate the influence of prestroke PA, age, sex, smoking, diabetes, and protective treatments such as statin and hypertension treatments on acute stroke severity. We hypothesized that prestroke PA would positively influence acute stroke severity.13 and the local stroke register, Väststroke. The personal identification numbers used in Sweden allow for linkage between registries, and anonymous data were received . The reason for combining 2 registries was that one register could not deliver all variables. The study was a cross-sectional, nonexperimental study in which data on PA were collected retrospectively.The data used in this study were based on the Physical Activity Pre-Stroke In GOThenburg population, with data from the Swedish Stroke Register (Riksstroke)We extracted clinical data from 2,233 patients  with stroke who were admitted to Sahlgrenska University Hospital from November 1, 2014, to April 30, 2016. The inclusion criteria were first-time stroke, receipt of care at stroke units at Sahlgrenska University Hospital in Gothenburg, and assessment with measures of stroke severity and PA. The exclusion criteria were former stroke or incomplete data.ICD-10 was used to identify patients with diagnoses of either intracerebral hemorrhage or ischemic stroke. Information about the number of days of inpatient care at the stroke unit and occurrence of new stroke or myocardial infarction during inpatient stay were retrieved from the registries.The 14 The cutoff scores were mild stroke (0–5), moderate stroke (6–14), severe stroke (15–24), and very severe stroke (≥25).15 The NIHSS was dichotomized into mild stroke (NIHSS score 0–5) and moderate to severe stroke (NIHSS score 6–42) in the regression analyses.Stroke severity at hospital admittance was assessed with the National Institutes of Health Stroke Scale , for which a higher score indicates greater stroke severity.17 The SGPALS is a self-reported 4-level scale with the following levels: (1) physically inactive, (2) some PA for at least 4 h/wk (light PA), (3) regular PA and training for at least 2–3 h/wk (moderate PA), and (4) regular hard physical training for competition sports several times per week (vigorous PA).17 Patients were asked about their PA at their first encounter with a physical therapist at the stroke unit with the following question: “How much did you move or exercise during leisure time before stroke? Try to estimate an average.” The physical therapist asked the patient to be precise about the previous activity level with the help of follow-up questions about intensity, duration, and type of activity, and relatives were asked to confirm the PA level whenever needed. The SGPALS was dichotomized into physically inactive patients (level 1) and light, moderate, and vigorous physically active patients (level 2–4) in the regression analyses. The SGPALS was divided into 3 groups (levels 3 and 4 were merged) to discover differences between different levels of PA in negative binomial regression analyses.17The independent variables selected were PA, age, sex, smoking, diabetes, and protective treatments such as statin or hypertension treatments. Prestroke PA was assessed with the Saltin-Grimby Physical Activity Level Scale (SGPALS).p < 0.002 according to the Bonferroni correction. Differences between patients included in the study and patients with missing assessments (NIHSS and SGPALS) were analyzed with independent samples t tests for age and χ2 tests for sex. Nonparametric statistics were used because of the non-normal distribution of the data.IBM SPSS Statistics 24 was used for statistical analyses. Descriptive data are presented as mean with SD for continuous variables or number (n) and percentage (%) for categorical variables. The significance level was adjusted to Negative binomial regression analyses with log link were performed to explore relationships between the levels of prestroke PA (SGPALS) and stroke severity (NIHSS). The NIHSS was defined as the dependent variable , and the SGPALS score divided into 3 levels were defined as factors.19Cross-tabulation was performed between each independent variable and the dependent variable to detect small or redundant groups, followed by dichotomization when needed.Logistic regression  was performed separately between each independent variable and the dependent variable.p < 0.25. The least significant variables were removed one at a time until a model with significant independent variables was found.Logistic regression  was performed backward with the remaining independent variables with 20 determination coefficient, R2, was registered.The predictor model with the fewest independent variables was chosen, and Nagelkerke'sLogistic regression was used to assess the effect of a set of predictors  on the dependent variable, defined as stroke severity (NIHSS). The following steps for the multiple regression model building were followed.The goodness of explanatory variables was calculated with the Hosmer and Lemeshow test, and the area under the curve in a receiver operating characteristic curve was computed. A 2-way analysis of variance (ANOVA) was conducted to explore the possible interaction between independent variables in the final model on stroke severity (NIHSS).The Swedish Data Protection Authority allows handling of data generated within the framework of quality registers as an exception from the general rule of patient consent in view of public interest because it provides improvement of the quality of care and treatment. Furthermore, according to the Personal Data Act (Swedish law No. SFS 1998:204), no informed consent is needed to collect data from medical charts for clinical purposes and quality control. The study was approved by the Regional Ethical Board of Gothenburg on May 4, 2016 (registration number: 346-16).ks.sunnerhagen@neuro.gu.se). According to the Swedish regulation (epn.se/en/start/regulations/), the permission to use data can only be according to application and approval from the ethical board.Data from registries are subject to the Personal Data Act (Swedish law No. SFS 1998:204). Data may be available to researchers upon request, after review of secrecy  reported that they were physically inactive before their stroke  and 45.2% women. Ischemic stroke was the most common cause of stroke (93.8%), and 5.8% had cerebral hemorrhage. A majority of the patients were assessed as having a mild stroke (79.8%) and 34.2% had 0 points on the NIHSS after admission to hospital. The cutoff scores for the NIHSS were mild stroke (0–5), moderate stroke (6–14), severe stroke (15–24), and very severe stroke (≥25).r stroke .p = 0.001) and contained more women  than the included group.Excluded patients with missing NIHSS and SGPALS assessments were compared with patients included in the study. The excluded patients were older with a mean age of 76 years (p < 0.001) and the moderate PA group  when analyzed with negative binomial regression. There was no significant difference between the light PA group (level 2) and the moderate PA group (levels 3 and 4) on stroke severity as measured with the NIHSS (p = 0.140)  .p < 0.25) were included in a multivariate analysis. In the multivariate analysis, the variables sex and hypertension were excluded. In the final model, PA and age remained. Patients who had been physically active before their stroke and were younger in age were more likely to have a mild stroke (odds ratio = 2.02 for PA and odds ratio = 0.97 for age). The model predicted 6.8% of stroke severity (p = 0.166) in a 2-way ANOVA analysis. Patients were divided into 4 groups according to their age , and prestroke PA was dichotomized.After analysis with univariate logistic regression, the variables PA, age, sex, and hypertension treatment .8 while a third shows no dose dependency for frequency.11 Therefore, it is difficult to compare results between studies because frequency, intensity, and type of PA differ. However, these results presented here suggest that both light and moderate PAs are beneficial.No dose dependency for intensity was found in this study, with no significant difference in stroke severity between the light PA group  and the moderate PA groups (training 2–3 h/wk). Two previous studies report dose dependency for duration of PA and mild stroke severity,22 Very few patients in this study had myocardial infarction or new stroke during their hospital stay; therefore, only descriptive statistics were produced for those outcomes , and availability of predefined variables and measurements only, thereby reducing the number of available variables. A specific limitation of this study is the potentially limited utility of retrospective self-reported questions such as the SGPALS, although several studies have shown that self-report is a reproducible and reliable way to assess the activity level.30 Whether these associations are truly caused by PA is not fully proven, nor the possible neuroprotective mechanisms, because most of such studies are conducted on animal models involving small sample sizes and constrained study design.31 Thus, there is a need for further research in this area to understand the possible mechanisms by which prestroke PA influences stroke severity.Some possible explanations for the benefits of prestroke PA for stroke severity could be reduction in inflammation or increase in the level of growth factors (which stimulates neurogenesis and angiogenesis).32 Another possible research area is to investigate if and how prestroke PA has an effect on patients after stroke in a longer time perspective.The exploration of the link between prestroke PA and poststroke cognitive function has scope for future research. A recent study from Sweden showed an association between a high cardiovascular fitness in midlife women and decreased risk of dementia.33 Regular PA has been shown to reduce the risk of several noncommunicable diseases in addition to stroke, and increased PA is considered essential for maintaining good health. This can be achieved through education and recommendations regarding PA.34 However, the evidence of the neuroprotective effects of PA is conflicting, and studies with homogenous populations and standardized outcome analyses are needed.35 Health care should provide PA counseling and support for increased PA among individuals with risk factors.Physical inactivity should be monitored as a risk factor in clinical medical practice.This study shows that prestroke PA and younger age are partially associated with less severe acute stroke. Both light PA such as walking at least 4 h/wk and moderate PA 2–3 h/wk appear to be beneficial.


Postvaccination fever is a mild adverse event that naturally improves without complications, but is highly prevalent and can be accompanied by febrile convulsions in some cases. These adverse effects may cause parents to delay or avoid vaccinating their children.This study aimed to identify postvaccination fever patterns and the ability of antipyretics to affect changes in these patterns from data collected from a mobile app named Fever Coach.Data provided by parents of feverish children derived from a mobile app, Fever Coach, were used to identify postvaccination fever patterns according to vaccinations and the use of antipyretic drugs. We selected single vaccination records that contained five or more body temperature readings performed within 48 hours of vaccination, and we analyzed postvaccination fever onset, offset, duration, and maximum body temperature. Through observing the postvaccination fever response to vaccination, we identified the effects of antipyretic drugs on postvaccination fever onset, offset, and duration times; the extent of fever; and the rate of decline. We also performed logistic regression analysis to determine demographic variables  involved in relatively high fevers (body temperature ≥39°C).P<.001). In nonantipyretic cases, offset times and duration times were 9.9 and 10.1 hours shorter, respectively, than in antipyretic cases. Body temperatures also decreased faster in nonantipyretic cases. Influenza vaccine-associated fevers lasted relatively longer, whereas pneumococcus vaccine-associated fevers were relatively short-lived.The total number of Fever Coach users was 25,037, with 3834 users having entered single vaccination records, including 4448 vaccinations and 55,783 body temperature records. Most records were obtained from children receiving the following vaccinations: pneumococcus (n=2069); Japanese encephalitis (n=911); influenza (n=669); diphtheria, tetanus, and pertussis (n=403); and hepatitis A (n=252). According to the 4448 vaccination records, 3427 (77.05%) children had taken antipyretic drugs, and 3238 (89.15%) children took antibiotics at body temperatures above 38°C. The number of children taking antipyretics at a body temperature of 38°C was more than four times that of those taking antipyretics at 37.9°C (307 vs 67 cases). The number of instances in which this temperature threshold was reached was more than four times greater than the number when the temperature was 37.9°C. A comparative analysis of antipyretic and nonantipyretic cases showed there was no difference in onset time; however, offset and duration times were significantly shorter in nonantipyretic cases than in antipyretic cases (These findings suggest that postvaccination fever has its own fever pattern, which is dependent on vaccine type and the presence of antipyretic drugs, and that postvaccination temperature monitoring may ease fever phobia and reduce the unnecessary use of antipyretics in medical care. The World Health Organization and the Korean Centers for Disease Control and Prevention recommend at least 10 and 14 vaccines, respectively, for routine immunization of children ,3. The iPostvaccination fever is a mild adverse event that is usually self-limiting over a few days without any specific complications. However, it is highly prevalent and, in some cases, accompanied with febrile convulsions. Furthermore, postvaccination fever may serve as an indicator of infectious disease. For these reasons, postvaccination fever can cause excessive anxiety in parents and caregivers -12. Prev2 within the hypothalamus [Although fever can occur after every vaccination, a higher incidence of fever has been observed in patients receiving pneumococcal and DTaP  vaccines than in those receiving other vaccines -19, indithalamus . HoweverPatient-generated health data (PGHD) acquired through high-mobility mediums such as mobile phones, Internet of Things, wearable devices, and mobile apps have recently emerged as alternative methods for collecting data -31. SomeFever Coach is a mobile health care app developed by Mobile Doctor for parents with a feverish child. The app is based on pediatric thermal standards; it assesses a child’s condition based on user input and provides guidelines for antipyretic use. The app provides services that support parents’ effective and accurate control of common fever symptoms. Fever Coach provides several data services related to managing fever in children, such as microdust concentration status, body temperature information depending on geographical area, disease epidemic alerts, and pediatric health information. The app was made available as a free download from the Google Play Store and Apple App Store. As of June 31, 2017, 197,555 people had registered their child with the app.We collected vaccination records and records of subsequent postvaccination fever responses and the antipyretics administered. To identify vaccination and antipyretic effects on postvaccination fever response patterns, we analyzed the logs of all users who signed up and registered their child between July 2015 and June 2017. Postvaccination fever usually lasts less than 48 hours ; therefoOnset time was defined as the time between vaccination and the point at which body temperature exceeded 38.0°C. If the first record was above 38.0°C, the time from vaccination to the registration of the data was defined as the onset time. Similarly, offset time was defined as the time between vaccination and the last point at which the body temperature fell below 38.0°C. The duration time was defined as the time elapsed between onset and offset points . Body teThis study was approved by the Institutional Review Board of the Asan Medical Center . The need for informed consent was waived by the Ethics Committee because this study used routinely collected log data that were anonymously managed at all stages, including during data cleaning and statistical analyses.Haemophilusinfluenzae type B (Hib), polio, rotavirus, and chickenpox were excluded from the analysis because the number of vaccination records was less than 100; therefore, only data regarding DTaP, Japanese encephalitis, pneumococcus, hepatitis A, and influenza were analyzed. Although DTaP and MMR vaccines are combination vaccines, we considered these combination vaccines to be single vaccinations because they are single shots that can be distinguished from multiple vaccinations that involve multiple shots and that vaccines contain a variety of ingredients that can cause side effects [The app allows users to enter data regarding vaccinations, body temperatures, and antipyretic drug administration history. The vaccination record consists of the type and time of vaccine given to a particular child. The body temperature record consists of body temperature and measurement time for a specific child. The antipyretic drug administration record consists of antipyretic agent type, dose, and time.  effects .The analysis was undertaken by grouping the data into 3238 cases with antipyretic records and 982 cases with no antipyretic records. For each vaccination record, the sex and age of the child were also collected.t test was used to determine the degree of difference, while the P value for a two-sided test was used to test for significance. To observe the process of fever for each vaccine record, the onset time was defined as reference time 0, and body temperature values recorded over the previous 3 hours and the following 24 hours were obtained. Cases were grouped according to whether they were given antipyretic drugs and were reclassified according to vaccine type. Additionally, an ANOVA test was performed to compare maximum temperature and fever duration among the vaccine types. We used a Dunnett T3 post hoc test because the P value of the Levene test was less than .001, which indicated a violation of the assumption of homogeneity of variance. Lastly, binary logistic regression analysis was performed to determine demographic variables  involved in relatively high fevers (body temperature ≥39°C). We performed multiple regression tests to identify multicollinearity between age and weight. If the variance inflation factor (VIF) was less than 10, it was considered that there was no multicollinearity for those variables. Data were processed and analyzed using R version 3.5.0, SPSS 21.0, and Python 3.6 .To confirm the postvaccination fever differences between vaccination and antipyretic drug administration, we performed statistical analysis. To compare differences in onset times, offset times, duration times, and maximum temperatures, an independent sample During the 24 months of the Fever Coach operation, a total of 25,037 users recorded 65,894 vaccinations involving 387,613 body temperature records for 25,608 children. Of the 64,003 vaccination records for which a child’s basic information was available, 15,538 (24.27%) were single vaccination records. Of the total number of enrolled children, there were 3834 (14.97%) children with five or more body temperature records at onset and offset, with 4448 vaccination records and 55,783 body temperature records . The ageAfter vaccination, 14.66% (475/3238) of children were administered the first antipyretic drug within 1 hour, and more than 50% of children were administered antipyretic drugs within 10 hours. By 10 hours, the number of children treated with antipyretic drugs increased gradually, but decreased from 11 hours to less than 10% at 41 hours. A total of 2887 (89.16%) children used antipyretics when the postvaccination body temperature was 38.0°C or above . The numThere were significant differences in offset times, duration times, and maximum temperatures between groups that had taken antipyretics and those that had not, but there was no significant difference in onset times . DiffereP<.001)  . Offset P<.001). In the postanalysis grouping, maximum temperature and fever duration decreased in the following order: hepatitis A, influenza, Japanese encephalitis, DTaP, and pneumococcus . In the antipyretic use group, the mean body temperature was 37.5°C or above even after 24 hours, but in the nonantipyretic use group, the mean body temperature dropped below 37.5°C before 20 hours for all vaccines except hepatitis A. In comparing vaccines, we found that postvaccination fever associated with the influenza vaccine tended to be relatively long-lasting, and that the pneumococcus vaccine showed a relatively rapid decline.P=.12), but the mean offset time and duration were significantly different . The maximum body temperatures at onset time were mean 38.8°C (SD 0.6°C) in the antipyretic group and mean 38.3°C (SD 0.5°C) in the nonantipyretic group, with a statistically significant difference between the two groups (P<.001).P<.001). We were also able to clearly identify differences among postvaccination fevers with and without antipyretic administration; offset, duration times, and maximum temperature were significantly different between groups (P<.001). Additionally, we presented new evidence from large-scale PGHD concerning fever duration and maximum temperatures reached after a single vaccination. The postvaccination fever duration was generally 48 hours but, for all single vaccinations, the longest postvaccination fever duration was mean 16.4 (SD 12.0) hours, and the highest maximum temperature was mean 39.0°C (SD 0.6°C). In terms of informatics, an additional contribution of this study is that the majority of users verified the pediatric fever level based on the PGHD. This study is original compared with previous studies for the following reasons: we used actual postvaccination fever response data without any clinical intervention; we analyzed large-scale postvaccination fever responses based on real-world data rather than relying on data from a small, clinical-based group; and our study used data derived from anyone using a mobile phone and not just from a specific hospital or specific patient area, which allowed us to analyze individual data covering a larger area than from data from a single institution or specific area.We identified different postvaccination fever responses for each vaccine using data collected through the mobile app Fever Coach. For example, in relation to the hepatitis A vaccine, the postvaccination fever maximum temperature was statistically significantly higher and was of a longer duration than that for other vaccines  to 15.3 hours (hepatitis A vaccine), a mean offset time of 12.5 hours  to 20.1 hours (hepatitis A vaccine), and a mean duration time of 2.5 hours (DTaP vaccine) to 6.1 hours (influenza vaccine) . Therefore, it is possible that if the period of decrease to the fever onset temperature (38°C) is prolonged, antipyretics could be responsible for inhibiting the immune response and extending the duration. It appears that the use of antipyretic drugs influences the offset time rather than the onset time, shortening the duration to offset time. Antipyretics were associated with a trend toward prolonged duration of illness in a group infected with Shigellasonnei and the influenza virus [The duration of postvaccination fever in children who were administered antipyretics tended to be more prolonged than for those who were not administered antipyretics in this study. However, the actual body temperature at onset time and the maximum temperature were significantly higher in children who were administered antipyretics than in those who were not administered antipyretics , questions can be raised concerning the accuracy of the body temperature data. Furthermore, the app is dependent on the user entering the data correctly and consistently.Despite these limitations, the five vaccines included in this study were more frequently recorded compared to the vaccines that we excluded. It is possible that postvaccination fever is actually more likely to occur due to these vaccines than others, and parents may also have believed that postvaccination fever in relation to these vaccines is more common than with others. Therefore, they may have been more likely to perform body temperature measurements. These limitations could be overcome through wearing thermometers and body temperature recording applications that continuously measure body temperatures before and after vaccination.In practice, multiple vaccinations are recommended rather than single vaccinations, and more people are having multiple vaccinations, since there are no challenges in obtaining immunogenicity, no increase in side effects, and immunization schedules can be simplified . HoweverIn our study, an analysis of the use of antipyretic drugs was only conducted after vaccination. However, the postvaccination fever pattern can vary depending on when an antipyretic drug is administered. More detailed research on postvaccination fever patterns is needed to determine when an antipyretic drug is to be administered.Through creating a model that can predict future progress, based on the natural course of postvaccination fever , postvacPostvaccination fever has its own fever pattern depending on the type of vaccine administered. The pattern of postvaccination fever can be altered using antipyretic drugs, making the diagnosis of postvaccination fever difficult. This study showed that antipyretic drugs may prolong the duration of postvaccination fever, due to routine use or overuse. Postvaccination body temperature observation and comparison with postvaccination fever patterns indicated here may reduce the unnecessary use of antipyretics.


Acipenser ruthenus) has fastest reproductive cycle; thus, this species can be used for surrogate production in sturgeons. Primordial germ cells are the origin of all germ cells in developing embryos. Dnd1 is essential for formation and migration of primordial germ cells and its inactivation results in sterility in fish. In our study, we have used a cutting-edge genome editing technology known as CRISPR/Cas9 to knockout dnd1 and to prepare a sterile sterlet host. CRISPR/Cas9 knocked-out embryos lacked primordial germ cells and can be used as a sterile host for surrogate production in sturgeons.Sturgeons, also called archaic giants, are critically endangered fish species due to overfishing for caviar and interference in their natural habitats. Some sturgeon species have life spans of over 100 years and sexual maturity is attained between 20 to 25 years. Sterlet (Acipenser ruthenus) due to its rapid reproductive cycle and small body size can be used as a sterile host for surrogate production for late maturing and large sturgeon species. Dead end protein (dnd1) is essential for migration of Primordial Germ Cells (PGCs), the origin of all germ cells in developing embryos. Knockout or knockdown of dnd1 can be done in order to mismigrate PGCs. Previously we have used MO and UV for the aforementioned purpose, and in our present study we have used CRISPR/Cas9 technology to knockout dnd1. No or a smaller number of PGCs were detected in crispants, and we also observed malformations in some CRISPR/Cas9 injected embryos. Furthermore, we compared three established methods to achieve sterility in sterlet, and we found higher embryo survival and hatching rates in CRISPR/Cas9, UV and MO, respectively.Sturgeons also known as living fossils are facing threats to their survival due to overfishing and interference in natural habitats. Sterlet ( Acipenser ruthenus) has the fastest reproductive cycle; males mature over 3 to 7 years and females over 5 to 9 years [Sturgeons are an ancient fish species that have existed for at least 200 million years and are famous for their caviar . Their v 9 years ,14,15,16 9 years ,18,19. Ndead end (dnd1) that is essential for formation and migration of PGCs should be done to achieve sterility experimentally, as was already shown in zebrafish, medaka, loach, goldfish and sturgeons [dnd1 in mouse also results in germ cell free sterile gonads [piwil1, piwil2 and vasa that start germ cells formation in homozygous mutant fish for aforementioned genes; however, the loss of germ cells occurs at later stages [dnd1 when both maternal and zygotic dnd1 mRNA is lost during early development [dnd1 RNA in salmon was found to be required for germ cells migration to gonads [nanos3 gene resulted in germ cell free gonads, therefore further strengthening the notion that maternally contributed factors cannot rescue germ cell development and survival [dnd1 knock out leads to all-male offspring. It is still unclear whether or not the absence of germ cells is relevant for the differentiation of sex in sturgeons.Inactivation of mRNAs such as turgeons ,22,23,24turgeons ,26. Losse gonads ; howevere gonads . This fer stages ,30,31. Celopment ,22,32. Zo gonads . In tilasurvival . Loss ofsurvival ,35. In mdnd1 in sterlet to achieve sterility [dnd1 in sterlet by applying CRISPR/Cas9 technology.The latest techniques of genome editing can insert, delete and/or alter DNA sequences of cells and organisms, thus enabling scientists to dissect functions of specific genes . In CRISterility  and deplterility ; howeverAll animal experiments were conducted in accordance with the Animal Research Committee of Faculty of Fisheries and Protection of Waters in Vodňany, University of South Bohemia in České Budějovice, Czech Republic. All experimental fish were maintained according to principles based on the European Union (EU) Harmonized Animal Welfare Act of the Czech Republic, and Principles of Laboratory Animal Care and National Laws 246/1992 “Animal Welfare” on the protection of animals were followed. Experiments were approved by the Ministry of Agriculture of the Czech Republic (reference number: MSMT-12550/2016-3).Acipenser ruthenus) of five to nine years of age were transferred from outdoor ponds into recirculating aquaculture system installed indoors. Fish were kept in tanks of 4000-L and water temperature was increased to 15 °C. In order to induce spermiation, male sterlet were injected with single intra-muscular injection of carp pituitary extract at 4 mg/kg body weight (BW) in 0.9% NaCl. Sperm collection was done 48 h after hormonal injection and were kept at ice at 4 °C until fertilization. Light microscopy was used to assess motility of spermatozoa that was found to be more than 80% and were used for the fertilization. Carp pituitary extract was used to stimulate the ovulation by intra-muscular injection in two doses, i.e., first dose at 0.5 mg/kg BW and second at 4.5 mg/kg BW, 12 h after the first injection. Collection of ovulated eggs was done from three different females from 18 to 20 h after the second injection, and eggs were inseminated with sperm from two different males at 15 °C in dechlorinated water. To remove stickiness, eggs were three times rinsed in 0.04% tannic acid. One hour later after the fertilization, chorion membrane (outer layer of eggs) was removed by using forceps. Chorion removed eggs were then transferred into 100 mL dechlorinated tap water with 0.01% penicillin and streptomycin in the glass petri dishes. Embryos were incubated at 15 °C in incubator. Temperature regulation was done at 15 ± 1 °C throughout the experimental period and water was changed on daily basis. Embryos were used for injection of sgRNAs and Cas9 complex to knockout dnd1, isothiocyanate (FITC)-biotin-dextran  in order to label PGCs [During the spawning season (March to June 2017), females and males of adult sterlet  gene. Target sites were, sgSRNA1: GGGGGGAATGCAGTCCAACC; sgRNA2: GGGGGAATGCAGTCCAACC; sgRNA3: TTCAATCATTTTCTTTCTTA; sgRNA4: TGGTTTAAAACCGTAAAGAT and sgRNA5: ATTTTCTGAGTCCATGTTTC. Oligos for sgRNAs were ordered from Macrogen Company  and were annealed according to references [® mySPEC spectrophotometer) was used for sgRNAs quantification. All sgRNAs were diluted and aliquoted. Cas9 protein was purchased from PNA Bio and was re-suspended as per manufacturers’ instructions, aliquoted and stored at −80 °C.Five single guide RNAs (sgRNAs) were designed to target ferences ,42  in animal pole using a glass capillary needle. 1% FITC-biotin-dextran  was co-injected in vegetal pole to label PGCs, and in the control group only 1% FITC-biotic-dextran was injected according to Saito et al. . Glass mArdnd1 gene spanning the target sites. Mutation detection assay was performed according to [Control sterlet embryos and sgRNA+Cas9 ribonucleoprotein injected embryos were individually collected after 4 dpf and 22 dpf of injection. Total genomic DNA was isolated using a PureLink Genomic DNA Mini Kit (Invitrogen). Primers  were used to amplify the region of rding to ,45 and Mrding to  was usedIn order to perform the mutation detection assay , PCR on the genomic DNA was run over 35 cycles using KOD FX Neo (TOYOBO) and PCR products were analyzed using a microchip electrophoresis system  according to Shigeta et al., 2016 .p < 0.05). Statistical tests were performed using R programming language software .Statistical significance of treatments on number of PGCs was analyzed by Wilcoxon test and Kruskal-Wallis test. If there was a significant difference, thereafter we used Dunn’s Post-Hoc test to find out which groups were different  according to Saito et al., [We have already successfully used the  et al., . Dnd1-MO embryos A; howeve embryos A as comp et al., . In grouArdnd1 , sugarcane (Saccharum spp. hybrids), Camelina sativa and Arabidopsis thaliana [Ardnd1 to achieve sterility in sterlet (Acipenser ruthenus) to prepare a host for surrogate production in critically endangered sturgeon species. This study is consistent with Chen et al., [The majority of the genome editing studies have been conducted on polyploid species in plants such as rice  and zebrafish (Danio rerio), complete removal of PGCs by using MO against dnd1 led to production of all-male population in treated embryos [Misgurnus anguillicaudatus) and goldfish (Carassius auratus), sterilized embryos developed into both the sexes [dnd1 in sturgeons to generate sterile host for surrogate production; however, sex determination after PGCs removal is still unclear [Dnd1-MO was injected as it is toxic to embryos [We observed a healthy development of embryos after double injection until 22 dpf. Nevertheless, to guarantee the success of production of gametes via surrogacy, it is essential to check the development of sturgeons until the adult stage. It is important to confirm that complete gonads are formed in both of the sexes. In medaka  fish were germ cell free in the F0 [dnd1 gene caused complete loss of the germ cells in F0 generation. Interestingly, maternal RNA was unable to compensate for loss of zygotic gene in F0 generation [Several previous studies in fish species have studied the functions of n the F0 . Thus, ineration . In sturHost derived sperm or egg generation is not permitted in surrogate production technique to avoid the possibility of hybrids among offspring and consequent genetic contamination of target species. Due to complications in chromosomal pairing, interspecific hybrids of distantly related species are generally sterile. Nevertheless, hybrids of sturgeons from various species crosses tend to be viable and also fertile, even with different ploidy levels . TherefoAcipenser ruthenus) in order to prepare a host for surrogate production in sturgeons. The dnd1 is essential for the formation and migration of PGCs in vertebrates, and its knock out in sterlet led to mismigration and/or absence of PGCs in the sterlet embryos, thus these sterile embryos can be potentially used as hosts for surrogacy in sturgeons.In our present study, we have used CRISPR/Cas9 genome editing technology to achieve sterility in sterlet (viz., Dnd1-MO, UV irradiation and CRISPR/Cas9 that have been established in our lab to sterile sterlet. In terms of removal of PGCs, no significant difference was found among the compared methods; however, higher survival and hatching rates were found in CRISPR/Cas9, UV and MO, respectively.Thereafter, we also compared three methods


Tetranychus urticae Koch (Acari: Tetranychidae), is one of the most polyphagous herbivores, feeding on more than 1,100 plant species. Its wide host range suggests that TSSM has an extraordinary ability to modulate its digestive and xenobiotic physiology. The analysis of the TSSM genome revealed the expansion of gene families that encode proteins involved in digestion and detoxification, many of which were associated with mite responses to host shifts. The majority of plant defense compounds that directly impact mite fitness are ingested. They interface mite compounds aimed at counteracting their effect in the gut. Despite several detailed ultrastructural studies, our knowledge of the TSSM digestive tract that is needed to support the functional analysis of digestive and detoxification physiology is lacking. Here, using a variety of histological and microscopy techniques, and a diversity of tracer dyes, we describe the organization and properties of the TSSM alimentary system. We define the cellular nature of floating vesicles in the midgut lumen that are proposed to be the site of intracellular digestion of plant macromolecules. In addition, by following the TSSM's ability to intake compounds of defined sizes, we determine a cut off size for the ingestible particles. Moreover, we demonstrate the existence of a distinct filtering function between midgut compartments which enables separation of molecules by size. Furthermore, we broadly define the spatial distribution of the expression domains of genes involved in digestion and detoxification. Finally, we discuss the relative simplicity of the spider mite digestive system in the context of mite's digestive and xenobiotic physiology, and consequences it has on the effectiveness of plant defenses.The two-spotted spider mite (TSSM),  Plant-pest interactions represent an elaborate interplay between organisms whose complexity reflects a constant evolutionary arms-race between a plant and a guild of its pests. Plants evolved a myriad of strategies to deter herbivory, ranging from the manipulation of multitrophic interactions through the recruitment of pest natural enemies, to the placement of physical or chemical barriers that repel herbivores and prevent consumption of plant tissues. In addition, as herbivores feed on plants to acquire nutrients, they also ingest a wide range of plant defense compounds that interfere with essential herbivore physiological processes  and the black swallowtail (Papilio polyxenes) to feed on furanocoumarin-containing plants , is a chelicerate herbivore with an exceptionally wide host range , The two-spotted spider mite was named for its two distinct dark spots that originate from the internal gut content visible through the semitransparent cuticle Figures . TSSMs, The analysis of the TSSM genome revealed that its herbivorous pest adaptations at least partially associate with the expansion of gene families that encode proteins involved in digestion and detoxification  for more than 10 years. Bean plants were grown in a peat–vermiculite growing mix . The mites and their host plants were maintained at 26°C, under 100–150 μmol m−2 s−1 cool-white fluorescent light and a 16/8 h (light/dark) photoperiod.The London mite population was maintained on the bean, 2O). Further dehydration and paraffin embedding were performed in tissue processor (Leica ASP300TP). Embedded mites were sectioned on a microtome (Leica RM2255 Microtome) at a thickness of 5 μm. Sections were dewaxed in two 10 min changes of 100% xylene and were progressively rehydrated. The following histological dyes were used as general staining: 0.1% Safranin O  and 0.05% Fast Green , 1% hematoxylin  solution and 1% eosin , and Movats' pentachrome solution. Images were obtained using a Zeiss AxioCam Color HRc CCD Camera 412-312 and Zeiss Axioplan II microscope. Finally, 300 nM of DAPI  mixed in 10 mM PBS was used to visualize nuclei under epifluorescence Zeiss Axioplan II microscope.Spider mite adult females were collected and fixed overnight at 4°C in 4% formaldehyde in 10 mM phosphate buffer saline (PBS), pH 7.4 with 1% (v/v) Triton X-100. Mites were washed twice in 10 mM PBS and were subsequently dehydrated in an ethanol series  and stored at −80°C. Sections of approximately 10 μm in thickness were cut using the Leica CM3050 S cryostat . Sections were stained with 0.5% Oil Red O .2O), for 15 min in each solution. Mites were embedded in LR White resin  and were cured overnight at 55°C. Specimens were cut with a Reichert Ultracut S ultramicrotome  into 1 μm serial sections, using a glass knife. Sections were stained with toluidine blue, 0.5% (w/v) in 0.1% (w/v) Na2CO3, for 5 min on a slide warmer at 60°C and dried overnight at room temperature.Adult female mites were fixed in solution of 2.5% (v/v) glutaraldehyde in 100 mM sodium phosphate buffer at pH 7. After 24 h, mites were washed in 100 mM sodium phosphate buffer at pH 7 and dehydrated in a graded alcohol series: 25, 50, 75, 95, 100 and 100%  in 10 mM PBS, was applied to deposits. Slides were examined under an epifluorescence Zeiss Axioplan II microscope fitted with a DAPI filter cube. Images were taken using AxioCam Color HRc CCD Camera 412-312. Images captured were further refined using the mask layer option from Adobe Photoshop® to highlight nucleus in digestive cells  glycerol with 10 μM of DAPI  in 10 mM PBS. Images were captured using an epifluorescence Zeiss Axioplan II microscope fitted with a DAPI filter cube  and an AxioCam Color HRc CCD Camera 412-312. For the collection of feces and guanine pellets, a small square arena of about 1.5 cm Figures ; overlay Figures .Guanine particles localized in digestive cells and the posterior midgut were visualized with confocal microscopy using an argon laser at a 543 nm wavelength excitation and a LP of 650 nm. Chlorophyll and its degradation products were visualized with a He-Ne laser using a 633 nm excitation wavelength and a LP of 650 nm. A red false color was used to show the chlorophyll and its degradation product while a green color was used to highlight guanine crystals. Epifluorescence microscopy was also used to visualize expelled guanine crystals, using UV light with a FITC filter cube . Images were taken with a Zeiss Axioplan II microscope using AxioCam Color HRc CCD Camera 412-312.To assess the size range of particles that can be ingested by adult female spider mites, a device for delivering water (control) or an aqueous suspension of polystyrene fluorescent microspheres  was designed using a 0.2-mL tube cap filled with either 50 μL of water or 2.5% (w/v) aqueous suspension of FMs and covered with stretched Parafilm®  and a fluorescent tracer dye  were delivered using the method described above. In addition, fluorescein-labeled dextran of various sizes  were delivered to adult female mites as mixtures with 6% blue dye  using the leaf coating method  in 10 mM PBS, overnight at 4°C  glycerol diluted in 10 mM PBS and 0.1% (v/v) Tween 20 was gently applied to the back of an adult female mite with a fine brush to improve the transparency of the cuticle. Movement within the TSSM gut was recorded using a Canon EOS Rebel T5i camera .T. urticae genome using STAR aligner version 2.5.3a   TSSMs ingest food via the stylet  comprises the greatest volume of the alimentary tract, filling almost the entire opisthosoma. Of five caeca, three project cranially  generative cells .The Malpighian tubules mark the transition between the midgut and the hindgut in insect digestive tracts Nation, . They seArabidopsis indole glucosinolate defense compounds  whose digestion is at least initially extracellular and restricted to the gut lumen  intracellularly  (see Figures in situ localization of transcripts/proteins of biosynthetic enzymes should help resolve the management of nitrogen metabolism in TSSM.Furthermore, remnants of undigested food and byproducts of digestive processes also accumulate in the gut. Guanine, the end product of nitrogen metabolism McEnroe,  is easilTrichoplusia ni (Herde and Howe, Anopheles gambiae larvae revealed that even though the expression of detoxification genes occurs throughout the gut, detoxification functions dominate in the anterior midgut and the hindgut/Malpighian tubes (Neira Oviedo et al., Adaptation to xenobiotic compounds encompasses many strategies including avoidance, target site modification, metabolism/detoxification, transport/excretion and sequestration (Després et al., Here, we described how the TSSM digestive physiology deviates from the canonical processes as characterized in insects. This work builds on earlier ultrastructural analyses of the TSSM gut and provides a cell biology context for TSSM-plant interaction studies. Combining this knowledge with genomic and reverse genetics tools will allow a functional dissection of the TSSM gut and the identification of the specific features that enabled the evolution of an extreme generalist feeding strategy characteristic of two-spotted spider mites.NB, VZ, TS, MG, and VG conceived and planned the study. NB, CO, and SY performed experimental procedures and collected data. NB, VZ, TS, MG, and VG performed analysis and wrote the manuscript.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.


Previous results obtained from serum samples of late-stage, high-grade serous ovarian carcinoma patients showed large alterations in lipid metabolism. To validate and extend the results, we studied lipidomic changes in early-stage ovarian tumours. In addition to serous ovarian cancer, we investigated whether these changes occur in mucinous and endometrioid histological subtypes as well.N = 138) or borderline (N = 25) ovarian tumours, and 191 controls with benign pathology. These results were compared to previously published data.Altogether, 354 serum or plasma samples were collected from three centres, one from Germany and two from Finland. We performed lipidomic analysis of samples from patients with malignant (We found 39 lipids that showed consistent alteration both in early- and late-stage ovarian cancer patients as well as in pre- and postmenopausal women. Most of these changes were already significant at an early stage and progressed with increasing stage. Furthermore, 23 lipids showed similar alterations in all investigated histological subtypes.Changes in lipid metabolism due to ovarian cancer occur in early-stage disease but intensify with increasing stage. These changes occur also in other histological subtypes besides high-grade serous carcinoma. Understanding lipid metabolism in ovarian cancer may lead to new therapeutic and diagnostic alternatives. Current diagnostic methods primarily include vaginal ultrasound combined with the blood test to measure cancer antigen 125 (CA 125) levels. These methods lack specificity and sensitivity, especially in non-advanced ovarian cancer.2 In ovarian cancer, the metabolic alterations in tissues and body fluids have been investigated by metabolic profiling to identify biomarkers for early detection and reliable prognosis.5 Recently, using liquid chromatography-mass spectrometry (LC-MS), Gaul et al.6 found from serum 16 diagnostic metabolites, including many lipids and fatty acids, that distinguish early-stage ovarian cancer samples from healthy control samples. In a lipidomic study, Buas et al.7 showed 34 significantly altered metabolites between serous ovarian carcinoma and benign serous ovarian tumour patients, and the plasma levels of the lipids were reduced in patients with a malignant disease. Recently, our metabolomic analyses of tumour and blood samples from high-grade serous ovarian carcinoma (HGSOC) patients showed elevated concentrations of hydroxybutyric acids, implicating that these molecules could act as diagnostic and prognostic biomarkers.8 Subsequently, lipidomic profiling of the same samples showed an overall reduction in the levels of most of the lipid species but elevations in specific ceramide (Cer) and triacylglycerol (TAG) lipids in metastatic ovarian cancer patients.9Malignant tumours, including ovarian cancer, adopt many metabolic abnormalities to meet the increased energy demand associated with increased cellular proliferation and tumour growth.9 to analyse blood samples from patients with early-stage ovarian cancers. These results were subsequently compared to the results obtained from patients with benign gynaecological disease. Our further aim was to investigate whether the lipidomic alterations found in patients with HGSOC can be applied to other histological subtypes, i.e., to mucinous and endometrioid ovarian carcinoma.Despite several studies showing lipidomic alterations in ovarian cancer, we are not aware of any studies that confirm which lipid species are the most consistently altered. To this end, as well as to validate our published lipidomic results and extend the analyses to low malignant potential (borderline) ovarian tumours and early-stage ovarian cancers, we applied the same previously used methodologyN = 189) and another from Finland  and Oulu (N = 54) University Hospitals). In addition, we used data from an independent, previously published study9 referred herein as the Charité discovery (N = 250). The Charité discovery study included 5 additional samples from patients with endometrioid tumours that were excluded from the original publication.9 Clinical characteristics of these three study cohorts are shown in Table 10 The gynae-pathologists at the respective hospitals  did the histological analyses, and immunohistochemistry was used when needed. The Charité samples were collected at the Tumor Bank—Ovarian Cancer Network (www.toc-network.de) at the Charitè Medical University  between July 2013 and September 2016. The Finnish samples, from Tampere University Hospital and Oulu University Hospital, were collected between February 2011 and November 2014 and between January 2009 and December 2015, respectively.We performed lipidomic profiling on two study cohorts, one from Charitè N = 89 and an9 Briefly, lipidomic analyses were performed using two platforms, a global screening method and a phosphosphingolipid platform. For the screening method, 10 µl of sample was needed for the extraction of the lipids using a modified Folch extraction.11 For the phosphosphingolipid method, 25 µl of sample was needed for the extraction of lipids using protein precipitation in methanol.The samples were randomised within each cohort before lipidomic analysis. The lipidomic analysis has been previously described in detail.12 and MRM for phosphosphingolipids. The lipidomic data were processed using Analyst and MultiQuant 3.0 software (AB Sciex), and the area or height ratios of the analyte and its corresponding internal standard (IS) peak were normalised with the IS amount and the sample volume. The details of the chromatography and mass spectrometry conditions have been previously described.9Lipidomic screening and phosphosphingolipid platforms were both analysed on a hybrid triple quadrupole/linear ion trap mass spectrometer  equipped with ultra-high-performance liquid chromatography . Chromatographic separation of the lipidomic screening platform was performed on an Acquity BEH C18, 2.1 × 50 mm id. 1.7 µm column . Chromatographic separation of the phosphosphingolipid platform was performed on an AQUASIL C18, 2.1 × 50 mm, 5 µm  column set at 60 °C. For the MS analysis, a targeted approach in the positive ion mode was used for both platforms. The data were collected using a scheduled multiple reaction monitoring (sMRM™) algorithm for the lipidomics screening platform9The number of lipids and the mean coefficient of variation for each lipid class, determined from the quality control samples (6 in each 96-well plate), are shown in Supplementary Table p-values were determined by parametric t-tests on log-transformed concentrations. R version 3.4.2 was used for all statistical analyses. Tableau 10.1 was used for heatmap visualisations. For diagnostic calculations, logistic regression models were developed using all samples in the Charité cohort and tested in the Finnish cohort. The area under the curve (AUC) values were determined using the pROC package.13 The top models presented in the article were selected by calculating the sum of the AUC values in both cohorts, and selecting the models with the highest values.Group comparisons (patients vs. controls) were performed by calculating the mean relative difference between the groups, and the To validate the lipidomic alterations detected in ovarian cancer patients, we determined which lipids were similarly altered between the patients and the controls in the two study cohorts (Charité and Finland), in addition to the previously published Charité discovery cohort Table , provideTo identify which lipids have the best diagnostic potential, or those already altered in early-stage (I/II) cancer, we selected lipids that showed consistent increase or decrease both in stage I/II vs. controls and stage III/IV vs. controls, including all cohorts and histological subtypes. In addition, the lipids had to be significantly altered at least in stage III/IV patients in the Charité and Finnish cohorts. This approach resulted in 39 lipids, which are shown in a heatmap in Fig. 9 we investigated whether some changes in lipid species are also significant in patients with other histological subtypes (mucinous and endometrioid). Thus, we selected lipids showing the same direction of alteration in all histological subtypes of the Charité and Finnish cohorts. In addition, the selected lipids had to be significant in either mucinous or endometrioid subtypes in either of the cohorts. Twenty-one of 23 lipids were decreased in all histological subtypes . For endometrioid histology, none of the lipids were significant in the Charité cohort (N = 9), whereas the Finnish cohort, with a slightly greater number of cases (N = 14), showed significant alterations.As the previous results were derived from HGSOC patients only,We also analysed whether the observed lipidome alterations are present in borderline ovarian tumours. When only those lipids that were altered in the same direction in both cohorts and significant in at least one of them were selected, there were only a few significant alterations Fig. . Thus, i14 we investigated combinations of all lipids and lipid ratios together with CA 125. The lipids used for this analysis are shown in Fig. t-test p < 0.05 and mean relative change > 10%) between control samples of the Charité and Finland cohorts. The models were generated using all subjects in the Charité cohort, and tested in the stage I/II and III/IV ovarian cancer patients separately, in addition to the validation in the Finnish cohort. As an example, the models with the highest improvement in both the Charité and Finnish cohorts are shown in Table Finally, we investigated whether lipids can improve the diagnostic value of CA 125. As lipid ratios have shown diagnostic value in other diseases,7 and glycerophospholipids16 in ovarian cancer patients. The intensification of lipid changes in the advanced-stage ovarian cancer patients suggests that the tumours are exploiting circulating lipids and lipoproteins with proportion to their size. The overall decrease of PCs may be associated with reduction of high-density lipoprotein (HDL) cholesterol and ApoA1 in the ovarian cancer patients,18 as PCs are known to be abundant especially in the HDL particles.19 However, this phenomenon cannot be used to explain the increase of lipid species in ovarian cancer patients. It has been suggested that changes in lipid metabolism during ovarian cancer pathogenesis reflect higher levels of cell division,20 enhanced fatty acid β-oxidation,5 and increased cellular proliferation or motility due to increased PI3-kinase activity,21 yet there are likely to be additional mechanisms explaining the alterations of specific lipids.The present global lipidomics study investigating early- and advanced-stage ovarian cancer of various histological subtypes was performed to validate and extend our previous results on lipid changes in HGSOC patients. Altered lipid metabolism seems to be linked to ovarian cancer, but specific findings are still strikingly variable. Our data are in line with those earlier studies showing an overall decrease in the serum/plasma concentration of lipid metabolites9 Moreover, the phenomenon is evident at the early stages of disease development, i.e. stage I/II, but was found to become more pronounced with disease progression. In addition to HGSOC, Cer(d18:1/18:0) and TAG(18:1/18:1/20:4) were also significantly increased in mucinous and endometrioid ovarian cancer samples from the Finnish cohort. However, the number of mucinous and endometrioid carcinoma samples was likely too low in the Charitè cohort to show any significant difference. Interestingly, Cer(d18:1/18:0) and its precursor Cer(d18:0/18:0) have been associated with the development of insulin resistance and type 2 diabetes.24 Taken together, these alterations to the lipid profile and other metabolic changes, such as increase of ketone bodies,8 suggest that the metabolic profile of ovarian cancer patients resemble a diabetic phenotype.These results confirm our previous report describing an increase in the serum concentration of Cer(d18:1/18:0), Cer(d18:0/18:0) and TAG(18:1/18:1/20:4) in ovarian cancer patients.25 but results appear vary depending on the type of tumour.26 Cers are considered to have anticancer properties, to act as second messengers for cell apoptosis25 and to modulate cell growth.27 Another sphingolipid, sphingosine-1-phosphate (S1P), has opposing cellular effects to Cers.26 The role of sphingolipid metabolism in ovarian cancer has been investigated in a recent study in which 74 women with HGSOC were found to have significantly elevated plasma and tissue concentrations of C16-Cer, C18:1-Cer and C18-Cer compared to those of healthy controls,28 which is in line with our results. The researchers speculated that the increased amounts of Cers would be associated with particularly aggressive epithelial ovarian cancer cases and that the increased Cer concentrations would lead to increased conversion to S1P, as they found an elevated S1P concentration in tumour tissue. However, congruent with our data, elevation of S1P could not be observed in blood.Sphingolipids, especially Cers, have been linked to the development and progression of cancer,7 have shown reduction of all measured TAGs in the plasma of ovarian cancer patients. However, in a lipidomic analysis of low and highly aggressive ovarian cancer cell lines, TAGs increased dramatically along aggressiveness of the cells and were assumed to be the largest source of cellular energy.29 In a mouse model of HGSOC, compared to healthy mice, the serum levels of LPE(16:0) and PIs were decreased, while TAG(55:7) was significantly increased at early-stage cancer development.30 On the other hand, decreased levels of TAGs in epithelial ovarian cancer patients have been shown to predict early recurrence of cancer.31 In our study, only the concentrations of TAGs with longer fatty acid chains were increased or not altered, while those TAGs with short fatty acid chains were decreased. Our former study proposed that this result could be explained by genetics via low expression of the ABCD1 gene,9 which is associated with transport of long-chain fatty acids into the peroxisome for β-oxidation.32Buas et al.33 In 2004, it was shown that plasma levels of lysophospholipids varied between healthy controls and ovarian cancer patients, as well as pre- and postoperatively.34 Moreover, in a pathway analysis, glycerophospholipid (LPCs and PCs) metabolism was a main dysregulated pathway in the pathogenesis of ovarian carcinoma.35 Alteration of LPC levels may be caused by the binding and activation of specific cell surface G protein-coupled receptors, which can activate cell growth and proliferation.36 Altered LPCs and lysophosphatidylethanolamines (LPEs) contribute to genetic instability and cancer initiation via enhanced phospholipase A2 activity37 and inflammation.3 Phospholipids are needed in cancer cells to generate the cellular membrane and maintain membrane integrity.3 A large metabolic profiling study3 of 448 plasma samples from epithelial ovarian cancer patients identified 53 specific metabolites that distinguished early- and late-stage ovarian cancer with an AUC of 0.88. These metabolites included LPCs and LPEs, which were elevated in localised ovarian cancer but reduced in metastasised ovarian cancer. A potential explanation for the reduced levels of LPCs and LPEs in advanced cancer could be that rapidly proliferating tumours consume more phospholipids in their attempt to maintain membrane integrity, leading to an exhaustion of substrates.30 Also lysophosphatidic acid (LPA) has been purported to be a possible biomarker because some studies have shown LPA to be elevated in plasma samples of ovarian cancer patients.,38 but we could not confirm this as we did not monitor LPAs in our lipidomic method.Phospho- and sphingolipids are the most studied lipids in regard to the pathogenesis of ovarian cancer.39 Denkert et al.20 found significantly different metabolite levels (including metabolites from glycerolipid metabolism and free fatty acids) in borderline ovarian tumour tissues compared to invasive ovarian carcinomas using gas chromatography/time-of-flight mass spectrometry. However, they had only nine borderline tumours in their study. Based on the present study, lipid metabolism in borderline ovarian tumours differs from that in invasive cancers. The Charité cohort had more borderline ovarian tumours (N = 18) than the Finnish cohort. These samples were mainly serous epithelial tumours. Significant differences were observed only for occasional plasmalogens as compared to benign controls.Borderline ovarian tumours have low malignant potential and elevated mitotic activity without stromal invasion. They commonly occur in younger women compared to ovarian cancer patients and have lower recurrence rates.40 Our study lacks data on possible hormone replacement or hormone therapy in the Charitè premenopausal group. The samples from the Finnish cohort were postmenopausal with no current hormone therapy.In the Charitè cohort, the results were evaluated by menopausal status. Greater alterations in Cer d16:1, d18:0 and d18:1 were observed in postmenopausal women. However, in some PC lipids, premenopausal changes were stronger. A serum lipidomics study of ovariectomised healthy rats showed that Cers and phospholipids increased in response to oestrogen deficiency while TAGs decreased, which was contrary to earlier studies.9 suggest that age does not explain the differences in lipid metabolism observed in ovarian cancer patients. Fourth, the blood samples were not collected during a fasting condition, which may affect the results. However, it is worth noting that there were no differences between groups and that it is expected that fasting samples might have given a better separation between the ovarian cancer patients and the subjects with benign disease.Our study had some limitations. First, changes in lipoprotein levels can at least partly explain the overall decrease of lipids among cancer patients, but unfortunately, we did not have lipoprotein levels available from the patients. Neither did we have the information on body mass index, which may also affect lipid levels. Second, in the Finnish cohort, the sample sets contained both serum and plasma samples, which may affect the lipid levels. However, it is worth noting that the lipid changes were consistent with the two other data sets, and thus, it can be assumed that the difference does not significantly affect the results. Moreover, the logistic regression models developed in the Charité cohort showed high AUC values in the Finnish cohort, which also supports the validity of the results. Third, there was an age imbalance in the cohorts, as the Charité cohort patients were older than the controls. However, the results were consistent with the Finnish cohort, where the controls were older than the patients. This finding and our previous age-adjusted lipidomic analysesWe have shown that blood lipidomic changes occur in several patient cohorts and already at the early-stage ovarian cancer, but intensify with the progression of the disease. Many of the lipid changes are similar in patients with serous, mucinous and endometrioid ovarian carcinoma, suggesting that rewiring of lipid metabolism is an integral part of ovarian carcinogenesis. The results provide an excellent basis for further development of diagnostics and the future investigations should also explore the potential of exploiting the altered ovarian cancer lipid metabolism for therapeutic purposes.Supplementary Table S1Supplementary Table S2


Alternanthera philoxeroides, upon root flooding, to nutrient versus dissolved oxygen deficiency in terms of plant growth, biomass allocation, root production, root efficiency (plant growth sustained per unit root surface area), and root aerenchyma formation. Both nutrient and dissolved oxygen deficiency hampered the growth of root-flooded plants. As expected, plants experiencing nutrient deficiency increased biomass allocation to roots and exhibited lower root efficiency; in contrast, plants experiencing dissolved oxygen deficiency decreased biomass allocation to roots but achieved higher root efficiency. The diameter of aerenchyma channels in roots were enlarged in plants experiencing dissolved oxygen deficiency but did not change in plants experiencing nutrient deficiency. The widening of aerenchyma channels in roots could have improved the oxygen status and thereby the nutrient absorption capability of roots in low oxygen environments, which might benefit the plants to tolerate flooding.Terrestrial plants may experience nutrient and oxygen stress when they are submerged, and increases in flooding are anticipated with climate change. It has been well reported that plants usually shift biomass allocation and produce more roots in response to nutrient deficiency. However, it is unclear whether plants experiencing oxygen deficiency stimulate biomass allocation to roots to enhance nutrient absorption, similar to how plants experiencing nutrient deficiency behave. We investigated the responses of the terrestrial species  Resource availability and disturbances have been regarded as the major factors driving plant functioning  and traiRoot nutrient uptake is generally an active, energy-dependent process  and reguUpon flooding, some flooding-tolerant terrestrial plants tend to produce aquatic roots , which gAlternanthera philoxeroides (Mart.) Griseb., a flooding-tolerant terrestrial plant  Griseb., a perennial terrestrial plant of the Amaranthaceae family, originates in South America, but has spread to many parts of the world and is considered an invasive species in the United States, Australia, New Zealand, Thailand, and China. This species is found to have good tolerance to submergence, upon which it usually produces a number of aquatic roots on the nodes of the submerged stem . Unbranched plants with stem length of ca. 25 cm were selected and cut at the stem base. All cuttings were taken back to the laboratory immediately, healthy and vigorous cuttings were selected and used in the experiments as described below, each cutting was regarded as a plant.The A. philoxeroides plants to nutrient versus dissolved oxygen shortage, one with nutrient treatments, the other with dissolved oxygen treatments. 30 plant cuttings were used in the nutrient experiment  and another 30 plant cuttings were used in the dissolved oxygen experiment , respectively. Before flooding treatment, the stem height and total fresh weight of each selected plant cutting were measured. All plant cuttings  did not have any roots before flooding treatment, i.e., all roots of the treated plants were aquatic roots formed on the stem nodes during the treatment. For brevity we will often refer to them as “roots” below.Two experiments were conducted in this study to investigate the responses of root-flooded +, 14.5 NO3-, 2 NH4+, 2 Mg2+, 4 Ca2+, 2 H2PO4-, 2 SO42-, 0.0046 BO33-, 0.0005 Mn2+, 0.045 Cl-, 0.0002 Zn2+, 0.0001 MoO42-, 0.0002 Cu2+, 0.045 Fe3+. The solution was adjusted to pH 6.5 using NaOH.) were applied for root-flooded A. philoxeroides plants. Each plant (viz. plant cutting) was put in a plastic bottle (volume: 1000 ml) which had been fully filled with Hoagland’s solution of one of the three strengths, part of the plant stem including four stem nodes was submerged in the nutrient solution. The bottle was covered by a lid which had two holes, one hole was used to let the stem of the plant pass through the lid and hold the plant upright during the experiment, the other hole was for introducing a tube into the nutrient solution to bubble air. The bottle was wrapped with aluminum foil thereby keeping the submerged stem part and the nutrient solution in darkness, and then placed in a growth chamber under the condition of 12 h light  and 12 h dark (25.0°C). Ten replicate plants were randomly assigned to each nutrient level. For each plant, the nutrient solution was renewed every 2 days until the end of the experiment. During the experiment, all bottles were gently bubbled with air to keep the nutrient solution saturated with air. The partial pressures of oxygen in the nutrient solutions were 20.5 ± 0.053 kPa , the pH of the solutions was 6.5 ± 0.11 during the experiment. All plants were randomly repositioned within the growth chamber once a day.In nutrient treatment experiment, three nutrient levels, 100, 50, and 5% strength standard Hoagland’s solution (The nutrient composition of the full strength solution in deionized (DI) water was (mM): 6.5 KA. philoxeroides plants were randomly selected and put in 100% strength standard Hoagland’s solutions with different dissolved oxygen levels. Three dissolved oxygen levels  were set, which were equivalent to oxygen partial pressure of 20.6 ± 0.102, 10.2 ± 0.038, and 1.04 ± 0.029 kPa in solutions during the experiment, respectively. The dissolved oxygen levels were obtained by aerating the solutions with air/N2 mixtures containing 100, 50 and 5% air, respectively. Ten replicate plants were randomly assigned to each dissolved oxygen level, each of the plants was placed in a plastic bottle fully filled with Hoagland’s solution, with part of its stem including four stem nodes submerged. The pH of the solutions was 6.4 ± 0.09 during the experiment. The growth conditions including illumination and temperature, the dissolved oxygen monitoring of solution, the solution renewal frequency, and the plant repositioning frequency were the same as those in nutrient treatment experiment.In dissolved oxygen treatment experiment, Each experiment lasted 23 days after which all plants were harvested. Stem height, total fresh weight of plant, and fresh weight of all roots (produced during treatment) were measured. In addition, the root number (the number of first order roots), total root length, root surface area and root volume of each harvested plant were measured by a Root Analysis System . 2–3 roots with approximately same size (diameter) were randomly chosen from each harvested plant for root porosity and aerenchyma investigation in nutrient treatment as well as dissolved oxygen treatment. For each root, a free hand transverse section of the root was made at the position approximately 0.5–1 mm away from its attachment point to the stem, the transverse section area of the root and the total area of aerenchyma in the root were measured by using a stereo-microscope  and software , and the proportion of total area of aerenchyma to the transverse section area of the root was calculated to indicate root porosity. Moreover, the transverse section of each root was divided into four quadrants, rows of aerenchyma channels aligning from the central vascular cylinder to the epidermis of the root in each quadrant were randomly selected and the transverse section area, the longest and shortest diameter of each aerenchyma channel were measured .FW) and stem height (RGRH) were calculated for each plant by using the following formula:The relative growth rates of fresh weight  was calculated as: root fresh weight increment/fresh weight increment of the whole plant. Ratio of root surface area to root volume (cm2 cm-3) of each plant was calculated as: root surface area/root volume. Linear regression analyses were performed to investigate the relationship between the root surface area and plant fresh weight for plants under each treatment level.Plant biomass allocation to roots  and plant stem height (RGRH) of plants growing in 5% strength standard Hoagland’s solution were smaller than those of plants growing in 50 and 100% strength solutions. Plants in 50 and 100% strength solutions did not show a difference in RGRFW or RGRH. With decreasing nutrient levels, A. philoxeroides tended to allocate proportionally more of its newly gained biomass to roots  had smaller RGRFW and RGRH than plants in solutions with 50 and 100% air saturation ; plants in solutions with 50 and 100% air saturation did not show significant differences in RGRFW and RGRH   . Howeverncreased , 2. ThesA. philoxeroides   , the plaClimate change will impact most ecosystems extensively . Apart fWe have realized through this study that plant biomass allocation to roots was not only affected by nutrient level in the environment, but also might be affected by nutrient absorption capability per unit root. The relation between biomass allocation to roots and nutrient level may be not as certain as it is usually supposed to be. In other words, higher nutrient availability in the environment does not necessarily lead to lower biomass allocation to roots, and conversely, lower nutrient availability in environments will not always result in higher biomass allocation to roots. When the nutrient absorption capability of roots is affected by factors such as aerenchyma morphology, oxygen availability, etc., the relation between plant biomass allocation to roots and nutrient level in environments may be changed. This alerts us that when we investigate the effects of nutrient supply on plant growth and biomass allocation, nutrient absorption capability of roots needs to be considered.BZ conceived the original research plans and supervised the experiments. QA performed most of the experiments. KY, FL, and XZ provided assistance for some experiments. QA and BZ designed the experiments, analyzed the data, and wrote the article with contributions from PvB and JC.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.