Individuals differ in the way they judge ambiguous information: some individuals interpret ambiguous information in a more optimistic, and others in a more pessimistic way. Over the past two decades, such “optimistic” and “pessimistic” cognitive judgment biases (CJBs) have been utilized in animal welfare science as indicators of animals’ emotional states. However, empirical studies on their ecological and evolutionary relevance are still lacking. We, therefore, aimed at transferring the concept of “optimism” and “pessimism” to behavioral ecology and investigated the role of genetic and environmental factors in modulating CJB in mice. In addition, we assessed the temporal stability of individual differences in CJB. We show that the chosen genotypes (C57BL/6J and B6D2F1N) and environments (“scarce” and “complex”) did not have a statistically significant influence on the responses in the CJB test. By contrast, they influenced anxiety-like behavior with C57BL/6J mice and mice from the “complex” environment displaying less anxiety-like behavior than B6D2F1N mice and mice from the “scarce” environment. As the selected genotypes and environments did not explain the existing differences in CJB, future studies might investigate the impact of other genotypes and environmental conditions on CJB, and additionally, elucidate the role of other potential causes like endocrine profiles and epigenetic modifications. Furthermore, we show that individual differences in CJB were repeatable over a period of seven weeks, suggesting that CJB represents a temporally stable trait in laboratory mice. Therefore, we encourage the further study of CJB within an animal personality framework. Individuals differ in the way they perceive the world. From human psychological research, it is known that these differences become particularly evident in ambiguous situations in which individuals have to decide between different options. Symbolic for such situations is the often-quoted question: “Is the glass half-full or half-empty?”. Some individuals  interpret ambiguous information in a more positive, and others  in a more negative way; a phenomenon referred to as cognitive judgment bias (CJB) in the scientific literature . This CJWhile CJB assessment has become a key technique in animal welfare research, the ecological and evolutionary relevance of CJBs has so far not been properly addressed experimentally  were housed in two different environmental conditions (“scarce” and “complex”) and we assessed their CJB using an automated, touchscreen-based active choice paradigm . Since pSecond, we explored whether CJB can be considered a stable trait. Therefore, we measured CJB four times across the course of seven weeks and calculated the repeatability of the mice’s responses as a measure of temporal stability. Based on the literature summarized above, we expected the animals’ responses to be repeatable across this period of several weeks.To confirm the effects of the selected strains and environments on the animals’ behavioral profiles, we assessed anxiety-like behavior and spatial learning in a battery of standardized tests. As others showed lower levels of anxiety in the C57BL/6J strain  and miceWe purchased 36 female C57BL/6J and 36 female B6D2F1N mice from a professional breeder  at the age of four weeks . Mice were housed in same-strain groups of three individuals per cage . To allow for individual identification within cages, all mice received partial ear punches upon arrival. This routine procedure induces only slight and momentary pain , hence, n = 11), “scarce” environment B6D2F1N , “complex” environment C57BL/6J , and “complex” environment B6D2F1N mice . To optimize sample sizes per group, we used a design in which mice from each housing cage participated in different sets of the following experimental phases: a touchscreen training phase, first CJB test phase, repeated CJB testing phase, and behavioral test phase . Thus, by using a two-by-two full factorial design, four different treatment groups were created : “scarceThe experiment lasted for one year (February 2019–March 2020) and was conducted in two independent batches, with the second batch starting three months after the first one. Each batch followed the same experimental procedure and consisted of four different phases: a touchscreen training phase, first CJB test phase, repeated CJB testing phase, and behavioral test phase. The three mice housed in the same cage belonged to the same treatment group but participated in different phases of the experiment . This created a split plot design with different sample sizes for each phase .3), with a variety of items that allowed mice to express an array of natural behaviors, such as climbing, gnawing, hiding, and digging , the connection tunnel was detached, and cages were returned to the rack. The tunnel connector was closed by a cap  when not in use.The limited access to the “playgrounds” was intended to maintain the novelty of this environment during the experimental phase. Maintaining novelty is one of the suggested methods to avoid habituation and loss of interest in enrichment during long-term exposure, by increasing intrinsic motivation to explore novel stimuli . To furtTo sustain the novelty of social enrichment, “playgrounds” were either separated by aluminum grid walls which allowed mice to see and sniff mice from other cages  or opaque red PVC walls that prevented such contact . Mice did not encounter the same condition for more than three days in a row . The system consisted of four independent chambers. Each chamber was equipped with a tone generator, an overhead illumination, an infrared-sensitive touchscreen at the front, and a reward dispenser with a well for reward collection at the rear end. As a reward, we used servings of diluted sweet condensed milk . The touchscreen itself was separated into three adjoining windows by a Perspex mask. The central window was used to display cues in the form of white bars (6 × 1 cm²) and the two side windows served as the response windows: mice needed to nose-poke a grey cross  displayed inside these windows in response to a cue presented in the central window. Data from the touchscreen training and cognitive judgment bias tests were automatically recorded by the ABET II software .During touchscreen training and CJB test phase, mice had one session approximately every 24 h with 1-2 days of a break after five sessions. They were transported individually to the touchscreen system from the housing room using a semi-transparent red transport box. Training sessions ended after max. 30 min or earlier if the scheduled number of trials was reached before that time , “middle” (3 cm below upper edge), and “near negative” (2 cm below upper edge). Using multiple ambiguous cues is recommended to achieve a robust CJB test . In totaMice could either react toward the ambiguous cues as if predicting the positive cue outcome by touching the side that was associated with the positive reference cue (“optimistic” choice), or as if predicting the negative cue outcome by touching the side that was associated with the negative reference cue (“pessimistic” choice). Based on their responses during five testing sessions (days), we calculated choice scores for each individual for each ambiguous cue . The CJB test phase, which itself lasts a week, was repeated three times, with always one non-testing week in-between  were tested in a battery of behavioral tests. The test battery included an elevated plus maze (EPM), an open field test (OFT), and a free exploration test (FET) to assess anxiety-like behavior and exploration . The subAll tests were performed in a room that met the same conditions as described above for the housing room. Tests were video recorded (Logitech Webcam Pro 9000) and automatically tracked by software . All setups were cleaned with 70% ethanol between consecutive tests.Mice were transported into the testing room either in a semi-transparent red transport box  or in their home cage covered with a black cloth . When the home cage was used, the test mice’s cage mate(s) were transferred into waiting cages, furnished the same way as their home cage. In the testing room, tested mice had 1 min of waiting time in the transport box to acclimate before being tested. After placing the mice into the start position, the experimenter started the tracking software and left the room . All tests were performed during the dark phase between 8.15 a.m. and noon.The apparatus was elevated by 50 cm from the ground and had four arms (30 × 5 cm² each) and a central area (5 × 5 cm²) where the four arms met , 1990. TThis allowed us to account for differences in the animals’ overall activity as well as the time they spent in the ambiguous center area. In addition, we assessed the total distance traveled.The apparatus was a plywood box with a square area (80 × 80 × 42 cm³) painted with white varnish . The are2 and was located at the right side of the apparatus (experimenter’s perspective), close to the back-right corner. The mice’s home cage was attached to the opening (during the accommodation time in a transport box) via a transparent tunnel (24 cm × 15 cm × 10 cm). Mice were placed in the home cage and had 15 min to freely explore the apparatus. The two cage mates were tested on consecutive days. We quantified latency to enter the apparatus, number of entries, time spent in the apparatus, and distance traveled.The apparatus was a modified version of the open field test which allowed mice to enter the apparatus by choice . Light iThe apparatus (40 cm × 24 cm) was divided by transparent walls 15 cm in height, forming a labyrinth that offered the mice’s home cage as the goal . Light iMice improved their performance in the second trial of all three behavioral parameters . Due to F-statistics and P-values for fixed factors, ANOVA type III tables were produced with sum-contrast coding of fixed factors and the Satterthwaite method for denominator degrees of freedom. Differences were considered statistically significant at P ≤ 0.05 . For better interpretability of main effect estimates, we centered factors with two levels to 0, coding the two levels as −0.5 (the reference level) and as +0.5 (the contrast level), respectively; thus, slope estimates from the model equal the treatment difference between the factor levels , resulting in a total sample size of at least 34 animals. Since sometimes a few animals in CJB tests drop out due to poor learning, we increased the number to 48 animals in total.We conducted an a priori sample-size calculation using the software G*Power . Data of touchscreen-based CJB tests for mice are very scarce, so it is difficult to properly estimate sample sizes on this basis. Therefore, we based our calculations on the behavioral tests we conducted. Previous data showed that effects of strain (C57BL/6J and B6D2F1N), as well as environmental enrichment on the parameters assessed in tests on anxiety-like behavior are rather large—e.g., effect sizes for large effects between  effects  and betw effects . We hereF = 0.17, P = 0.68), trial type , or training duration  had an effect on the choice score. Thus, these design effects that were not of interest were removed from the final model. In the final analysis, as stated above, we focused only on the ambiguous cues but a model including reference cues was also explored ; genotype and environment as fixed between-subject factors, including a three-way interaction between cue, genotype, and environment ; and individual and cage as random factors, with individual nested within cage. Before selecting the final model, treatments were assigned at random to the dataset to prevent bias while exploring different models and potential influence of design effects .Repeatability is the proportion of total variance in multiple measurements of a trait that is due to differences between individuals. It thus constitutes a useful tool to quantify the stability of individual differences over time . The repWe performed a principal component analysis (PCA) based on a total number of 13 behavioral parameters measured in the EPM, OFT, FET, and LM  explaining more than 72% of the total variance of the data were extracted via visual inspection of the scree plot . PC1 expTo investigate whether genotype and environment (interactively) influenced “anxiety” and “spatial learning”, we fitted linear models for PC1 and PC2 with touchscreen training (two levels: trained and non-trained), genotype (two levels: B6D2F1N and C57BL/6J), and environment (two levels: “complex” and “scarce”) as fixed between-subject factors, including a genotype-by-environment interaction, and with cage as a random factor . The model for PC2 violated the normality assumption of residuals, so square root transformation was used .Data analysis and plotting were done in R 4.0.0  with lmeF2,76.0 = 227.77, P < 0.001; for a response curve across all cues and a pairwise comparison see b = −0.02 ± 0.08, F1,19.0 = 0.06, P = 0.81), the two environments , or their interaction  on choice scores (b = −0.14 ± 0.08). There was also no statistically significant three-way interaction between cue, genotype, and environment , or interaction between cue and genotype , or between cue and environment  and at R = 0.23 for the “middle” cue , but at R = 0 for the “near negative” cue , the open field test (OFT), the free exploration test (FET), and the labyrinth maze (LM). PCA revealed two biologically meaningful components, PC1, reflecting “anxiety”, and PC2, reflecting “spatial learning”.F1,19.6 = 14.21, P = 0.001). Mice from the B6D2F1N strain scored higher on PC1 than mice from the C57BL/6J strain (b = 2.09 ± 0.56), indicating that B6D2F1N mice showed higher levels of anxiety-like behavior than did C57BL/6J mice . Mice from the “complex” environment scored lower on PC1 than mice from the “scarce” environment (b = −1.39 ± 0.56), indicating that mice from the “complex” environment showed less anxiety-like behavior than mice from the “scarce” environment .There was no statistically significant effect of touchscreen training on PC1  nor environments  had a statistically significant influence on PC2 . Trained mice scored higher on PC2 than non-trained mice (b = 0.40 ± 0.15), indicating that non-trained mice learned better than trained mice , which might not have been enough to induce a positive shift of the choice score in the CJB test. In addition, also the animals living in the “scarce” environment were provided with basic enrichment items in their cages . Thus, the difference between the “scarce” and the “complex” environment might have been smaller than intended, especially when considering that the access to the “playgrounds” was limited. However, mice from the “complex” environment showed lower levels of anxiety-like behavior, indicated by lower scores on PC1, which would suggest that the “playgrounds” still had a positive effect on the mice.Second, the limited access to the “playgrounds” might have induced a negative contrast effect. More precisely, since the animals were always taken out of the “playgrounds” after 1 h, they might have experienced a withdrawal of enrichment, potentially masking any positive enrichment effects .g of 0.2–0.6 from the individual studies). Thus, stronger manipulations and larger sample sizes might be needed to detect the effects of environmental factors. However, our results could also be explained by a recent theory that differences in CJB will emerge when there is a mismatch between the animals’ expectations and the actual event that follows  genetic and numerous, partly stochastic, environmental influences, which cannot be easily disentangled . This asR = 0.30 for the “near positive” and R = 0.23 for the “middle” cue, indicating moderately stable individual differences in CJB over seven weeks. To our knowledge, this is the longest period for which the repeatability of CJB tests has so far been estimated . The reason for this is not clear but might be due to a potentially lower response accuracy toward this cue. Mice showed a lower response accuracy toward the negative than toward the positive reference cue, in our study . C57BL/6J mice scored lower on PC1 than B6D2F1N mice, which indicates lower levels of anxiety-like behavior in C57BL/6J compared to B6D2F1N mice. This is in accordance with a previous study comparing anxiety-like behavior between these two strains . FurtherFurthermore, mice from the complex environment scored lower on PC1, which indicates reduced anxiety-like behavior compared with mice from the “scarce” environment. Again, this is in line with previous studies showing positive effects of environmental enrichment on anxiety .Regarding “spatial learning” (PC2), neither genotype nor environment significantly influenced performance in the labyrinth maze. Concerning genotype, to our knowledge, the only study that compared the same two strains in a spatial learning task showed that B6D2F1 mice outperformed both parental strains . HoweverAlbeit not in the focus of this study, we controlled for touchscreen training in the statistical models run for PC1 and PC2, since not all mice received training . We founWe systematically investigated the influence of two genotypes and two environments on cognitive judgment bias, anxiety-like behavior, and spatial learning in laboratory mice. We found that although the selected genotypes and environments influenced some aspects of anxiety-like behaviors, there was no influence of genotype and/or environment on the choice score from the CJB test and spatial learning. Similar discrepancies between CJB and anxiety-like behaviors have already been reported in other studies, indicating that CJB and anxiety may represent distinct systems. Consequently, a “pessimistic” individual might not necessarily be an anxious one.Furthermore, we provide the first evidence for CJB to be repeatable across several weeks in laboratory mice, indicating that behavioral responses in CJB tests represent a stable trait. On this basis, we suggest that individual differences in CJB reflect not only emotional states but also personality differences in decision-making under ambiguity. However, the proximate causes of these individual differences still need to be elucidated.Future research should thus aim to identify and quantify the specific drivers of individual differences in CJB. Furthermore, the study of “optimism” and “pessimism” from the perspective of animal personality could provide valuable insights into ecological and evolutionary processes. In particular, empirical studies may answer the question, whether optimists and pessimists do indeed adjust differently to a given environment, and whether they have different social and environmental preferences. Eventually, future studies can contribute to our understanding of the fitness consequences resulting from being an optimist or a pessimist.http://www.beheco.oxfordjournals.org/Supplementary material can be found at Supplementary Video 1: Playground system for providing complex environmentSupplementary Video 2: Touchscreen-based cognitive judgment bias testSupplementary Figure S1: Scree plot from PCA on 13 behavioral parameters from the battery of behavioral testsSupplementary Figure S2: Choice score for each cue in cognitive judgment bias (CJB) test pooled across treatment groupsSupplementary Figure S3: Individual choice scores across repeated cognitive judgment bias (CJB) tests for each cueSupplementary Table S1: Discrimination training stepsSupplementary Table S2: Spatial learning in the labyrinth maze (LM)Supplementary Table S3: Summary statistic and pairwise comparison of choice scores for each cue in the first CJB test phaseSupplementary Table S4: Statistical analysis of CJB test and behavioral test batterySupplementary Table S5: Trait loadings on principal components and component importance from principal component analysis (PCA) of the behavioral test batteryAnalysis of behavioral test battery without PCAarac040_suppl_Supplementary_Video_1Click here for additional data file.arac040_suppl_Supplementary_Video_2Click here for additional data file.arac040_suppl_Supplementary_MaterialClick here for additional data file.


Pyroptosis plays a crucial role in bronchopulmonary dysplasia (BPD) and is associated with various lung injury illnesses. However, the function of pyroptosis-related genes (PRGs) in BPD remains poorly understood. The gene expression omnibus (GEO) database was searched for information on genes associated with BPD. Twenty-five BPD-related DE-PRGs were identified, all of which were closely associated with pyroptosis regulation and immunological response. LASSO and SVM-RFE algorithms identified CHMP7, NLRC4, NLRP2, NLRP6, and NLRP9 among the 25 differentially expressed PRGs as marker genes with acceptable diagnostic capabilities. Using these five genes, we also generated a nomogram with excellent predictive power. Annotation enrichment analyses revealed that these five genes may be implicated in BPD and numerous BPD-related pathways. In addition, the ceRNA network showed an intricate regulatory link based on the marker genes. In addition, CIBERSORT-based studies revealed that alterations in the immunological microenvironment of BPD patients may be associated with the marker genes. We constructed a diagnostic nomogram and gave insight into the mechanism of BPD. Its diagnostic value for BPD must be evaluated in further research before it can be used in clinical practice. Bronchopulmonary dysplasia (BPD) is the leading chronic respiratory disease in preterm newborns and is associated with a high mortality rate . BPD is Pyroptosis, also known as gasdermin-mediated programmed cell death, is triggered by inflammasomes and induced by the activation of caspase-1 and caspase-4/5/11 in canonical and noncanonical pathways, respectively, resulting in DNA cleavage, cell and organelle swelling, membrane rupture, and release of endocellular proinflammatory cytokines, including IL-1 and IL-18 . Pyroptovia the mechanism of mediating inflammation  as biomarkers for BPD. Additionally, bioinformatics was used to delve into the roles of these genes in the immune microenvironment. This study provides new insight into the pathophysiology of BPD as well as the development of therapeutic interventions for patients who suffer from this condition.via G*Power software (We retrieved the gene expression data for BPD and control samples from the Gene Expression Omnibus (GEO) database. GSE32472 provides microarray profiles of blood samples from neonates with BPD, including microarray analyses of gene expression on the 5th, 14th, and 28th days of life. On day 28, after a more definitive diagnosis of BPD was established, 100 blood samples were chosen at random to verify the reliability of the selection process. These samples were collected from 38 controls and 62 patients with BPD. We calculated the Effect size d to evaluate the statistical power of the sample size software , which csoftware .p-value less than 0.05 was chosen to serve as the criterion for statistical significance.Expression data for 55 PRGs were initially retrieved from the GSE32472 dataset for control and BPD samples. Afterward, we run the Wilcoxon test in the R software to identify the differentially expressed PRGs (DE-PRGs) between the two samples. A http://metascape.org) was employed to explore the biological significance of the differentially expressed genes  models, and the average misjudgment rates of their 10-fold cross-validation were compared. In addition, optimum gene biomarkers for BPD were discovered using biomarkers produced from both algorithms that overlapped. The diagnostic capabilities of the optimal gene biomarkers were evaluated by generating the receiver operating characteristic (ROC) curve and determining the area under the curve (AUC), sensitivity, specificity, and accuracy. Also, the prediction function in the R package was used for the purpose of developing a logistic regression model based on the five marker genes. This was carried out to predict the types of samples present in the GSE32472 dataset. We used ROC curves to assess the logistic regression model’s diagnostic power.The GSEA R package was used to perform this analysis. To further investigate the linked pathways of the five marker genes, the correlation between these marker genes and the other genes in the GSE32472 dataset was evaluated. The genes were then ranked, from highest to lowest, based on their correlations, and the ranked genes were referred to as the test set. The KEGG pathway set served as a predetermined set to discover its enrichment in the gene set.p < 0.05 and |t| > 2. When t was more than zero, the pathway was considered to be enriched in the group that had high expression levels; conversely, when t was less than zero, the pathway was considered enriched in the group that had low expression levels.The GSVA R package was used for this analysis. The KEGG pathway enrichment set was used as the background gene set to analyze each marker gene. Using the limma program, we simultaneously performed an analysis of the difference in GSVA scores that existed between groups that had high and low-expression levels of the marker genes. The screening condition difference were CIBERSORT is a technique for determining the cell fraction of complex tissues based on gene expression profiles . We usedmRNA–miRNA interaction pairs based on the five marker genes were predicted using the starBase database . RNA seqA diagnostic nomogram based on selected candidate PRGs was established using the “rms” package in the R software. The consistency between our predicted values and reality was assessed using a calibration curve. Clinical impact curve analysis and decision curve analysis (DCA) were carried out to determine whether or not the decisions made based on the model were favorable to the patients.http://pga.mgh.harvard.edu/primerbank/) and synthesized by Generay . The sequences were as follows: NLRC4, forward 5′-TGCATCATTGAAGGGGAATCTG-3′ and reverse 5′- GATTGTGCCAGGTATATCCAGG-3′; CHMP7, forward 5′- AAGCCTCTCAAGTGGACTCTT-3′ and reverse 5′- ACAGACGATACACCTCCTCAG-3′; NLRP2, forward 5′- TGGCCTGGAGATAGCAAAGAG -3′ and reverse 5′- CACCACCGTGTATGAGAAGGG -3′; NLRP6, forward 5′- CCTACCAGTTCATCGACCAGA-3′ and reverse 5′- CTCAGCAGTCCGAAGAGGAA -3′; NLRP9, forward 5′- TTTGGCTTGTTGTGGTATCTGAA -3′ and reverse 5′- CTGGGTAATGTTTGTCCAGCA-3′; and GAPDH, forward 5′- GGAGCGAGATCCCTCCAAAAT-3′ and reverse 5′- GGCTGTTGTCATACTTCTCATGG -3′.The JCRB Cell Bank provided the human lung adenocarcinoma epithelial cell line, A549. It was grown in DMEM with 1% penicillin/streptomycin solution and 10% fetal bovine serum . Experiments on hyperoxia were carried out in a Modular Incubator Chamber  in accordance with the manufacturer’s instructions. In details, the hyperoxia group was cultivated in a MIC-101 incubator with 85% O2 and 5% CO2, whereas the normoxia (NO) group continued to be cultured in an incubator with 21% O2 and 5% CO2. The RNeasy Mini Kit was used to extract total RNA . Utilizing ReverTra Ace, RNA was reverse transcribed into cDNA . Using the LightCycler 480 , quantitative real-time PCR was carried out using SYBR Premix Ex Taq II . Primers used were designed in PrimerBank  and 118 edges were present in the network . Five lnBPD is a complex condition with a substantial hereditary predisposition. Various pathways and genetic variations, according to some researchers, may be connected with BPD susceptibility. Each genetic variation plays a role in the onset and progression of BPD, and these variants result in the dysregulation of biological processes that occur in the lungs of preterm newborns . Therefovia a KLF2-mediated mechanism (Pyroptosis is a caspase-1/4/5/11-dependent programmed cell death that results from inflammasome activation and is accompanied by rupture of the cell membrane, pore formation, and leakage of the cell content. It is involved in the pathogenesis of respiratory diseases, such as asthma, acute lung injury, BPD, pulmonary fibrosis, and chronic obstructive pulmonary disease . In the echanism . 18β-Glyechanism . It is cThis research identified a total of five different genes associated with pyroptosis, including CHMP7, NLRC4, NLRP2, NLRP6, and NLRP9. The ROC curve revealed that all five genes had AUC values higher than 0.6, indicating that the five genes can distinguish between BPD and control samples with a high degree of accuracy and specificity. The three highest AUC values were for CHMP7, NLRC4, and NLRP6. Excessive activation of CHMP7 lowers translation efficiency and may result in severe membrane deformation, in addition to affecting cell development, metabolism, cell division, and the internal environment stability . Cancer + T cells, and NK cells protect against viral infection and can also play a role in immunopathology due to their contact-dependent effector actions. INF-α and TNF-α are believed to be the major agents responsible for T cell-mediated lung damage (Emerging evidence suggests that immune cells play a crucial role in chronic lung illness in preterm infants. TNF receptors, TNF-α, CD8g damage . Using tin vivo experiments and not enough in vitro experiments to verify the level of expression of the genes identified, thus reducing the precision of our study. Consequently, further validations are required to make the findings more convincing.Our research has some limitations. First, we discovered one of the largest BPD datasets for analysis, but the number of included samples was still small; hence, a large cohort is required to corroborate our results. There were no Various bioinformatics approaches and online databases were used in this study. Five marker PRGs were used to establish a diagnostic nomogram for BPD. We also performed immune infiltration analysis and constructed a ceRNA network. The findings of our study may provide new insights into the molecular mechanisms of BPD.


High quality, readable health information is vital to mitigate the impact of the COVID-19 pandemic. The aim of this study was to assess the quality and readability of online COVID-19 information using 6 validated tools. This is a cross-sectional study. “COVID-19” was searched across the three most popular English language search engines. Quality was evaluated using the DISCERN score, Journal of the American Medical Association benchmark criteria and Health On the Net Foundation Code of Conduct. Readability was assessed using the Flesch Reading Ease Score, Flesch-Kincaid Grade Level and Gunning-Fog Index. 41 websites were suitable for analysis. 9.8% fulfilled all JAMA criteria. Only one website was HONCode certified. Mean DISCERN score was 47.8/80 (“fair”). This was highest in websites published by a professional society/medical journal/healthcare provider. Readability varied from an 8th to 12th grade level. The overall quality of online COVID-19 information was “fair”. Much of this information was above the recommended 5th to 6th grade level, impeding access for many. Coronavirus disease 2019 (COVID-19) was first described in Wuhan, China in December 2019. As of June 3rd 2021 it has caused more than 3.5 million deaths, with 171 million confirmed cases [Journal of the American Medical Association [JAMA] benchmark criteria, DISCERN criteria, HONcode certification) and readability  of online healthcare information.Historically healthcare professionals have represented the primary source of health care information for the public. However, recent years have seen major changes in terms of the availability of health information through other sources. There has been an increase in internet usage worldwide, as well as an increase in the depth and breadth of online content , 4. The Guidelines specify health information intended for consumption by the general public should be at a 5th to 6th grade reading level ages 10 to 12) . Previou to 12 . COVID-19”, “2019-nCoV”, “novel coronavirus”, “COVID”, “Coronavirus”, “Coronavirus disease” and “SARS-CoV-2”. Those terms were searched across Google, Bing, and Yahoo! These search engines account for over 97% of United Kingdom (UK) searches  + 100 x [complex words/words]).The FKGL and GFI formulae both produce scores as US educational system grade levels, thereby being more readily interpretable than scores produced by FRES. By way of basic guide, US grade 1 approximates to ages 6–7, while grade 12 corresponds to ages 17–18 .These readability scores were chosen as they are validated systems which have been shown to be consistent in terms of their results , 30, whip < 0.05.Data analyses were performed using Prism 7 . Normally distributed continuous data are presented as the mean and standard deviation ; non-nor“COVID-19”, “2019-nCoV”, “novel coronavirus”, “COVID”, “coronavirus”, “coronavirus disease” and “SARS-CoV-2” were searched across the three most commonly used English language search engines. The search term “COVID-19” provided the most results, with a combined total of 13,827,000,000. The three next highest scoring search terms were “COVID” , “novel coronavirus”  and “2019-nCoV” . The search term “COVID-19” was therefore selected for analysis.On July 13th 2020 the search terms The 25 top-ranking websites from each search engine were initially included. Of these 75 websites, 34 were excluded. These were 30 duplicate websites, 2 websites providing financial information as opposed to healthcare information, 2 non-readable websites . Therefore, 41 websites were included for analysis. The internet search strategy is summarized in Fig.  and pay Of the 41 websites accessed 17 were governmental organisations. Given the UK search settings, unsurprisingly there was a predominance of UK sites among these (n = 8). Otherwise there was a relatively even distribution, albeit with mostly Anglophone countries featuring, including two United States governmental sites, two European Union and one each from Canada, Australia, New Zealand, South Africa and Nigeria. There were 10 non-governmental organization sites, including the World Health Organization, Wikipedia and Afghanaid. All except one of the 7 for profit corporation websites were news outlets, including the Daily Mail, CNN and the South China Morning Post. The 7 sites in the professional society/medical journal/healthcare provider category also accounted for a relatively broad geographical distribution, including the Lancet, the European Respiratory Society and Johns Hopkins Medical.JAMA benchmark criteria: four from the professional society/medical journal/healthcare provider category and two non-governmental organisations.Six of 41 (9.8%) websites fulfilled all four Overall, the name of the website author was reported in 14/41 websites (34.1%). 22/41 (53.7%) were compliant with the attribution criterion (listing of references and sources for content), website ownership, sponsorship, advertising and commercial funding arrangements were disclosed in 20/41 websites (48.8%), while the website currency criterion (listing of dates at which content was posted and updated) was adhered to in 39/41 websites (95.1%).JAMA score varied significantly according to publishing organisation type (H(3) = 11.27; p = 0.0103). Websites produced by governmental organisations had a significantly lower score (median = 2) compared with those produced by professional societies/medical journals/healthcare providers (median = 4) (p = 0.0077). Pairwise comparisons did not otherwise identify significant differences . Websites of a professional society/medical journal/healthcare provider origin were seen to have a significantly higher DISCERN score  than those produced by the other 3 categories of publisher: governmental organisation , non-governmental organisation  and for profit organisation  .[Only one of the 41 websites (2.4%) was HONcode certified. This webpage was OVID-19).The mean FRES of all websites (n = 41) was 53.9 (SD ± 14.3), indicative of a 10th to 12th grade reading level, mean FKGL was 7.6 (SD ± 2.1) equating to an 8th grade reading level, while mean GFI was 7.3 (SD ± 2.7) also indicating an 8th grade reading level.2 = 0.2108; p = 0.031). The mean GFI of websites produced by for profit organisations (n = 7) was 4.69, equating to a 5th grade reading level. This was significantly lower than that seen for government organisations (p = 0.0324) and websites produced by professional societies/medical journals/healthcare providers (p = 0.0450)  and FKGL  scores by organisation type  published the guideline ‘Communicating risk in public health emergencies’ . This woOverall this analysis showed that COVID-19 online information readability levels—as assessed by all three scoring systems—were greater than the recommended 5th to 6th grade level [This study had limitations. While the readability evaluation tools FRES, FKGL and GFI are objective and reproducible for text websites, they do not analyse audio, image or video based information. This limitation has been identified in other similar studies . ConversThis novel study identifies several objective deficiencies in both the quality and readability of online COVID-19 information. Much of the content is difficult to understand for a substantial proportion of the population, and the quality of that information is variable and often poor. A knowledge of these quality and readability assessment tools can assist clinicians in identifying the best sources of online information for patients, as well as providing a means through which authors can optimise the quality and readability of patient information prior to publication. Furthermore, ensuring health information quality and readability protects the rights of the individual to freedom of expression and opinion, as well as safeguarding against false information, particularly in times of public health emergency.


Monosodium glutamate (MSG) is a flavoring agent added to various foods. This experimental study investigated MSG effects on chicken embryos morphology and the possible ameliorative effects of pomegranate peel extracts (PPE) at different incubation periods.Seven hundred and twenty fertilized chicken eggs were used and divided into six groups: control, PPE, MSG, PPE + MSG, preventive (PPE–MSG) and therapeutic (MSG–PPE) groups. Fertile chicken eggs were injected with MSG (0.1 ml) and/or PPE (0.3 ml) twice before incubation at days 0, 1. Embryos were extracted at days 7, 10, 12, 14 and 16. Effects of MSG and/ or PPE on embryo development during different incubation periods were studied.MSG injected into embryos led to congenital anomalies that appeared mainly in MSG and MSG + PPE groups. These anomalies included growth retardation, absent eye, abdominal swelling and hernia. Mortality rate was the highest in MSG, then in MSG + PPE and MSG–PPE groups. PPE treatment reduced MSG toxic effects and these results were better in MSG–PPE and PPE–MSG groups than MSG + PPE group.MSG injection affected chicken embryonic development causing growth retardation and decline in total body length, break length, and total body weight in all the treated groups. These harmful actions can be ameliorated with PPE treatment depending on embryo age. GlutamaMSG leads to generation of free radicals, activation of proteases, phospholipases and endonucleases. It also causes transcriptional activation of apoptotic programs and genotoxicity in mice and rats . This ocA major advantage of using chicken embryo as a model in experimental biology is that one can window the egg, examine embryo, and precisely target exposure to its specific developmental stages . AdminisPunica granatum L.) is a fruit of tropical and subtropical regions. It originated in India and the Middle East and has been used for centuries for medicinal purposes. Pomegranate is consumed fresh and in processed forms as juice, flavors, or extracts  during different incubation periods.22.1MSG solution was made by dissolving 30 mg of MSG powder into 0.1 ml of distal water. It was injected in a dose of 0.1 ml before incubation according to 2.2control group injected with 0.1 ml of distilled water during the first week of incubation at days 0, 1 and was opened at days 7, 10, 12, 14 and 16 of incubation, each age consisted of 24 eggs. Pomegranate peel extracts treated group (PPE) was injected with PPE at days 0, 1 at a dose of 0.3 ml/egg (Seven hundred and twenty fresh fertilized chicken eggs were used (weight range was 60–62 g). These eggs were divided into six main groups (120 eggs each) as following: 3 ml/egg . Mono so3 ml/egg . Group tThe focus had been on determining the injection period in three stages, which were pre–incubation , at beginning of internal organs formation (day 1) and during completion of internal organs formation (days 3 and 4), because these periods are considered sensitive to any external effects that affects embryonic formation process. Selection of opening times was based on the end of the sensitive period at day 7 of the first week that is considered the end of embryonic formation, and was identified at days 10, 12, 14 of the second week of embryo formation to monitor the continued organs growth. Monitoring the occurrence of possible effects level was determined at day 16 of third week of incubation after completion of embryonic organ development to study the effects of experiment materials on organs maturity.2.3At days 7, 10, 12, 14 and 16, the eggs were opened at blunt end and the embryos were extracted, cleaned by washing with saline solution. Mortality rate was determined. Embryos were then dried and weighed. The external shape of embryos was observed, reported and photographed.2.4All embryos were photographed by digital camera (Canon Eso 600 D); a ruler was put near the embryo which was used as a scale when conducting morphometric using the photos. The distance between specimen and camera was the same for all the body photos.2.5http://compdent.uthscsa.edu/dig/itdesc.html)  . Total b2.6P < 0.05.Data was analyzed using SPSS version 19. Differences of the measured parameters in different studied groups versus control group were tested using One Way ANOVA test followed by least significant difference (LSD) test for multiple comparison, Significance was considered at 33.1Virtual examination of embryos revealed the presence of visible birth defects in a number of embryos of the studied groups at days 7, 10, 12, 14 and 16. In MSG group, morphological changes were found at day 7 such as small sized embryo, abdominal edema, abdominal hernia, exit of abdominal contents, small sized eye, congestion of brain region and increase in transverse region of brain in head. At day 10, malformations included small sized embryo, abdominal hernia, exit of abdominal contents, absent eye and increase in transverse region of brain. At day 12, malformations were small sized embryo, anomalies of extremities, abdominal distension, exit of abdominal contents, abdominal hernia and increase in transverse region of brain in head region. At day 16, there were abdominal edema, hernia and exit of abdominal contents . In MSG 3.2There were no deaths in the control, PPE and PPE–MSG groups. However, deaths were observed in MSG, MSG + PPE and MSG–PPE groups. The mortality rate was the highest among MSG group , then MSG + PPE , and lastly MSG–PPE  .Table 1C3.3P < 0.0001) and highest decrease was in MSG . Total length decease in PPE–MSG group was  and in MSG–PPE group was . A significant decrease in embryo body length was observed in MSG + PPE group versus control at day 7  and at days 10, 12, 14 and 16   versus control, with the least decrease was in PPE  .Table 2C3.4P > 0.05). Significant decrease was observed in embryo total body weight at day 10 of incubation in PPE , MSG , MSG + PPE , PPE–MSG  and MSG–PPE ; at day 12 of incubation in PPE , MSG , MSG + PPE , PPE–MSG  and MSG–PPE ; at day 14 of incubation in PPE , MSG , MSG + PPE , PPE–MSG  and MSG–PPE  and at day 16 of incubation in PPE , MSG , MSG + PPE , PPE–MSG  and MSG–PPE .The results showed in 3.5P = 0.033), MSG , MSG + PPE , PPE–MSG  and MSG–PPE ; at day 12 of incubation in PPE , MSG , MSG + PPE , PPE–MSG  and MSG–PPE ; at day 14 of incubation in PPE , MSG , MSG + PPE , PPE–MSG  and MSG–PPE ; at day 16 of incubation in PPE , MSG , MSG + PPE , PPE–MSG  and MSG–PPE . Meanwhile, at day 10 of incubation significant decrease was observed in embryo neck length versus control in MSG  and MSG + PPE   .Table 4C3.6P < 0.0001; mean difference = 0.095, P < 0.0001, respectively); at day 10 ; at day 12, 14 and 16   .Table 5C3.7P = 0.002; mean difference = −0.060, P < 0.0001, respectively). Meanwhile, significant decrease was observed in embryo eye diameter versus control at day 12 of incubation in MSG , MSG + PPE , PPE–MSG  and MSG–PPE ; and at day 14 of incubation in MSG , MSG + PPE , PPE–MSG  and MSG–PPE   .Table 6C4The results of the present study showed congenital malformations in the examined embryos mostly in MSG group followed by MSG + PPE and MSG–PPE groups. These malformations included retarded growth, body congestion and small eye size. With increased embryo age, loss of eyes and more malformations were reported as brain hemorrhage, widening in brain spaces. However, abdominal hernia and exit of abdominal contents were found in all age groups. No malformations were observed in PPE–MSG and PPE groups. MSG side effects vary from delay in cell division to morphological abnormalities. In consistence with our results, others  reportedIn consistence with MSG + PPE and MSG–PPE groups were less affected by MSG, while PPE and PPE–MSG groups showed no malformations. This is due to due to antioxidant substances, vitamins, organic and inorganic amino acids present in PPE that led to balance in embryos growth and inhibition of malformation. Antioxidant substances present in PPE remove free oxygen radicals that result from MSG administration. Vit A present in PPE has an important role in embryos growth, helps epithelial cell differentiation, enhances hematopoiesis and brain growth and prevents lipid peroxidation and DNA destruction. Vitamin C increases immunity and enhances proteins formation . So, PPEThe results of the present study indicated that the mortality rate was the highest in MSG group, followed by MSG + PPE group, then MSG–PPE. Death resulted from direct MSG effect on cell metabolism in embryos . IncreasIn this study, significant decrease in embryo body length, neck length and total body weight was observed in all studied groups at different periods versus control group with the least significant decrease was observed in PPE then PPE–MSG group. However, the highest significant decrease was observed in MSG followed by MSG + PPE and lastly MSG–PPE group. Significant decrease in embryo beak length was observed only in MSG and MSG + PPE groups at different studied periods versus control. This agrees with In this respect, In contrast, MSG may induce increase in total body weight and body fat accompanied by insulin resistance as MSG molecules block leptin hormone receptors in hypothalamus, preventing its action and so increase palatability of food rich in taste enhancements, resulting in obesity . Also, BThe results of this study showed that the eye diameters in MSG and MSG + PPE groups at age of (7–10) days were greater than control. This increase is caused by an increase in retinal and eye outer membrane thickness as a result of brain swelling in MSG treated embryos. On the other hand, eye diameter in MSG and MSG + PPE groups at days 12 and 14 were smaller versus control and this was consistent with On the other hand, many scientific researches mentioned the important role of pomegranate peel in weight loss, and found positive results not only on obesity but also on other health aspects. Pomegranate decreases weight by inhibition of pancreatic lipase enzyme activity that digests fats, and reduce amount of calories consumed, in addition to its role as an antioxidant and anti–inflammatory . Sayed-A5MSG had different side effects on embryos, especially in early ages, and these effects include decrease in total body weights and lengths of embryos, decreased growth, slow maturity, and defect in the formation of two or more body organs as a result of the failure to develop and complete the body's defense systems. It was observed that some embryos showed resistance to damage resulting from MSG treatment when treated with PPE. Meanwhile, in some groups, PPE did not improve MSG side effects especially in MSG + PPE and MSG–PPE groups. The variation in the rates of weights and lengths of embryos in experimental groups was due to different incubation period and time of PPE administration. As a result, MSG and PPE resulted in different effects.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Plant response to osmotic stress is a complex issue and includes a wide range of physiological and biochemical processes. Extensive studies of known cultivars and their reaction to drought or salinity stress are very important for future breeding of new and tolerant cultivars. Our study focused on the antioxidant activity, accumulations of osmotica, and the content of abscisic acid in apple  and cherry  cultivated in vitro on media containing different levels of polyethylene glycol PEG-6000. Our results indicated that the studied genotypes responded differently to osmotic stress manifested as reduction in the leaf relative water content (RWC) and increment in the activities of antioxidant enzymes, proline, sugars, and abscisic acid content. Overall, cherry cultivars showed a smaller decrease in percentage RWC and enzymatic activities, but enhanced proline content compared to the apple plants cultivars. Cultivars “Rubinstep”, “Napoleonova”, and “Kaštánka” exhibited higher antioxidant capacity and accumulation of osmoprotectants like proline and sorbitol that can be associated with the drought-tolerance system. Plants are constantly exposed to various types of stress, of which the most severe is stress produced by water imbalance in the ecosystem. Both decreasing rainfall and rising average temperatures reduce water storage in soil and cause drought stress. Moreover, irrigation with high ion content water and overuse of chemical fertilizers significantly contributes to soil salinization. Drought and high soil salinity lead to osmotic stress in plants and represent the major abiotic stress factor that affects the growth of plants and agricultural production worldwide . ResponsIn general, osmotic stress causes a wide range of morphological, physiological, and biochemical changes that are crucial for mitigating the negative effect of water deficiency. One of the known mechanisms that plants use to avoid water deficit in salt or drought stress environment is osmotic adjustments which are crucial for maintaining cell turgor and plant metabolic activity. Plants synthetize osmotica such as proline or soluble sugars to establish osmotic balance at the cellular level . Proline2O2 and superoxide ·O2−, in plant cells. Overproduction of ROS is toxic for plants because they cause the oxidation of lipids, proteins, nucleic acids, and carbohydrates that can result in irreversible changes in plants or lethality. Plants have evolved complex antioxidant system to protect cellular membranes and organelles from the damaging effect of ROS that consists of both enzymatic and non-enzymatic mechanisms of detoxification. Non-enzymatic antioxidant system includes both water soluble low molecular mass compounds, such as ascorbic acid or glutathione, and lipid soluble antioxidants such as α-tocopherol and β-carotene  × 100, where Acontrol is the absorbance of the solution without extract and Asample is the absorption of the solution with extract.Antioxidant capacity of plant extracts was assayed using previously reported  discolor−1, pH 7.0). The homogenates were centrifuged at 14,000 rpm for 15 min at 4 °C. The SOD activity (EC 1.15.1.1) was determined following the manufacturer instructions of SOD assay kit  and expressed as inhibition rate (%). Superoxide anions generated by oxidation of xanthine reduce nitroblue tetrazolium to nitroblue tetrazolium formazan. The presence of SOD reduces the level of superoxide, which also decreases the level of formazan derivative. The activities of CAT (EC 1.11.1.6) and APX (EC 1.11.1.1) were measured at 240 and 290 nm following the decomposition of H2O2 and oxidation of ascorbic acid, respectively, and expressed as µmol mg−1 proteins and nmol mg−1 proteins, respectively [For detection of enzymatic activities, 200 mg of fresh leaves was homogenized in cooled potassium phosphate buffer  and a StrataTM-X-C 33 µm column  via elution with methanol. The desired fraction was evaporated to dryness and subsequently analysed using UHPLC-MS. Concentration of abscisic acid in all samples was determined using UHPLC Infinity II 1290 system coupled to a 6470 Series Triple Quadrupole mass spectrometer (Agilent Technologies) with negative electrospray ionization (ESI). The method included a Zorbax RRHD Eclipse plus C18 column , column temperature 40 °C, injection volume 1 µL, the mobile phase flow rate 0.40 mL min−1, and a gradient elution program 0 min–10% A, 1.0 min–10% A, 4.0 min–95% A, 5.0 min 94% A, 5.1 min–10% A, and 6.0 min–10% A in B, where A was Milli-Q water and B was acetonitrile. MS conditions were as follow: gas temperature 300 °C, gas flow 6 L min−1, nebulizer 40 psi, sheath gas temperature 400 °C, sheath gas flow 12 L min−1, capillary voltage 2000 V, and nozzle voltage 1200 V. Two MRM transitions were monitored: 263.1→219.1, 153.1.Fresh plant material (100 mg) was immediately frozen in liquid nitrogen and stored at −80 °C until used. The extraction of abscisic acid (ABA) was carried out as described previously . Brieflyp < 0.05) using Minitab v. 19 software . Number of replications (n) in tables/figures denotes number of individual samples measured for each parameter.Statistical differences among the treatments and cultivars were evaluated using analysis of variance (ANOVA) followed by Tukey test  . OverallIncreasing concentration of PEG in medium stimulated the proline accumulation in all tested apple and cherry cultivars . The maxIn case of the tested apple explants, a sharp increment in monitored sugars content after the application of the two lowest doses of PEG was observed in almost all varieties . SubsequWe followed the changes in the concentration of two sugar alcohols. Glycerol maximum was recorded for apple cultivars only at higher PEG concentrations, specifically in PEG25 for “Malinové holovouské” (3.7 times) and in PEG50 for “Rubinstep” (more than 2.5 times). The maxima in cherries at a PEG5concentration ranged to “P-HL-C” (3.5-fold) and “Kaštánka” with more than 2.5-fold increase. A more than 3-fold increase in second sugar alcohol sorbitol was observed in the varieties “Rubinstep”, “Idared”, and “Napoleonova”. The maxima differed between both individual sugars and varieties. Thus, we could not determine unambiguously in which variety the synthesis of sugars was most stimulated. However, the above results show that the most often mentioned apple cultivar included “Rubinstep”, and cherry cultivars “Napoleonova” or “Kaštánka”.Our results show that the plants synthesized a sufficient amount of antioxidants and their total antioxidant capacity, measured as scavenging of DPPH radical, decreased only slightly with the increasing PEG concentration. The most significant differences were noticed in apple cultivars “Fragrance” and “Malinové holovouské” and in cherry cultivars “Regina” and “Napoleonova” .A simultaneous increase in activities of monitored enzymes with increasing PEG concentration was noticed in tested apple and cherry cultivars . The higThere was a sharp increase in APX activity in apples after application of the lowest PEG dose that further enhanced with increasing PEG concentration. The lowest values were observed again in the cultivar “Malinové holovouské” where the rose was also the highest (up to 3.5 times). APX activity was significantly stimulated in “Idared” as well (up to 3-fold) and “Car Alexander” (2.5-fold). APX activity values within cherry cultivars displayed a similar accumulation trend as in activity of CAT. Overall, the values were lower compared to apples and the increase in activity was gradual. The lowest values of PEG0 and PEG-treated plants were observed in the cultivar “Regina”. The most stimulated enzymatic activity was recorded in “Sunburst” (more than 3.5-fold).The most significant increment in SOD activity in apple explants was recorded in cultivars “Rubinstep” and “Car Alexander” (approximately 2-fold). At the same time, these varieties had the lowest enzymatic activity seen for PEG0 plants. On the contrary, the highest values in PEG0 plants were typical of cultivars “Fragrance” and “Idared” and the lowest increment in activity was recorded due to an increase in concentration of PEG in the medium. The increment in SOD activity was less pronounced in cherries. The highest values were recorded for the “Napoleonova”, but only a 1.2-fold increase. At the same time, the lowest activity of PEG0 plants was observed in this case. Overall, it can be noted that SOD activity, both with and without the addition of PEG, was significantly lower in cherries.The addition of PEG in the medium was associated with a significant increase in ABA content, compared to control (PEG0). The increase of PEG concentration over 5% for apple and over 10% for cherry explants caused a significant reduction of ABA content. The highest increase, up to 27 times, was observed for apple cultivar “Malinové holovouské”. For cherry cultivars “Napoleonova” and “Kaštánka” more than 20-fold rose was monitored. Overall, higher levels in PEG0 plants were in the “Rubinstep”, “Idared”, and “P-HL-C”, while simultaneously the lowest increase in ABA content of only 2.5–3.5-fold after the PEG application was observed .Apples and cherries are among the most cultivated fruit trees both in the Czech Republic and Europe. The total yield of these trees is affected by water availability. The growing number of dry days during the vegetative season and worldwide increasing shortages of water led to the search for drought-resistant cultivars. The response of plants to limited water supply are monitored at multiple levels . In the Prunus rootstock [Leaf water potential and RWC are usually used as important and reliable indices of plant water deficit or water status. In agreement with our finding, various studies reported a significant decline in RWC in several fruit species including apple rootstock , pistachootstock , as wellootstock ,37. Moreootstock  pointed ootstock ,39 that,ootstock .The influence of abiotic and biotic stress stimuli, including water deficit, leads to an imbalance between ROS production and degradation. Their elevated accumulation affects plant metabolism in different ways and leads to cellular damage. The antioxidant machinery in plants is a result of both sequential and simultaneous actions of various antioxidant molecules together with antioxidant enzymes including SOD, CAT, and APX ,10. Our In our results, it is not possible to state quite clearly which varieties are more tolerant due to changes in the accumulation of the above enzymes. Due to the sharp increase in the activities of CAT, APX, and SOD, “Malinové holovouské” and “Car Alexander” appeared as more tolerant apple cultivars. However, these findings contradict the declared low or very low drought resistance, respectively ,39. The It is well known that plants accumulate wide scale of osmoregulators such as inorganic ions, soluble sugars, proline or glycine betaine in the vacuole and cytosol that contribute to membrane stability thus reducing the negative effect of water stress . We follPrunus and safflower cultivars in comparison with a drought-sensitive one [In addition to its osmoprotective role, proline also acted as a ROS scavenger and molecular chaperone contributing to stabilization of protein structure and protection of the cells from damage caused by adverse environments . Therefotive one ,45. Thustive one .Prunus rootstock [Carbohydrates, besides their important function as energy rich molecules and building block for plant growth, play a crucial role in processes maintaining cell turgor under osmotic stress, e.g., under drought and salinity. Changes in the concentrations of water-soluble carbohydrates, mostly sugars such as glucose, fructose, sucrose, sorbitol, and mannitol, can be a result of the regulation of sugar metabolism and export ,48. Leveootstock . On the ootstock  and sorbootstock  in sorbiootstock .Owing to the putative role of proline and sorbitol as antioxidants, they could be ameliorating deleterious effects of drought-induced oxidative stress by protecting membranes and enzymes. These osmoprotectants, together with higher activity of antioxidant enzymes, show these genotypes as drought resistant ,52.Abscisic acid is regarded as a stress hormone as a result of its rapid accumulation under unfavourable environmental conditions. Therefore, overall higher ABA concentrations in plants can be associated with improved stress tolerance that helps the plant to survive . In the Potentially drought-sensitive cultivars of apple  and peppOur work focused on the relative water content, antioxidant activity, accumulations of osmotica, and the content of abscisic acid in apple and cherry cultivated in vitro on media containing increasing levels of polyethylene glycol and causing different levels of osmotic stress in tested plants. Our results confirmed that studied genotypes responded differently to osmotic stress manifested as reduction in the leaf relative water content and increment in the activities of antioxidant enzymes, as well as contents of proline, sugars, and abscisic acid. Generally, cherry cultivars exhibited a lower decrease in RWC and enzymatic activities but a higher proline content than the apple plants. Based on our findings, we concluded that apple cultivar “Rubinstep” and cherry cultivars “Napoleonova” and “Kaštánka” could be evaluated as drought tolerant since they exhibited the major features including higher antioxidant capacity and accumulation of osmoprotectants that are crucial for plant stress-tolerance. To our best knowledge, such a comprehensive analysis of mentioned responses to osmotic stress in apple and cherry in vitro culture, as we produced, has never been published. It provides fundamental information for breeders. Our study also suggests a fast method for prediction of the plant tolerance under controlled laboratory conditions that can be used for the initial screening of fruit cultivars.


The contribution of vitamin D to thyroid disorders has received paramount attention; however, results are mixed. Hence, we designed a systematic review and meta-analysis to obtain a definitive conclusion.The search included PubMed, ISI Web of Science, Scopus, and Google Scholar databases up to March 2021 to collect available papers reporting the relationship between serum levels of vitamin D and thyroid disorders. The pooled effect was reported as weighted mean difference (WMD) and 95% confidence interval (CI).P = 0.013; I2 = 99.9%), Hashimoto’s thyroiditis (HT)  and hypothyroidism patients , but not in subjects with Graves’ disease (GD) .Out of 6123 datasets, 42 were eligible to get into this systematic review and meta-analysis. Serum vitamin D was markedly lower in autoimmune thyroid diseases (AITD) (WMD − 3.1 ng/dl; 95% CI, − 5.57 to − 0.66; Our findings suggested lower vitamin D levels in patients with hypothyroidism, AITD, and HT compared to healthy subjects. However, the link between serum vitamin D and GD was only significant among subjects ≥40 years old. Vitamin D is an essential fat-soluble nutrient with hormone-like activity , 2. VitaOver the last decades, numerous studies have indicated that low serum vitamin D levels are associated with a series of diseases such as high blood pressure, heart disease, diabetes, cancer, mood disorders, multiple sclerosis, and autoimmune diseases , 9. ThisThe thyroid gland is one of the largest endocrine glands with many roles for homeostatic control, including growth, energy expenditure, and metabolism , 13. AnyAfter registration on the Prospero database (CRD42020187237), this dataset was conceived pursuant to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement .3” AND “hypothyroidism” OR “hypothyroid” OR “thyroid disorder” OR “triiodothyronine” OR “thyroxin” OR “thyroid stimulating hormone” OR “thyroid hormones” OR “hyperthyroidism” OR “hyperthyroid” OR “autoimmune thyroiditis” OR “anti-thyroid peroxidase” OR “Graves’, disease”, OR “Hashimotoʼs thyroiditis”. The authors also screened the reference sections of the contained articles to spot any other eligible studies not captured via the online database search.We performed a systematic literature search up to March 2021 in four databases, including PubMed, ISI Web of Science, Scopus, and Google Scholar to find papers reporting the relationship between serum levels of vitamin D and thyroid disorders. The terms used for the database search were “vitamin D” OR “25-hydroxyvitamin D” OR “1,25-dihydroxyvitamin D” OR “cholecalciferol” OR “ergocalciferol” OR “calcitriol” OR “vitamin DBefore the screening process, all search results were exported into the EndNote X7 software . Original observational human studies  about the association between serum vitamin D and thyroid disorders with reported means, medians, or odds ratios (ORs), and the corresponding 95% confidence intervals (CIs) were included. Studies that recruited women during pregnancy or lactation, non-human datasets, reviews, case reports, editorials, poster abstracts, non-original, or irrelevant datasets were excluded. Two investigators were responsible for the selection process to lower the potential error (S. T & A. A). The difference of opinion was dealt with the cooperation of a third reviewer (G. A).The data of interest were extracted from each study using a predefined excel form in a duplicate and blinded manner. For each study, the extracted information was as follows: first author’s name, publication year, location, sample size, sex, age, body mass index (BMI), study design, vitamin D assay method, the season of sample collection, and population health status. Two independent reviewers conducted the processes of data extraction (S. T & A. A). The difference of opinion on a given study was dealt with via discussion, and if necessary, arbitration by a third reviewer (G. A).According to the Newcastle-Ottawa Quality Assessment Scale for observational studies , authorsP-values < 0.05 considered as statistically significant. Heterogeneity was checked using the I2 index. I2 index equal to 25, 50, or 75% represents low, moderate, and high heterogeneity, respectively [Authors run a meta-analysis, including weighted mean difference (WMD) with 95% CIs plus a random effect model, to estimate the quantitative summary of the association between vitamin D and thyroid disorders, including hypothyroidism, AITD, HT, and GD. Mean ± standard deviation (SD) was extracted to estimate the pooled effects using WMD. For datasets with a standard error (SE), SD was determined using the following equation: SE × √n. Related equations were used to obtain the preferred data out of the median ± range or interquartile range . If a stectively . The funectively .Our initial search through databases identified a total of 6123 papers. With duplicates removed, the 3062 remaining articles were examined based on the review of the titles and abstracts by two independent reviewers. Authors retrieved and reviewed 98 articles based on the full text, and finally, 35 studies were eligible to get into the current study. Moreover, the works of Bouillon et al. , Unal etA total of 35 studies 42 arms), including 28 case-control , 25–51 a arms, inP = 0.013) with significant heterogeneity  .Thirteen datasets comprising 12,916 participants, inspected the association between serum levels of 25(OH) D and AITD status among 1886 AITD diagnosed and 11,030 non-AITD individuals , 58, 62.P = 0.06) and heterogeneity was significant  .Eight citations addressed the link between serum levels of 25(OH) D and GD status among 604 GD diagnosed and 2827 non-GD individuals –47, 51. 01) Fig. . SubgrouP < 0.001) with significant heterogeneity (I2 = 91.0% P < 0.001) .Twelve studies with 2440 participants examined the association between 25(OH) D and HT status among 1375 HT diagnosed and 1065 healthy subjects , 36–44. P = 0.03); patients with hypothyroidism presented with lower serum vitamin D compared to healthy subjects. There was significant heterogeneity among the included studies  , possible un-detected studies were tracked by trim and fill analysis, but this method could not add any dataset to our included ones.Nine studies examined the relationship between serum levels of 25(OH) D and hypothyroidism among 372 hypothyroid patients and 802 healthy individuals –35, 61. As far as we know, this is the first comprehensive attempt to examine the association between serum levels of vitamin D and thyroid disorders in form of a systematic review and meta-analysis. It revealed that vitamin D was significantly associated with hypothyroidism, AITD, and HT. Furthermore, vitamin D was significantly lower among ≥40-year-old GD subjects. Some issues should be considered when elucidating the results. First of all, there were substantial inter-assay differences in the performance of commercially available kits for serum vitamin D assay . This noPursuant to previous reports, the role of vitamin D in immunity is through producing anti-inflammatory and immune-regulatory markers via VDR expression in the nucleus of cells . VDR is The limitations of this study are as follows. There was significant heterogeneity in our study that may have affected the results and diminished the generalizability of the outcomes. The probable sources of heterogeneity might be differences in age, gender, BMI, study design, vitamin D assay methods and kits, the season of sample collection, geographical variation, and quality of the studies. Moreover, the nature of cross-sectional studies makes it impossible to draw a causal link between variables. Furthermore, there was no information regarding dietary vitamin D intake that could have affected serum levels of vitamin D.In the present systematic review and meta-analysis, vitamin D levels were significantly lower in hypothyroidism, AITD, and HT patients compared to healthy people. However, there was no significant association between serum vitamin D and GD, except among subjects ≥40 years old. The effect sizes of the included studies showed significant heterogeneity which might affect the interpretation of results. Further well-designed prospective cohort studies and clinical trials are needed for a better understanding of the relationship between vitamin D and thyroid disorders.


Six main constitutes in MCs were quantified by high performance liquid chromatography (HPLC) and its anti-oxidant activities were evaluated by DPPH and ABTS free radical scavenging methods. Then grey relational analysis was employed to predict the key components acting on anti-oxidative activity based on the chem-bio results. The predicted components and its mechanisms on anti-oxidation were uncovered by network pharmacology approach and cell test, respectively. The content of paeonol and paeoniflorin accounts for more than 80% the whole content of detected components. However, the two main ingredients showed a great variety among MCs. The antioxidant capacities of MCs also showed a great discrepancy based on DPPH and ABTS methods. The key components acting on anti-oxidation were identified to be paeonol, gallic acid and benzoylpaeoniflorin, and their potential therapeutic targets were predicted and verified, respectively. The present results reveal that MC has a significant antioxidant activity and the compounds of paeonol, gallic acid and benzoylpaeoniflorin could be considered as the promising antioxidant candidates with the property of suppressing oxidative stress and apoptosis.The present study determines the potential antioxidants in Moutan Cortex (MC) and predicts its targets of anti-oxidative activities. The quantitative analysis and the free radical scavenging assays were conducted to detect the main components in MC and assess its anti-oxidant activities. The grey relational analysis and the  Free radicals play an important role in maintaining homeostasis at the cellular level in the normal healthy tissues. However, when the body are exposed to the different physicochemical conditions, more free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) will be generated and thus disturb the balance of ROS generation and antioxidant defense systems, generally resulting in the oxidative stress  and subsPaeonia suffruticosa Andr, belonging to Paeoniaceae family, is a deciduous shrub with nearly worldwide distribution. The dried bark of Paeonia suffruticosa Andr, commonly called Moutan Cortex (MC), has been used in China for a long history. Many studies have identified and reported more than one hundred of ingredients from MC, including phenols, monoterpenes, monoterpene glycosides, flavonoids, tannins, and triterpenoids  is an analytical method based on the development trend of the curve shape on each factor . This meNetwork pharmacology is a new discipline based on the basic theories of systems biology. It conducts a comprehensive analysis of biological systems and further find the specific node with multiple targets . This paIn previous study, we had established a quantitative HPLC-MS method and identified main constituents in MC . In the The standards  of paeonol, paeoniflorin, benzoylpaeoniflorin, paeonolide, gallic acid and oxypaeoniflorin were purchased from Chengdu Herbpurify co, LTD. ABTS  and DPPH  were obtained from Sigma Aldrich, USA. HPLC grade acetonitrile was got from Tedia Company, Inc, USA . Formic acid with HPLC grade was obtained from Chongqing Chuandong Chemical co, Ltd. Methanol for extraction was purchased from Chendu Jinshan Chemical Reagent co, Ltd. Folin & Clocalteu’s phenol reagents were purchased from Shanghai Macklin Biochemical Co., Ltd.45 batches of MC samples were purchased from Chongqing herbal medicine market. The samples were from eight different production areas, in which 3 samples were collected from Guangxi province (GX1-3); 10 batches were from Chongqing (CQ1-10); 13 batches were from Anhui province (AH1-13); 10 batches were from Sichuan province (SC1-10); the other nine samples were from Henan province (HN1-3), Hubei (HB1-3), Shanxi (SXI) and Shandong province (SD1-2), respectively. The voucher samples were deposited at college of pharmaceutical sciences and Chinese medicine, Southwest University.Duramens were removed from MC samples before be used. Then samples were pulverized and filtered through an 80-mesh sieve. 0.50 g dried powder sample was extracted with 50 ml 70% methanol aqueous solution for 30 min in an ultrasonic water bath . After extraction, add solvent to bring the volume to 50 ml. Then the extract was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm syringe filter before analysis. All samples were analyzed in triplicates.18 . The eluent solvents consist of the deionized water with 0.2% formic acid (A) and acetonitrile (B) using a gradient program of 0–3 min, 90–89.2% A; 3–5 min, 89.2–88% A; 5–9 min, 88–87.8% A; 9–10 min, 87.8–87.7% A; 10–15 min, 87.7–87% A; 15–18 min, 87–85% A; 18–21 min, 85–84.2% A; 21–26 min, 84.2–26% A; 26–30 min, 26–10% A; 30–35 min, 10% A. The elution was performed with the eluent solvent at a flow rate of 1.0 ml/min, and 10 µl of sample solution was injected into the LC system for analysis. Two ultraviolet spectra were monitored for acquiring chromatograms of six components, at 230 nm for paeoniflorin and benzoylpaeoniflorin and 274 nm for gallic acid, paeonol, paeonolide and oxypaeoniflorin, respectively. In this study, the established method was applied to analyze the contents of paeonol, paeoniflorin, benzoylpaeoniflorin, paeoniflorin, gallic acid and oxypaeoniflorin of MCs to reflect its chemical properties.In previous study, we found paeonol, paeoniflorin, benzoylpaeoniflorin, paeonolide, gallic acid and oxypaeoniflorin  which we0 and A1 represent the absorbance of negative control and sample, respectively.The antioxidant activity of MC extracts was determined by using DPPH free radical scavenging assay with minor modification . BrieflyThe scavenging activity of ABTS radical was determined according to the reported method with slight modifications . The ABTThe grey relational analysis (GRA) was utilized as an evaluation system to assess the effects of the diverse existing compounds on antioxidant activity in this study . The conFirstly, the raw data of associated factors are normalized and then the deviation sequences are determined. Finally, the grey relational coefficients are calculated by the following equations:The correlation coefficient is defined as the following equation:n is the number of performance characteristics.The grey relation grade (GRG) is formulated as follows:The influence degree of the factors including the contents of components and antioxidant activities on the research object was estimated by comparing their GRG value. The higher GRG value between two associated factors is, the closer sequence of the two factors would be.In order to further recognize the mechanisms of the underlying the antioxidant effects on the targeted compounds from GRA, the network pharmacological approach was used, including the evaluation of the targeted compounds’ Absorption, Distribution, Metabolism, and Excretion (ADME) properties, prediction of compounds-related targets, and recognition of core functions and modules via the protein-protein interaction (PPI) network approach. We identified a core modulatory network and found the main pathway that involved in the antioxidant activities of the targeted compounds.http://www.drugbank.ca/, version 4.3) and the Online Mendelian Inheritance in Man (OMIM) database21 (http://www.omim.org/). After overlap analysis of these collected targets, the putative CM target-known therapeutic targets of the antioxidant network were constructed using the links between putative targets of CM and known antioxidant targets.Firstly, the structurally similar drugs of the selected components of CM were screened by using MedChem studio, and then the antioxidant targets of these drugs were obtained from the DrugBank database and considered as the putative targets of the selected components of CM. Next, we collected all the known therapeutic targets of drugs contributed to antioxidant effects from DrugBank20  and based on the pathway data obtained from the Kyoto Encyclopedia of Genes and Genomes database .To further clarify the pathways involved in the putative CM targets, the pathway enrichment analysis was performed by using the database Visualization and Integrated Discovery software32  was immediately added to the wells. The reagents were thoroughly mixed and assayed at 540 nm on a SYNERGY H1 microplate reader.The detection of intracellular ROS levels was conducted according to the reported methods . RAW 264Total RNA was extracted using a Qiagen RNeasy Mini kit . and cDNA was synthesized using a PrimeScript™ RT reagent Kit . The reverse transcription-polymerase chain reaction (RT-PCR) was applied to evaluate the mRNA expression of TNF, ALB, VEGFA and Caspase3. RT-PCR primers for these genes were followed: sense (5′-CCCTCACACTCACAAACCAC-3′) and antisense (5′- CACCACAGGGCAAAGGAGAT-3′) for TNF; sense (5′-AA-GACGTGTGTTGCCGATGA-3′) and antisense (5′-GGCCTTTCAAATGGTGG-CAG-3′) for ALB; sense (5′- GGGAGTCTGTGCTCTGGGAT-3′) and antisense (5′-GGTGTCTGTCTGTCTGTCCG-3′) for VEGFA; sense (5′-GGGGAGCTTGGAACGCTAAG-3′) and antisense (5′- CCGTACCAGAGCGAGATGAC-3′) for Caspase3; sense (5′- TGCTCCTCCCTGTTCCAGAG-3′) and antisense (5′- CTCGTGGTTCACACCCATCA-3′) for GAPDH. The following PCR conditions were applied at 95°C for 2 min, 40 cycles of 95°C for 15 s and 60°C for 1 min.The experimental data were presented as means of six replicates determination ±standard deviation. All statistical analyses were carried out using a SPSS20 software, Graphpad Prism software or online software.As shown in The antioxidant activity assays based on DPPH and ABTS free radical scavenging activities were applied in the present study . The resThe experimental data was combined from the quantitative analysis and anti-oxidative activity assay and then was imported it into a SPSS statistics 20 software for further analysis. Firstly, the raw data was normalized and then was used for HCA. As shown in monoterpene glycoside, also has been proved as a strong antioxidant from related studies  acting on anti-oxidative properties, we used network pharmacology strategy to predict the putative targets of these ingredients. First, 535 genes were selected and predicted as the putative targets of the three compounds, including 177 genes of paeonol, 258 genes of gallic acid and 100 genes of benzoylpaeoniflorin, and 408 targets were yielded after deletion of duplicates. Detailed information about the putative targets of the compounds was provided in Then the known therapeutic targets of drugs in the treatment of oxidation related diseases were collected. After removing redundant entries, 1,034 known therapeutic targets for the antioxidant activities were used for the further data analysis.The known therapeutic targets of the antioxidant activity networks and putative compounds-target networks were then constructed. The interaction between the target proteins was shown in The predicted targets from PPI network mainly responded to many biological process, such as intrinsic apoptotic signaling regulation, DNA damage, peptidyl-tyrosine autophosphorylation, proteolysis, and nitric oxide biosynthetic process . The celp < 0.05). The t-BHP-induced intracellular ROS accumulation was monitored within cells using a DCFH2-DA fluorescence intensity analysis. The results from cellular ROS level further indicated that these components could clearly inhibit the generation of cellular ROS induced by t-BHP. Among the components, the treatment of gallic acid and benzoylpaeoniflorin showed a dose-effect relationship, and paeonol treatment presented the most ROS inhibition at the concentration of 20 μM . Therefore, it can be suggested that the enrichment of paeonol, gallic acid and benzoylpaeoniflorin in MC could present as potential antioxidants.The genes of TNF, ALB, VEGFA and Caspase3 were predicted as the oxidative relative targets of the three components through network pharmacology analysis. Thus, we considered that the protective effects of the components against t-BHP-stimulated oxidative stress related to those target genes. The results from gene expression assay demonstrated that these ingredients could lead to the most significant decrease in the expression of the TNF, ALB, VEGFA and Caspase3 genes . This rePaeonia suffruticosa Andr, in which monoterpene glycosides and phenols were the predominant constituents (Previous study had revealed that more than 100 compounds were isolated and identified from tituents . In additituents . In the tituents . Howevertituents . Besidesin vivo (The components of paeonol, gallic acid and benzoylpaeoniflorin obtained high grades based on the chemical and bioactive evaluation by grey relational analysis and thus these compounds were predicted as key components of MC that acting on anti-oxidative activities. It is well known phenolic acids present strong antioxidant capacities , so therin vivo .Network pharmacology strategy is a potent tool to reveal the putative mechanisms of drugs like herbal medicine, which always are a complex chemical composition. In the present study, we had selected three key anti-oxidative components based on grey relational analysis. Thus, clarifying the mechanism of antioxidant action of these compounds by network pharmacology approach is a key imperative. Consequently, the compound-putative target network, PPI network with common targets for antioxidant, compound-disease were built to systematically analyze the mechanism of antioxidant action of the selected compounds. This network pharmacology study predicted the following four potential targets: ALB, TNF, VEGFA and Caspase3. Among these, ALB functions in the regulation of blood plasma colloid osmotic pressure and acts as a carrier protein for a wide range of endogenous molecules. Human serum albumin appears to reduce oxidative stress via NADPH oxidase inhibition in the human vascular smooth muscle, indicating that the serum level may be a critical determinant of vascular oxidative stress in some human diseases . TNF is By KEGG enrichment analysis, the top 10 pathways were mainly related to cancer and immune system. As we know, one theory of tumorigenesis is from oxidative stress that activates inflammatory pathways leading to transformation of a normal cell to tumor cell. Oxidative stress can activate a variety of transcription factors including NF-κB, AP-1, p53, HIF-1α, PPAR-γ, β-catenin/Wnt, and Nrf2. Activation of these transcription factors can lead to the expression of over 500 different genes including inflammatory cytokines . ExtensiTaken together, the present results revealed that MC had significant antioxidant activity, and the compounds of paeonol, gallic acid and benzoylpaeoniflorin could be considered as promising antioxidant candidates of MC and markers for quality control of MC. Furthermore, a comprehensive method based on chemical analysis, bioactivity activity assays coupled with grey relational analysis was established to identify antioxidant candidates from MC, and network pharmacology strategy was proved to be an efficient tool for uncovering pharmacological mechanism of active ingredients.


Background and Objective:ClinicalTrials.gov is a centralized venue for monitoring clinical research and allows access to information on publicly and privately funded studies. To better recognize influential institutions in the field of headache, we identified major organizations conducting clinical trials in migraine research. Furthermore, we examined the frequency of different study designs.Methods: Utilizing the ClinicalTrials.gov application programming interface, we extracted studies including individuals with migraine from February 29, 2000, to July 28, 2020, for the following: (1) host organization, (2) study type, (3) primary purpose, (4) intervention model, and (5) allocation.Results: We included 921 entries encompassing 423 organizations. Thirty-two organizations produced ≥5 entries each and 40.0% of all entries. Most, 86%, were interventional studies while 13.6% were observational studies. The most common study design had a randomized allocation of participants. The most frequent primary purpose was treatment (62.4%) followed by prevention (13.0%). There were 56.9% parallel assignment models, 15.2% single group assignment models, and 12.4% crossover assignment models.Conclusion: A minority of organizations contribute to a significant number of registrations of clinical migraine trials, suggesting that clinical research in migraine is oligarchic. The most common study is interventional and randomized, with parallel assignment of participants with treatment as the primary purpose. This likely reflects the need to evaluate novel putative pharmacological medications. Migraine directly affects more than 1 billion people worldwide and incurs significant individual and societal burden . The pasClinicalTrials.gov is a centralized database for monitoring clinical research. Created from the US Food and Drug Administration Amendments Act of 2007, ClinicalTrials.gov provides patients and healthcare professionals access to federal clinical trial registry of investigational drug applications for publicly and privately funded studies  host organization; (2) study type; (3) primary purpose; (4) intervention model; and (5) allocation. Our study consisted of four phases.We downloaded profiles of trials including subjects with migraine on Prior to any data extraction, we had to determine how many registration entries exist in the database. We conducted an exploratory inquiry to identify the number of entries containing the word “migraine” through the following search:https://clinicaltrials.gov/api/query/full_studies?expr=migraine&fmt=JSON&min_rnk=4000.ClinicalTrials.gov main page and obtaining the first 4,000 results. If this search returned ≤4,000 entries, then we would have identified all the relevant entries.This was equivalent to entering “migraine” into the “other terms” search field in the ClinicalTrials.gov API through Clojure, a functional programming language.After identifying all relevant entries, we extracted these entries through the We used the PRISMA paradigm for inclusion and exclusion of trials in our search strategy . For theOnce the two arms were identified, they were merged and duplicates were removed. The records were then screened and removed, with reasons, as well as assessed for eligibility. The inclusion criterion was at least one arm including subjects with migraine.We used custom backend codes in Haskell to identify the following:“Responsible Organization”—i.e., “Lead Sponsor” designated by “LeadSponsorName” field in each entry.“Study Type”—i.e., “interventional,” “observational,” “expanded access.”“Design Primary Purpose”-designated by either “treatment,” “prevention,” “basic science,” “supportive care,” “diagnostic,” “health services research,” “educational/counseling/training,” “screening,” device feasibility,” or “other.” Of note, “prevention” study designation is not prophylactic treatments of migraine but defined as “preventing the development of a specific disease or health condition” .“Design Intervention Model.”-The options were “parallel assignment,” “single group assignment,” “factorial assignment,” “sequential assignment,” or “crossover assignment.”“Design Allocation Count.”-The options were “randomized,” “non-randomized,” or “n/a.”“Overall Status.”-The options were “completed,” “recruiting,” “unknown status,” “terminated,” “not yet recruiting,” “withdrawn,” “active, not recruiting,” “enrolling by invitation,” “suspended,” and “no longer available.”“Clinical Trial Phase.”-The options ranged from “early phase 1” studies to “phase 4” studies.Of note, not all study entries identify the “Study Type,” “Design Primary Purpose,” “Design Intervention Model,” and “Design Allocation Count.” All entries should include the full name of the organization.The exploratory search inquiry identified 868 entries containing “migraine(s)” in the inclusion criteria and 857 containing “migraine(s)” in the title. There were 787 duplicates that contained both “migraine” in the title and “migraine.” After removal of duplicates, there were 928 unique entries. During the screening process, we excluded 7 entries. Our final data set consisted of 921 unique entries .We identified 423 unique trial organizations encompassing 921 studies. Fifty organizations registered for four or more studies, 28 registered for three studies, and 52 registered for two studies. The remaining 293 organizations (293/423 or 69.2%) registered only one study, accounting for 31.8% (293/921) of all studies included.Thirty-two organizations produced ≥5 entries encompassing 40.0% (368/921) of all entries . There wFor study type, 86.2% (794/921) were interventional studies, 13.6% (125/921) were observational studies, and 0.2% (2/921) expanded access studies. There were no missing entries.For design allocation, 69.1% (636/921) were randomized studies, 6.9% (64/921) were non-randomized studies, and 9.9% (91/921) were N/A. This sums to 791; thus, there were 14.1% (130/921) studies that did not contain this label and were unaccounted for.For design primary purpose, there were 62.4% (576/921) treatment studies, 13.0% (120/921) prevention studies, 3.8% (35/921) basic science studies, 0.1% (9/921) supportive care studies, 0.9% (8/921) diagnostic studies, 0.7% (6/921) health services research studies, 0.3% (3/921) educational/counseling/training studies, 0.4% (4/921) screening studies, and 0.1% (1/921) device feasibility study. Twenty studies were labeled as “other.” This amounted to 782; thus, there were 15.1% (139/921) studies that did not contain this label and were unaccounted for.For the design interventional model, there were 56.9% (524/921) parallel assignment models, 15.2% (140/921) single group assignment models, 12.4% (114/921) crossover assignment models, 1.2% (11/921) factorial assignment models, and 0.5% (5/921) sequential assignment models. This aggregated to 794 studies; there were 127 studies that did not contain this label and were unaccounted for.Of note, we reviewed the 42 studies registered by the Danish Headache Center, the highest ranking organization in our list. Of the registrations, 21 were crossover assignment and 8 studies were in parallel assignments. (The others were of different design or were unclassified).Of note, some of the studies included detailed descriptions of the design interventional model in addition to labeled as one of the above categories. These descriptions themselves were not considered here.There were 61.5% (566/921) “completed” trials, 11.3% (104/921) “recruiting” trials, 8.7% (80/921) “unknown status” trials, 5.5% (51/921) “terminated” studies, 5.3% (49/921) “not yet recruiting” studies, 3.3% (30/921) “withdrawn” studies, 2.9% (27/921) “active, not recruiting” studies, 0.9% (8/921) “enrolling by invitation” studies, 0.4% (4/921) “suspended” studies, and 0.2% (2/921) “no longer available” studies.There were 0.7% (6/921) “early phase 1” trials, 3.9% (36/921) “phase 1” trials, 10.2% (94/921) “phase 2” trials, 13.5% (124/921) “phase 3” trials, and 11.4% (105/921) “phase 4” trials. Therefore, 60.4% (556/921) studies did not include a phase designation.ClinicalTrials.gov. Furthermore, academic organizations accounted for just under half of the 32 most productive organizations. Our list of influential headache research institution is similar to a recent scientometric study on the most productive academic institution in headache (We demonstrate that the landscape for clinical research in migraine is oligarchic; 3% of organizations were responsible for about 40% of registrations on headache . Taken tClinical trial registrations in migraine research comprise mostly of interventional studies with a parallel assignment model. Since vigorous efficacy, tolerability, and safety data are a requirement from regulatory organs such as U.S. Food and Drug Administration and European Medicines Agency for approval of novel therapies, it is not surprising that most registrations of clinical trials were intervention studies. Although a randomized and parallel assignment design is the gold standard in drug development, these types of studies are expensive and cumbersome to conduct –11. SincAs in other fields, few academic institutions in our field appeared to achieve their position through reputation. For example, the Danish Headache Center was the highest ranked organization. Likely, this is due to the center being one of the first to offer a multidisciplinary approach collected under one institution . Indeed,ClinicalTrials.gov for determining prevention vs. treatment studies are accurately followed. Finally, a future study should assess whether data from all registered clinical trials are published and publicly available to increase transparency.It is surprising that most clinical trials did not contain a clinical trial phase designation. Presumably, these accounts for trials that were experimental or observational in nature. A future study may be able to elucidate this further. Moreover, approximately 9% of trials appeared to be either “terminated,” “withdrawn,” “suspended,” or “no longer available;” future studies to identify why these trials were terminated would be valuable for clinical trial research design. As a future direction, it would be interesting to ClinicalTrials.gov is an influential database. Each study is uniquely designated, allowing for the study of clinical trial characteristics otherwise not possible through traditional literature reviews. However, we did not include other regional databases such as the European Union Clinical Trial Registry or Japan Primary Registries Network; therefore, it is unclear how our results were biased toward, studies conducted in the US. However, it is worth noting that a significant number of our entries were not from American organizations.CinicalTrials.gov, although comprehensive, may suffer from poor reporting of clinical trial results as well as exclusions of basic science research . FurtherClinicalTrials.gov. The most common registration was a clinical study that is interventional and randomized, with parallel assignment for treatment purpose study design.Clinical research in migraine is dominated by the top 3% of organizations accounting for almost half of all migraine registrations on The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.PZ and TD conceptualized and designed the study. PZ conducted data collection. All the authors interpreted the data, contributed to the drafting of the manuscript, and approved it to be published. All authors agreed to be accountable for all aspects of the work for any issue related to the accuracy or integrity of any part of the work. PZ attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.Rutgers University supplied funding for publication fees.PZ has received honorarium from Lundbeck Biopharmaceuticals, Board Vitals, and Fieve Clinical Research. He collaborates with Headache Science Incorporated without receiving financial support. He has ownership interest in Cymbeline LLC. The remaining author declares 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. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.