Astragalus mongholicus Bunge (Fabaceae) is an important plant source of the herbal drug known as Radix Astragali, which is used worldwide as a medicinal ingredient and a component of food supplement. Russian Federation, Mongolia, Kazakhstan, and China are the main natural distribution areas of A. mongholicus in the world. However, the quality of medicinal plant varies among different locations. As for A. mongholicus, limited literature focused on its biodiversity mechanism. Here, we combined the chemometric analysis of chemical components with genetic variation, as well as climatic and edaphic traits, to reveal the biodiversity mechanism of A. mongholicus. Results showed that the detected chemical, genetic and climatic traits comprehensively contributed to the quality diversity of A. mongholicus. The eight main chemical components, as well as the inorganic elements of P, B and Na were all significant chemical factors. The precipitation and sunshine duration were the main distinguishing climatic factors. The inorganic elements As, Mn, P, Se and Pb were the distinguishing edaphic factors. The systematic method was firstly established for this medicinal plant in order to illustrate the formation of diversity in terms of quality, and provide scientific evidence for geographic indications and climatic adaptation in production and in the clinical application of herbal medicinal plants. Astragalus mongholicus Bunge (Fabaceae) is the main plant source of the herbal drug Radix Astragali, which is recorded by European Pharmacopoeia , British Pharmacopoeia , Japanese Pharmacopoeia  and Chinese Pharmacopoeia . A. mongholicus is locally adapted and chemically, climatically and genetically differentiated for traits related to quality. Russian Federation, Mongolia, Kazakhstan and Inner Mongolia, Shanxi and Gansu Provinces in China, are the major natural distribution areas for A. mongholicus in the world. A. mongholicus is used not only in the Chinese, Japanese, and Korean systems of medicine but also widely cultivated and used outside of Asia, such as Germany and the United States[Panax ginseng C.A. Mey., Araliaceae) root samples from fifteen different areas showed variations related to geographic location[Dendrobium candidum Wall. ex Lindl. (Orchidaceae) obtained from six different areas showed significantly different content of mannose and polysaccharide[ia BP, 203 versioned States. This phed States. There ieia JP, 1 version ed States. General location. The steia CP, 20 version.accharide–15.A. mongholicus, studies have tried to explain the differences in the molecular variation and soil properties. However, limited literature accounted for the mechanisms of biodiversity by systematical method and reciprocal correlation based on chemical, genetic, climatic and edaphic traits[A. mongholicus was used as a representative medicinal plant to interpret the mechanism of biodiversity and local adaptation that could be attributed to internal  and external (climatic and edaphic traits) causes based on chemometric and correlation analysis, which is essential for the geographic indications and further scientific development of herbal medicinal plants.For ic traits–19. In tA. mongholicus samples were collected from three locations, namely, the provinces of Inner Mongolia, Shanxi and Gansu, which are the main producing areas in the People’s Republic of China  and astragaloside were quantified by ultra-performance liquid chromatography (UPLC). The chromatographic conditions for determining the six flavonoids were: ACQUITY UPLC BEH C18 column ; column temperature was maintained at 30°C; and the mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile-isopropyl alcohol (7:3) containing 0.1% formic acid. The elution conditions were optimized as follows: 10%-20% B held for (0–1 min); 20%-25% B held for (1–6 min); 25%-30% B held for (6–8 min); 30%-40% B held for (8–10 min); 40%–100% B held for (10–12 min); 100%-10% B held for (12–13 min); 10% B held for (13–14 min); the injection volume was 2 μL; and the flow rate was set at 0.25 mL/min.2 was 25 psi. All experiments were performed in triplicate.The chromatographic conditions for determining the astragaloside content were: ACQUITY UPLC BEH C18 column ; the column temperature was maintained at 30°C; the mobile phase was acetonitrile–water (8:2) for isocratic elution; the injection volume was 2 μL; the flow rate was set at 0.25 mL/min; the ELSD condition was 45°C, and the pressure for NThe content of Astragalus polysaccharide was determined by the phenol–sulfuric acid method. The inog). The supernatant was removed and transferred into a new tube before adding 700 μL isopropanol and mixing for 15–20 min. The mixture was centrifuged in CB3 spin columns for 40 s at 12,000 rpm. The filtrate was discarded and 500 μL GD (adding quantitative anhydrous ethanol before use) was added before centrifugation at 12,000 rpm for 40 s. The filtrate was discarded, and 700 μL PW buffer (quantitative anhydrous ethanol was added before use) was used to wash the membrane before centrifugation for 40 s at 12,000 rpm. This step was repeated with 500 μL PW, followed by a final centrifugation step for 2 min at 12,000 rpm to remove the residual wash buffer. The spin column was dried at room temperature for 3–5 min and then centrifuged for 2 min at 12,000 rpm to obtain the total DNA.The material specimens were dried by natural methods, and 30 mg of dried plant material was used for DNA extraction. Samples were rubbed for 2 min at a frequency of 30 r/s in a FastPrep bead mill . The total genomic DNA was isolated from the crushed material according to the manufacturer’s instructions . We made the following modifications to the protocol: chloroform was diluted with isoamyl alcohol (24:1), and the buffer solution GP2 was diluted with isopropanol (same volume). The powdered sample, 700 μL of 65°C GP1 and 1 μL of β-mercaptoethanol were mixed for 10–20 s before the mixture was incubated for 60 min at 65°C. Subsequently, 700 μL of a chloroform: isoamyl alcohol mixture was added, and the solution was centrifuged for 5 min at 12, 000 rpm , and 1/1 μL of the forward/reverse (F/R) primers (2.5 μM). The reaction mixtures were amplified in a 9700 GeneAmp PCR system . Amplicons were visualized by electrophoresis on 1% agarose gels. The purified PCR products were sequenced in both directions by an ABI 3730XL sequencer .General PCR reaction conditions and universal DNA barcode primers were used for the ITS, ITS2, and http://cdc.cma.gov.cn/home.do). The data of 20 climatic factors from 1951 to 2012 were included in our analysis.The climatic data were collected from the China meteorological data sharing service system .The DNA sequencing peak diagrams were obtained and proofread before the contigs were assembled with CodonCode Aligner 5.0.1 . The complete ITS2 sequences were obtained via the HMMer annotation method based on the Hidden Markov model. All of A. mongholicus obtained from different locations were determined and analyzed  was performed for the chemical, molecular, climatic and edaphic factors . We aimepsbA-trnH intergenic region showed no significant differences in terms of genetic distance. Thus, we excluded this region in subsequent analysis. For the chemical and climatic factors, the main chemical components calycosin-7-glucoside, polysaccharides, kaempferol, quercetin, and ononin were strongly positively correlated with the climatic traits of mean air speed, sunshine duration, evaporation capacity, extreme wind speed and percentage of sunshine, but these components were strongly negatively correlated with the precipitation, mean relative humidity and mean minimum air temperature. Meanwhile, formononetin and calycosin were strongly negatively correlated with the mean air speed, sunshine duration, evaporation capacity, extreme wind speed and percentage of sunshine, but were strongly positively correlated with the precipitation, mean relative humidity and mean minimum air temperature. Astragaloside was weakly positively correlated with the mean air speed, sunshine duration, evaporation capacity, extreme wind speed and percentage of sunshine but was weakly negatively correlated with the precipitation, mean relative humidity and mean minimum air temperature. For the chemical and edaphic factors, polysaccharide, kaempferol and ononin were strongly positively correlated with the inorganic elements Pb, As and Mn, but were weakly negatively correlated with Mg. Formononetin and calycosin were strongly positively correlated with Pn, As and Mn but weakly negatively correlated with Mg. Calycosin-7-glucoside and quercetin had no correlation with Pb, As, Mn and Mg. The inorganic element of B in the roots was positively correlated with Pb, As and Mn, but negatively correlated with Mg. Conversely, P in the roots was negatively correlated with Mg, but positively correlated with Pb, As and Mn. The inorganic elements in the roots were not correlated with the climatic factors.Based on the loading diagram, PCA and ANOVA analysis for four factors, the correlation between four factors were conducted . For theA. mongholicus samples were collected from three main producing areas, namely, Shanxi, Inner Mongolia and Gansu provinces in China. The samples were analyzed in terms of their chemical, genetic, climatic and edaphic traits, which were the main factors of influence corresponding to the quality of herbal material. The eight main active constituents included six flavonoids, astragaloside and Astragalus polysaccharide[A. mongholicus produced in Shanxi province contained the highest total content. By contrast, the content of the inorganic elements in the roots were not significantly different. Remarkably, the content of Astragaloside was higher in the samples of Inner Mongolia than those from the other two locations. As an isolated active constituent, Astragaloside mainly confers a protective effect against ischemic-heart disease and brain injury. The content of flavonoid and Astragalus polysaccharide were higher in the samples from Shanxi province. Flavonoid and polysaccharide components of A. mongholicus are mainly used for anti-neoplastic properties and inhibition of atherosclerosis formation. The present results were consistent with those of previous studies[Different geographic locations, climatic conditions and edaphic traits may contribute to the different quality levels of medicinal plants. In this study, the accharide. The cons studies, 27.psbA-trnH intergenic region, to analyze A. mongholicus from three different locations. The ML tree showed that the samples from different locations were divided into different clusters. The results were consistent to a certain degree with those of previous studies[et al., the ITS sequence for A. mongholicus from different regions were highly conservative at an intra-specific level. In our study, we confirmed the performance of ITS and its partial sequence of ITS2 in A. mongholicus. The ML tree of ITS and ITS2 were in substantial agreement, because ITS2 was a part of the ITS sequence. Furthermore, we combined the chloroplast barcodes, psbA-trnH intergenic region, which performed well compared with ITS and ITS2 sequence in our previous studies[psbA-trnH intergenic region performed better than the other two barcodes because it divided the samples into three observable clusters. The results indicated that A. mongholicus from different locations possessed stable genetic variation in the psbA-trnH intergenic region, which could be used as a molecular marker to distinguish samples from different locations.To explore the genetic variation between different locations, we employed three commonly used DNA barcodes in medicinal plants, ITS, ITS2 and s studies, 29. Accs studies–32. The A. mongholicus from different locations had different climatic and edaphic conditions. The PCA results showed that the three locations were obviously divided into three clusters. Nonetheless, the quality of Radix Astragali is the result of comprehensive influences. The ecological environment, which comprises the climatic and edaphic traits, was the main external cause, whereas the chemical component and genetic variation were the main internal causes that influenced the quality of Radix Astragali. Therefore, we analyzed the correlation between the chemical components and the genetic variation as well as the climatic and edaphic traits.In addition, the analysis of climatic and edaphic traits demonstrated that A. mongholicus.The ITS2 barcode was strongly negatively correlated with the main chemical components of Astragalus polysaccharide, astragaloside, kaempferol, and ononin. By contrast, ITS was not correlated with the main chemical components. The results indicated that the variation of genetic distance was negatively correlated with the chemical components on a certain level. In terms of the edaphic inorganic element and chemical components, we found that the harmful elements of Pb and As were strongly negatively correlated with the content of Astragalus polysaccharide, astragaloside, kaempferol, and ononin, but were strongly positively correlated with the content of formononetin and calycosin. These results were interesting because several heavy metals, such as Cu and Zn, are essential trace minerals of plants. Meanwhile, some heavy metals, such as Pb, Cd and Hg, are trace minerals that are not necessary for the growth of plants, and a very small amount can cause toxic contamination of the plants. The inoA. mongholicus from different locations based on internal  and external (climatic and edaphic traits) causes. The correlation analysis proved that the genetic, climatic and edaphic traits were similarly closely correlated with the content of the chemical components. This proposed method presents useful data for estimating the quality of A. mongholicus. Thus, the findings reported here firstly establish a scientific method to clarify the mechanisms of the biodiversity and local adaptation for medicinal plants. Further study should be focused on genome-wide studies, and developing suitable methods for quantitative traits analysis, which is conducive to better understand the biodiversity and local adaptation in plant population, especially for medicinal plants.This work proposes a systematic and comprehensive method for estimating the quality of S1 Table(DOCX)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Therefore, this article was aimed to review and discuss the methods carried out and the phytochemistry and bioactivity of the extracts used and also conclusions provided in these publications. There are a number of published data indicating in vitro studies that lacked CC50, IC50, were excluded. Published articles both in English  and in Persian  databases, from 1966 to October 2014 were incorporated in this review. The in vitro. Seventeen out of 42 studies in which 23 kinds of medicinal plants were subjected to crude extraction were included. The review of data showed that some of the herbal extracts including Hyssopus officinalis methanolic extract, Melissa officinalis aqueous extract, Quercus persica L. hydroalcoholic extract and Securigeras ecuridaca methanolic extract with selective index (SI) of 234, 877, >778 and 250, respectively were highly effective against HSV in vitro.Only 42 published reports were found to examine Iranian herbs against HSV replication More comprehensive studies using more advanced methods are needed to be done to achieve promising anti-HSV agents from the bioactive compounds isolated from these herbs. Belonging to the family of Herpesviridae and subfamily of Alphaherpesvirinae, herpes simplex virus type-1 (HSV-1) and type 2 (HSV-2) are two of the most common human pathogens. Infections caused by these viruses are public health concern worldwide. These infections range from inapparent to severe life threatening infections such as encephalitis , Iran, and natural or herbal medicine, were used as keywords. The databases used were limited to those Iranian articles published both in English and in Persian. In this review, iin vitro inhibition of HSV replication were found. In this systematic review, 17 studies in which 23 kinds of medicinal plants were subjected to crude extraction were included and the remaining that used no standard methods to evaluate CC50, IC50, and SI, were excluded. The review of data showed that some of the herbal extracts including Hyssopus officinalis methanolic extract, Melissa officinalis aqueous extract, Quercus persica L. hydro alcoholic extract and Securigeras ecuridaca methanolic extract with SI of 234, 877, >778 and 250, respectively were highly effective against HSV, in vitro , the characteristics of some of the extracts isolated from some Iranian medicinal plants are discussed below.Avicenna marinaAvicenna marina (commonly known as grey mangrove or white mangrove), is one of the mangrove species plants which is native to Southern Africa  of the methanol extract showed significant anti-HSV activity. This fraction inhibited the HSV replication following entry of the virus into the target cells. The phytochemical examinations of this extract have shown that fraction D contains flavonoid compounds. It has been shown that A. marina contains active flavonoid compounds which inhibit the replication of HSV (KOS strain) following penetrating into the target cells  of 25, 25 and 5.3, respectively. The results indicated that LMEG was the most active sub-fraction of A. marina with inhibitory effect on the early stage of HSV-2 replication , monosaccharides, and polysaccharides (25% of the dry weight of the gel)  for treatment of various ailments . An oral dose of the extract (125 mg/kg) significantly delayed the onset of HSV-1 infection by over 50%. It also increased the mean survival time of infected mice by 55-65% compared to the non-treated mice (p<0.05). The mortality rate for mice treated with the extract was also significantly reduced by 90% in comparison with the non-treated mice that exhibited 100% mortality , a member of the Lamiaceae family  is a thyme-like plant and a member of Lamiaceae family that wildly grows in central and southern parts of Iran  before and after cellular attachment in concentrations of 1.02 and 0.257mg/ml, respectively  as tonic, carminative, digestive, antispasmodic, anti-inflammatory, anti-tussive and expectorant and for the treatment of common colds  is one of the most important plant families in which Securigeras ecuridacaSecurigeras ecuridaca (family Fabaceae), also called goat pea, is an annual herb used against a variety of diseases such as malaria, gastric influx, epilepsy, hypertension and hyperlipidemia in eastern folk medicine  were 2±0.12, 0.2±0.01, 0.1±0.01 and 0.1±0.01 μg/ml, respectively. Also, the calculated selective index (SI) for crude methanol extract, kaempferol and kaempferol-7-O-glycoside were 250, 300 and 2500, respectively  for a long time to treat gastrointestinal upset, arthritic pain, urinary tract infection, and liver ailments , alkaloids , volatile oils, polysaccharides, amino acids, lipids, vitamins , inorganic elements , etc. The beneficial properties of tea are attributed to its polyphenols. Its flavonoides have antioxidant, anti-inflammatory, anti-allergic and anti-microbial effects. The polyphenols content of green tea and black tea varies from 30% to 40% and 3% to 10%, respectively. Tea and particularly, green tea has been evaluated for its medicinal properties and a number of studies suggested that chemical constituents of tea play important role in human health (Sharangi, 2009in vitro replication of HSV-1 is inhibited by aqueous extract of C. sinensis with IC50 value and selectivity index of 50 and 20 μg/mL, respectively (Farahani, 2013Tea is a cultivated evergreen plant and its green, oolong and black types are all made from the same plant species, Euphorbia spinidensEuphorbia genus are known to contain substances with inhibitory action against some bacteria. This inhibitory action has been attributed to their large amounts of phenolic compounds (Li et al., 2009E. spinidens is an indigenous plant naturally growing in different geographical regions of Iran and is increasingly used in traditional medicine. Crude methanol extract of E. spinidens and the corresponding fractions n-Hexane, Chloroform, and n-Butanol, with SI values of 15.85, 2.062, 8.2, and 25.37, respectively, inhibited early stages of HSV-1 replication, in vitro (Mohammadi-Kamalabadi et al., 2014Plants in the family of Euphorbiaceae are well known for the chemical diversity of their isoprenoid constituents (Jury et al., 1987Searching for new bioactive compounds in order to develop new anti-viral agents seems to be urgent. Research on some medicinal plants may potentially lead to isolation of active compounds which could be effective against HSV. To achieve this goal, both experimental and clinical investigations are needed to provide a better understanding of mechanisms of action, therapeutic effects, and the safety profile of these compounds. Iran with a diverse climate and wide variety of herbal flora could be considered as potentially a great source of these herbs from which isolation of these components is possible. Therefore, this systematic review which covered the published results of research on some Iranian medicinal plants and the corresponding isolated active phytochemicals, conclusively, would highlight the need for more comprehensive studies using more advanced methodology to develop promising anti-HSV agents from these bioactive compounds.


This study investigated the accuracy of gender-specific stereotypes about movie-genre preferences for 17 genres. In Study 1, female and male participants rated the extent to which 17 movie genres are preferred by women or men. In Study 2, another sample of female and male participants rated their own preference for each genre. There were three notable results. First, Study 1 revealed the existence of gender stereotypes for the majority of genres . Second, Study 2 revealed the existence of actual gender differences in preferences for the majority of genres . Third, in order to assess the accuracy of gender stereotypes on movie preferences, we compared the results of both studies and found that the majority of gender stereotypes were accurate in direction, but inaccurate in size. In particular, the stereotypes overestimated actual gender differences for the majority of movie genres . Practical and theoretical implications of these findings are discussed. Many people have gender stereotypes. Gender stereotypes are beliefs concerning the abilities, attitudes, preferences and behaviors of a ‘typical’ man or a ‘typical’ woman . For exaEveryday observations suggest the existence of strong and well-known stereotypes about gender differences in movie preferences, which can be summarized as follows. Women are supposed to like romantic and melodramatic movies, which are snidely called “chick flicks” or “tearjerkers”, as well as comedy, but to dislike action and horror movies. In contrast, men are supposed to like action and horror movies, but to dislike romantic and melodramatic movies. Empirical research on gender differences in actual movie preferences has confirmed, at least to some degree, these popular stereotypes see . The samFurther results showed that men, in stark contrast to women, have a preference for programs with violent or sexual content. For example, Finally, strong gender differences have also been observed in the use and reception of pornographic material. A couple of studies revealed that men consume pornographic films and materials more frequently than women e.g., . However1 And we do not yet know much about the relative popularity of movie genres within and across both sexes, either. Finally, studies typically addressed the actual preferences of men and women for movie genres, whereas gender stereotypes in movie preferences have neither been studied nor have stereotypes been compared to the actual movie preferences of men and women.To summarize, previous studies on film and movie genre preferences have already discovered some reliable gender differences, as described above. However, most of these studies involved relatively small numbers of movie genres —the stereotypes of men and women about the movie preferences of men and women (Study 1). The second aim was to investigate gender differences in stereotypes about the movie preferences of men and women (Study 1). In other words, we wanted to investigate whether men and women have similar or different stereotypes about gender-specific movie preferences. The third aim of our study was to investigate the actual preferences of young adults for our set of movie genres (Study 2). Hence, we addressed more genres than most of the previous studies, and included genres that have not been investigated (often) before . Finally, the fourth aim of our study was assessing the accuracy of gender stereotypes concerning movie preferences by comparing the results of Study 1 to those of Study 2.3: (1) action movie, (2) adventure movie, (3) animation movie, (4) comedy movie, (5) crime movie, (6) drama, (7) erotic movie, (8) fantasy movie, (9) ‘heimat’ film, (10) historic movie, (11) horror movie, (12) mystery movie, (13) romance, (14) science fiction movie, (15) thriller movie, (16) war movie, (17) Western movie. Only the genre names were used in both studies. We did not present particular examples for each genre because we wanted to investigate how participants think about movie genres, and not how they think about particular movies, and we had several additional reasons for doing that. First, we were sure that our participants would recognize each of the 17 movie genres used in our study without providing examples. Second, the representativeness of a particular movie for a genre is always debatable. Third, example movies might be familiar to some participants, but not to all. Hence, providing no examples created equal conditions for all participants. Finally, we deliberately chose genres of movies that are typically presented in the cinema (and on TV). Hence, we excluded common TV formats like daily soaps, documentaries, sports, talk shows, sitcoms and so on.We assessed and compared presumed gender differences in movie preferences to actual gender differences in movie preferences for 17 movie genres. The term “genre” refers to a category of motion pictures that are similar with regard to particular features, including topic , geographical or historical background , stylistic issues  and the targeted audience  to men. We had no predictions regarding the remaining genres. Our second research question asked whether male and female participants would express similar or different views  about the movie preferences of men and women.The research reported in our manuscript meets the ethical guidelines of the German Society of Psychology  and is consistent with the principles of research ethics as published by the American Psychological Association (APA). Data collection was completely anonymous. That is, except for age and gender, we did not record personal information from the participants. All participants in the two studies reported here gave informed consent before filling out the questionnaires on movie preferences. The research reported here had not been approved by a local ethics committee because the ethical guidelines of the German Society of Psychology do not require ethical approval of basic psychological studies involving simple behavioral data.SD = 4.0) and 75 men . The large majority of participants were students (undergraduates) at a public university in Germany. The interviewers were 15 students , and each interviewed 5 men and 5 women.One hundred and fifty volunteers participated in Study 1. The sample consisted of 75 women . Then, items 5–21 asked the participant to indicate for each of 17 movie genres the degree to which the respective genre is, in her or his opinion, preferred by men or women. In particular, each item asked: “What do you think: Are (adventure) films preferred by men or by women?” Participants had to indicate their answer on an 11-point rating scale. The ticks were numbered from 5 to 0 and to 5 again. The central tick (0) was always marked as “equally preferred by men and women”. For half of the questionnaires  the leftmost tick was marked as “exclusively preferred by women”, whereas the rightmost tick was marked as “exclusively preferred by men”. For the other half of questionnaires  the leftmost tick was marked as “exclusively preferred by men”, whereas the rightmost tick was marked as “exclusively preferred by women”. The items 5–21 probed for the following movie genres (the German terms are given in brackets): adventure [Abenteuerfilm], action [Action-Film], animation [Animationsfilm], comedy [Filmkomödie], crime [Kriminalfilm], drama [Filmdrama], erotic [Erotikfilm], fantasy [Fantasy-Film], ‘heimat’ [Heimatfilm], history [Historienfilm], horror [Horrorfilm], romance [Liebesfilm], mystery [Mystery-Film], science fiction [Science-Fiction-Film], thriller [Thriller], war [Kriegsfilm], and Western [Westernfilm]. The items 22–24 asked our participants for some quantitative aspects of TV and film consumption. Item 22 asked how many hours per day, on average, the participant watched TV. Item 23 asked how many films, on average, the participant consumed per week. Finally, item 24 asked how often, on average, the participant went to the cinema per month.We developed a questionnaire with 24 itemsWe constructed four versions of our questionnaire in order to minimize (a) the effects of marker positions and (b) the effects of item order. As already said above, in half of the questionnaires the left pole of the rating scales referred to women, whereas the right pole referred to men, and the reverse was true for the other half of questionnaires. This manipulation was crossed with two different orders of movie genres. For one half of the questionnaires, items 5–21 were jumbled to produce a random order of genres. This (random) order was reversed for the other half of the questionnaires. We made sure that similar numbers of men and women filled out each version of the questionnaire.People were asked to participate in a study on the film preferences of men and women. Participants filled out the questionnaire on themselves, in order to minimize interviewer effects. If necessary, the interviewer provided a pen and a writing pad. Data were always collected in dyads of one interviewer and one participant. Participants took either part for free or received course credits.Figure 1. In the next step, we submitted the means to a four-way Analysis of Variance (ANOVA) with the within-subjects factor Genre, and the between-subject factors Gender of Participant, Gender of Interviewer, and Item Order5. In this analysis, Gender of Interviewer and Item Order had no significant effects  and, therefore, we dropped these variables from further analyses. A two-factorial ANOVA revealed significant main effects for Gender of Participant, F = 16.13, MSE = 3.55, p < 0.001, Genre, F = 216.14, MSE = 3.19, p < 0.001, F = 1.33, MSE = 3.19, p = 0.19, We first transformed the ratings into numbers ranging from 0 (exclusively preferred by women) to 10 (exclusively preferred by men). The arithmetic means of the ratings are depicted in Gender of Participant resulted from the fact that judgments were biased toward the own gender. That is, the women’s judgments for most genres were biased towards the “female” pole, whereas the men’s judgments for most genres were biased towards the “male” pole. In order to clarify the main effect of Genre, we compared the mean judgment for each genre to the neutral value of 5 . Because multiple tests increase the risk of making a type-1 error, we used Bonferroni’s method to reduce the significance criterion α from 0.05 to 0.003. A significant positive deviation from the neutral value of 5 indicates a ‘male’ genre. Ten genres were viewed as male genres : action, adventure, erotic, fantasy, history, horror, science fiction, thriller, war, and Western. A significant negative deviation from the neutral value of 5 indicates a ‘female’ genre. Five genres were viewed as female genres: animation, comedy, drama, ‘heimat’ films, and romantic movies. The remaining two genres  were perceived as gender neutral.The main effect of SD = 1.0), view 2.0 films per week (SD = 1.7), and go to the cinema once in a month (SD = 0.8). On average, the men in our sample watch TV for 2.0 h per day (SD = 1.2), view about 3.0 films per week (SD = 3.5), and also go to the cinema once in a month (SD = 1.1). The results for men and women were not significantly different, all ts(149) < 1.10, all ps > 0.25, all gs < 0.40.On average, the women in our sample watch TV for 1.9 h per day , whereas ten genres were stereotyped as ‘male’ genres . Two genres were considered as equally popular among men and women .Participants considered dramatic and romantic movies as ‘female’ genres, and they considered action-packed and violent movies  as ‘male’ genres. Thus, our data reflect the most popular stereotypes about movie preferences of men and women and are, furthermore, in line with the results of previous research on gender differences in movie preferences .In addition, we observed some less obvious or less familiar stereotypes. Animation and ‘heimat’ movies were stereotyped as female genres, whereas erotic, fantasy, history, and science fiction movies were stereotyped as male genres. The latter findings are possibly not so surprising because many fantasy, history, and science fiction movies contain action, competition, and violence—the features that are seen as more attractive to male viewers than to female viewers e.g., . Interesr = 0.99.When comparing the stereotypes of men and women about the movie preferences of men and women, we observed a striking degree of agreement. This agreement is reflected in the lack of a significant two-way interaction in the ANOVA. Moreover, across the 17 genres addressed in our study, the average judgments of men and women were correlated by In Study 2, we investigated the actual preferences of young adults with regard to the 17 movie genres, which had already been addressed in Study 1. Our interest was threefold. We were, firstly, interested in the relative popularity of different film genres. Secondly, we wanted to further explore gender differences in the popularity of movie genres. And, thirdly, we were interested in comparing gender differences in actual film preferences to the stereotypes about gender differences in film preferences as observed in Study 1.6 reported the only study that explored a relatively large set  of movie genres. They asked 560 participants for their favorite movie titles and then sorted the responses into genres. Results showed that film drama (31% of the titles), action movies (17%), romantic movies (13%), and comedy movies (11%) were the most popular genres across gender and age. In contrast, biblical films (0.5%) and documentaries (0.1%) turned out as the two least popular genres. Unfortunately, the procedure used by To our knowledge, Fischoff et al. (1998)SD = 3.7) and 80 men . The large majority of participants were again students (undergraduates) at a public university in Germany. The interviewers were six female students.One hundred and sixty volunteers participated in Study 2. The sample consisted of 80 women . Items 5–21 asked the participant, for each of 17 movie genres, to indicate on an 11-point rating scale how much they liked a particular genre. In particular, each item asked “How much do you like (horror) movies on a scale from 0  to 10 (extremely)? Please tick.” The items 5–21 probed for the same genres as in Study 1. The items 22–24 asked our participants for some quantitative aspects of TV and film consumption as in Study 1.We developed a new questionnaire with 24 itemsWe constructed two version of our questionnaire in order to minimize the effects of item order. For version A, items 5–21 were jumbled to produce a random order of genres. For version B, the random order from version A was reversed. Then we made sure that equal numbers of men and women filled out each version of the questionnaire.The procedure for Study 2 was the same as for Study 1.Fs < 1, all ps > 0.25). Hence, we excluded this variable from further analyses, and conducted a two-factorial ANOVA on the mean preference ratings, which involved the between-subject factor Gender of Participant and the within-subject factor Genre. There were significant main effects for Gender of Participant, F = 45.04, MSE = 12.45, p < 0.001, Genre, F = 46.03, MSE = 7.96, p < 0.001, F = 16.36, MSE = 7.96, p < 0.001, Figure 2 shows the corresponding means.A preliminary analysis revealed that item order  had no effects on the results  liked most of the 17 genres more than the female participants . In order to understand the main effect of Genre, we compared the mean rating for each genre to the neutral value of 5. Because multiple tests increase the risk of making a type-1 error, we again used Bonferroni’s method to reduce the significance criterion α from 0.05 to 0.003. A significant positive deviation from the neutral value of 5 reflects a popular genre . Five genres were popular: action, adventure, comedy, crime, and thriller. For nine genres, popularity did not differ from 5, and therefore these genres had average popularity: animation, fantasy, drama, history, horror, romance, mystery, science fiction, and war. A significant negative deviation from the neutral value of 5 signifies an unpopular genre . Three genres were unpopular: erotic, ‘heimat’ movies, and Western. The results of the corresponding t-tests are presented in Table 1 (column b).The main effect for Gender of Participant and Genre, we compared the average male rating to the average female rating for each genre. For these comparisons, we again reduced the significance level to 0.003 by applying Bonferroni’s method. Drama and romance were more strongly preferred by women than by men. Nine genres were more strongly preferred by men than by women: action, adventure, erotic, fantasy, horror, mystery, science fiction, war, and Western. Finally, six genres were equally popular among men and women: animation, comedy, crime, ‘heimat’, history, and thriller. The results of the corresponding t-tests are presented in Table 1 (column c).To uncover the source of the significant two-way interaction of SD = 1.2), view 2.2 films per week (SD = 1.9), and visit the cinema 1.3 times in a month (SD = 1.2). On average, the men in our sample watch TV for 1.9 hours per day (SD = 1.4), view about 2.5 films per week (SD = 2.0), and visit the cinema once in a month (SD = 0.9). The results for men and women were not significantly different, all ts(158) < 1.55, all ps > 0.10, all gs < 0.25.On average, the women in our sample watch TV for 2.0 h per day (Table 2 (fourth column) gives the concordance between stereotypical and actual gender differences in movie preferences for each genre in qualitative terms . The classification of genres as male, female or neutral from Study 1 agreed with the classification of genres from Study 2 for 11 of 17 genres . This agreement was larger for ‘male’ genres (8 from 10 genres) than for ‘female’ genres (2 out of 5 genres). The majority  of mismatches were overestimations, that is, participants presumed a gender gap in movie preferences that was actually not present.We compared presumed and actual movie preferences of men and women in qualitative and in quantitative terms. Figure 3 presents a quantitative picture of the concordance between stereotypical and actual gender differences in movie preferences. We compared the effect sizes . Each point represents the difference between the preference rating for a genre and the neutral value of 5. Hence, negative values indicate a presumed female preference for a genre, whereas positive values indicate a presumed male preference. The white points in Figure 3 show the effect sizes of the actual gender gap in movie preferences as observed in Study 2 . Each point represents the difference between the preference ratings of men and women for a genre. Hence, negative values indicate an actual female preference for a genre, whereas positive values indicate an actual male preference. The patterns appear quite consistent: In fact, the correlation between the presumed gender differences and actual gender differences (across genres) is very high: r = 0.92 (for effect sizes). However, there were numerical differences between the (effect size of) presumed gender differences and the (effect size of) actual gender differences in movie preferences.8. If the difference between presumed and actual gender difference was smaller than 0.4 in effect size, we counted the case as a match. According to this scheme, there were ten overestimations , one underestimation (mystery), and six matches .We classified a difference as an overestimation when the presumed gender gap exceeded the actual gap by more than 0.4 in effect size, and we classified a difference as an underestimation when the actual gender gap exceeded the presumed gap by more than 0.4 in effect sizeTable 2, rightmost column). When compared to the results of Study 2 investigated the popularity of 17 movie genres among young adults. There were three general findings. Across both genders, we observed marked differences in the relative popularity of the 17 genres. The popularity of five genres was significantly above the intermediate value of 5: comedy, thriller, crime, adventure, and action : science fiction, war, action, erotic, adventure, Western, horror, fantasy, and mystery. Six genres were equally popular among men and women (sorted by absolute popularity from high to low): thriller, animation, comedy, history, ‘heimat’ films, and crime. Only two movie genres were preferred by women: romance and drama.Study 2 also revealed gender differences in the popularity of most movie genres. Half of the genres  were preferred by men. The following list is ordered by the effect size of the gender gap from large to small  correctly predicted the existence or direction of a gender gap in movie preferences (as observed in Study 2), but overestimated the size of the gender gap. We discuss this finding in some more detail in the Section “General Discussion”.Table 2, second column). The findings reflect the most popular clichés about gender differences in movie preferences. Interestingly, male and female participants reported very similar gender stereotypes on film preferences. In fact, across the 17 genres, the average ratings from men and women correlated with r = 0.99, an outstanding correlation. Study 2 assessed the actual movie preferences in a new sample of men and women. As a first result, the study revealed gender-independent differences in the popularity of 17 movie genres . In addition, we observed strong gender differences in the popularity of many genres. In fact, a gender effect was found for 11 out of 17 genres . Only two genres were more strongly preferred by women, whereas nine genres were more strongly preferred by men. These findings replicate and extend previous findings on gender differences in movie preferences  and actual film preferences of men and women (Study 2) with regard to 17 movie genres. Study 1 revealed gender stereotypes for the large majority  of genres under investigation . Second, the participants overestimated the size of gender differences in movie preferences for the majority of genres: The predicted gender gap exceeded the actual gap by more than 0.4 effect sizes for 10 out of 17 (59%) movie genres. Third, male and female participants made very similar predictions about the movie preferences of men and women  and, hence, the stereotypes of men and women were similarly accurate. We may conclude then that gender stereotypes regarding movie preferences were accurate in direction, but inaccurate in size.This is the first empirical study that investigated the accuracy of gender stereotypes about movie preferences. Therefore, we assessed stereotypes about movie preferences of men and women in Study 1 and compared them to the actual movie preferences of men and women as observed in Study 2. This comparison revealed three main findings. First, the participants correctly predicted the Indeed, the finding that our participants correctly predicted the direction of gender differences in preferences for most movie genres under investigation also fits the majority of findings from previous studies on the accuracy of gender stereotypes. In fact, from their literature review, Our empirical results showed that several beliefs about gender differences in movie preferences are inaccurate in that they overestimate actual gender differences. These results could be used for reducing (or eliminating) gender stereotypes and, hence, for improving the relationship between groups e.g., . For exaOur results could also be informative for the movie industry. Modern movies and the advertisement for these movies are often conveying gender stereotypes, and depicting traditional gender roles e.g., . First, Our study is limited with regard to (a) the set of movie genres included in our study and (b) the population under investigation. Although we investigated a larger set of movie genres than previous studies, we still excluded some interesting movie genres, such as documentaries, sports movies or musicals. Moreover, we also excluded hybrid genres  and sub-genres  from our investigation. Thus, it is possible that our participants in fact thought of different sub-genres when faced with a particular genre name, and averaging across such heterogeneous categories should have increased the between-subjects variance in our data. It might be interesting to do follow-up investigations on the accuracy of gender stereotypes about preferences for different sub-types of a movie genre.Also, future research could use different questionnaire items. In our first study, for instance, participants where asked how much they thought a film of a certain genre was preferred by men or by women. Instead, one could ask participants for assumed gender-specific preference for certain film features . Thus, one could avoid the use of genre, because this term might serve as a strong label and thus a category that evokes gender stereotypes.Our two studies assessed movie preferences in two samples of young students, aged between 18 and 35 years, enrolled in different majors at a German University. There is evidence that movie preferences differ between age groups e.g., , and betFinally, future studies might investigate how participant’s gender role identity  abilities e.g., . FinallySS and PW planned the study, prepared the materials, and supervised data collection. PW analyzed the data. BL, SS, and PW wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human leucine-rich glioma-inactivated protein 1 encephalitis (LGI1) is an autoimmune limbic encephalitis in which serum and cerebrospinal fluid contain antibodies targeting LGI1, a protein of the voltage gated potassium channel (VGKC) complex. Recently, we showed that a feline model of limbic encephalitis with LGI1 antibodies, called feline complex partial seizures with orofacial involvement (FEPSO), is highly comparable to human LGI1 encephalitis. In human LGI1 encephalitis, neuropathological investigations are difficult because very little material is available. Taking advantage of this natural animal model to study pathological mechanisms will, therefore, contribute to a better understanding of its human counterpart. Here, we present a brain-wide histopathological analysis of FEPSO. We discovered that blood–brain barrier (BBB) leakage was present not only in all regions of the hippocampus but also in other limbic structures such as the subiculum, amygdale, and piriform lobe. However, in other regions, such as the cerebellum, no leakage was observed. In addition, this brain-region-specific immunoglobulin leakage was associated with the breakdown of endothelial tight junctions. Brain areas affected by BBB dysfunction also revealed immunoglobulin and complement deposition as well as neuronal cell death. These neuropathological findings were supported by magnetic resonance imaging showing signal and volume increase in the amygdala and the piriform lobe. Importantly, we could show that BBB disturbance in LGI1 encephalitis does not depend on T cell infiltrates, which were present brain-wide. This finding points toward another, so far unknown, mechanism of opening the BBB. The limbic predilection sites of immunoglobulin antibody leakage into the brain may explain why most patients with LGI1 antibodies have a limbic phenotype even though LGI1, the target protein, is ubiquitously distributed across the central nervous system. N-methyl-d-aspartate receptor (BR)  , or the tor (BR) . For thetor (BR) , 6. Patitor (BR) , 8.In human LGI1 encephalitis, magnetic resonance imaging (MRI) studies have shown increased volume and T2 signal of temporomesial structures with the hippocampus most prominently affected. Initial swelling of these structures is often followed by atrophy , 9. WherThe involvement of the hippocampus in human LGI1 encephalitis has been confirmed in histopathological studies. Parenchymal T cell infiltrates are present, but sparse. In addition, neurodegeneration caused by immunoglobulin G and complement deposition has been shown , 16. TwoRecently, we described an acute seizure disorder with orofacial involvement in cats. This disorder was coined feline complex partial seizures with orofacial involvement (FEPSO) . BesidesIn both human LGI1 encephalitis and in FEPSO, little is known about pathologic changes outside the hippocampus. Extrahippocampal pathology is expected since human LGI1 antibody-positive sera not only bind to the hippocampus but also to other regions, including the molecular layer of the cerebellum where LGI1 is expressed in high amounts , 21, 22.Taken together, although the limbic system displays the most frequent and most prominent signs and symptoms, recent publications point toward the involvement of other brain areas. The limited amount of human material, especially of brain structures other than the impaired hippocampus, poses a problem in the study of these extrahippocampal structures.Since a relatively high number of FEPSO brains are available, we took the opportunity to perform a detailed neuropathological analysis of limbic and non-limbic structures. Our analysis shows that inflammatory infiltrates can be found brain-wide. A disturbance of the blood–brain barrier (BBB), occurring with prominent loss of tight junctions and resulting in leakage of immunoglobulin and complement, however, was more restricted. This leakage was found in brain areas such as the hippocampus but also in the subiculum, amygdale, and piriform lobe and, to a lesser degree, in the basal ganglia and hypothalamus. Moreover, in these areas, immunoglobulin and complement deposition to neurons was present and was associated with severe neurodegeneration. Importantly, this finding suggests that BBB disturbance and immunoglobulin leakage does not depend on T cell inflammation but that other, yet unknown, mechanisms are responsible.In this study, 16 cats with FEPSO, characterized by facial seizures and hippocampal sclerosis or hippocampal necrosis , were inThe project was discussed and approved by the institutional ethics committee  in accordance with GSP guidelines and national legislation.In all cats, a general necropsy was performed, and brains were fixed in 4% neutral-buffered formalin, embedded in paraffin, and coronal sectioned. Immunohistochemistry was performed as shown previously  at the lWe investigated BBB damage in more detail by double-labeling for the tight junction marker zona occludens 1 (ZO-1) and cat immunoglobulin in FEPSO animals in the hippocampus, the amygdala, basal ganglia, cortex, and cerebellum. Additionally, we investigated the hippocampus in normal and epileptic controls. Cats showing an average degree of BBB leakage were selected and investigated. To check for endothelial cell integrity of the blood vessels, we performed a triple staining for ZO-1, cat immunoglobulin, and von Willebrand Factor (vWF) as endothelial marker. Antigen retrieval was done with proteinase for 15 min at 37°C, and standard staining procedures were followed as described previously  as described elsewhere  or 2.5 mm2 (40 grids in 400× magnification), depending on the number of brain slices containing the region of interest. C9neo+ cells were counted in 1.25 mm2 (20 grids in 400× magnification) for the cortex, cerebellum, and caudate nucleus. In the amygdala and the hippocampus, C9neo+ cells were counted in the whole area. For the determination of cell loss, the number of TUNEL+ cells among 100 cells was determined in the respective areas. For the determination of neuronal loss in the hippocampus, the number of NeuN+ cells was counted in 0.75 mm2 (3 grids in 200× magnification) of each hippocampal subfield in normal controls and FEPSO cats. The percentage of remaining NeuN+ cells in comparison with normal controls was calculated for each subfield. The statistical difference to 100%  was calculated.CD3For quantification of immunoglobulin in different brain areas as well as in the hippocampus between FEPSO and controls, all slides were incubated and developed for the final color reaction for the exact same time. Images were analyzed using ImageJ by digital optical densitometry, as shown previously .MR studies of two cats, acquired with a high-field MR unit , were retrospectively evaluated. In each case, transverse T2-weighted fluid attenuation inversion recovery (FLAIR), sagittal 3D T2-weighted turbo spin echo (T2), transverse 2D and sagittal 3D pre- (T1) and post-contrast T1-weighted turbo spin echo images (T1C) were available. Slice thickness was 0.8–3 mm.www.brainmaps.org . To this end, a Wilcoxon-signed rank test was performed. All graphical data are represented as medians with the interquartile ranges. Animals tested positive for LGI1 antibodies and animals with unknown status are graphically separated (data points and error bars). Data of LGI1 antibody positive animals indicated in red and animals with unknown status indicated in black. Results were considered statistically significant at p ≤ 0.05.For statistical analysis, GraphPad Prism 6 was used. First, we tested for differences between FEPSO animals positive for LGI1 antibodies and FEPSO animals with unknown LGI1 antibody status. To this end, a two-way ANOVA was used, but no differences were found. Therefore, datasets were pooled for further analysis, which was performed with Kruskal–Wallis tests with Dunn’s correction for multiple testing. Neuronal loss within the hippocampus was evaluated by the percentage of remaining neurons with regard to normal control hippocampal subareas (corresponding to 100% NeuN2). The hippocampus of epileptic controls showed higher numbers of T cells (2.4 cells/mm2). These T cell numbers were comparable to T cell numbers in hippocampi of FEPSO animals (3.2 cells/mm2). Statistical analysis of hippocampal T cell numbers between normal controls, epileptic controls, and FEPSO, possibly due to the high variance, however, revealed no significant difference. In brains of FEPSO animals, T cells were mostly found in perivascular cuffs and the meninges, with only moderate parenchymal infiltrating T cells. Parenchymal CD3+ T cells were found in all investigated regions of the brain, including the hippocampus, amygdala, basal ganglia, cortex, and cerebellum  to support our findings. Bilateral T2-weighted signal and volume increase changes were found in the hippocampus as well as at the level of the amygdala and the piriform lobe  subiculum, entorhinal cortex, and amygdala. In some animals, immunoglobulin deposition was observed in the prepiriform cortex, putamen, claustrum, olfactory tubercle, nucleus accumbens, tenia tecta, septo-olfactory junction, hypothalamus, and anterior commissure. amygdala.In order to analyze early constituents of the complement cascade, we stained for complement factor C1q. In normal controls, C1q was occasionally found on the surface of cortical neurons. However, in none of these animals, C1q was found on neurons in the hippocampus Figure A. A simi+ cells found . In CA1, 52.2% were lost, and in CA2 and CA3, 37.5 and 46.7% were lost, respectively. The dentate gyrus, with a median of 98.2% remaining neurons, did not show significant neuronal loss, although three FEPSO animals also showed severe neurodegeneration in the dentate gyrus with less than 50% of neurons remaining. Finally, CA4 showed significant neuronal loss compared with the dentate gyrus Figure . One recLGI1 encephalitis is a form of autoimmune epilepsy, an expanding group of disorders with antibodies against various neural antigens and epileptic seizures as one of the broad range of neurological signs. The similarities between human LGI1 encephalitis and FEPSO have only recently been discovered , 20. TheThe findings that human LGI1-positive sera show strong staining of the cerebellum and that human patients can (very rarely) develop cerebellar symptoms , 34 and Interestingly, in FEPSO, BBB disturbance was not purely restricted to the hippocampus but was also revealed in presubiculum, subiculum, amygdala, piriform lobe and, more rarely, in areas such as basal ganglia and hypothalamus. The larger limbic areas were also found to be affected in MR T2-weighted FLAIR scans showing signal as well as volume increase. In human LGI1 encephalitis, hippocampal and amygdaloidal swelling, followed by atrophy of the affected areas, have been observed in MRI studies , 36, 38.An important finding in this study of FEPSO is that BBB leakage was more restricted to specific areas associated with the limbic system, whereas T cell infiltrates were present brain wide. A clear finding in animal models of multiple sclerosis and neuromyelitis optica is that activated T cells can open the BBB and thereby enable antibodies to enter the CNS –44. HereTo summarize, in this naturally occurring animal model, we were able to show that BBB disturbance and neurodegeneration in FEPSO is restricted to the hippocampus and other limbic structures, but is not found in other areas such as the cerebellum despite the presence of T cell infiltrates. Our findings broaden the perspective on LGI1 encephalitis by not only looking at the target organ (the brain parenchyma) but also on the question of how the presumably pathogenic antibodies can enter it. This not only deepens the knowledge and understanding of LGI1 encephalitis but also leads to new questions regarding regional selectivity of dysfunctional BBB. These findings are not only of use in veterinary medicine but also are highly relevant to human disease. A better understanding of underlying disease mechanisms will help to ameliorate future treatments and hopefully minimize remaining cognitive deficits of patients.The project was discussed and approved by the institutional ethics committee  in accordance with GSP guidelines and national legislation. In addition, all animal owners have given their consent.JB and AP designed and supervised the project. AT and JB prepared the manuscript. AT performed the statistical analysis and, together with JB, the data interpretation. AT, MF, and LQ-G performed immunohistochemical staining, cell quantification, and imaging. AK performed the necropsies. SK provided MRI scans and interpretation of those. CB contributed to the study design. All authors reviewed the manuscript.CB gave scientific advice to Eisai  and UCB , undertook industry-funded travel with support of Eisai , UCB , Desitin , and Grifols , obtained honoraria for speaking engagements from Eisai , UCB , Desitin , Diamed , Fresenius Medical Care , Biogen , and Euroimmun . He received research support from Diamed  and Fresenius Medical Care . He is a consultant to the Laboratory Krone, Bad Salzuflen, Germany, regarding neural antibodies and therapeutic drug monitoring for antiepileptic drugs. The other authors declare no competing financial interests.


The relationship between body mass index (BMI) and mortality remains controversial. Furthermore, the association between BMI and cardiovascular events (CVE) is not conclusive and may differ by ethnicity. We aimed to estimate the associations between the BMI and mortality or cardiovascular disease in a general Korean population.This study was based on a sample cohort database released by the Korean National Health Insurance Service. We analyzed a total of 415,796 adults older than 30 years of age who had undergone a national health examination at least once from 2002 to 2012. Hazard ratios for death and cardiovascular events were calculated using Cox proportional hazards models.2. Compared with them, subjects with a BMI ≥ 30kg/m2, men with a BMI < 25 kg/m2, and women with a BMI < 22.5 kg/m2 showed significantly higher overall mortality. Additionally, men with a BMI < 22.5 kg/m2 and women with a BMI < 20 kg/m2 displayed an increased risk of cardiovascular mortality. Unlike the mortality trend, the CVD events trend showed a linearly positive association. The risk of a CVE was the lowest in men with a BMI ranging from 20 to 22.4 kg/m2 and in women with a BMI < 20 kg/m2.For both men and women, BMI and overall mortality showed a U-shaped association, with the lowest mortality rate among those with a BMI of 25–27.4 kg/mThe BMI showed a U-shaped association with overall mortality, where slightly obese subjects showed the lowest rate of mortality. The CVE exhibited a linear association with the BMI, where the lowest risk was observed for normal weight subjects in a general Korean population. The prevalence of individuals who are overweight or obese has increased worldwide. Obesity is closely associated with metabolic diseases, including type 2 diabetes mellitus (DM), hypertension (HTN), and cardiovascular diseases (CVD), which are the leading causes of death . Althoug2 displayed the lowest risk of overall mortality [The association between BMI and mortality could depend on ethnicity. In studies of East Asian populations, a U-shaped relationship between BMI and mortality was reported, and individuals with a BMI ranging from 18.5 to 27.5 kg/mortality –6. In a ortality , 8. Whenortality Cardiovascular disease is a leading cause of death, and HTN, type 2 DM, dyslipidemia, smoking, and obesity are considered cardiovascular risk factors. However, the relationships between the BMI and cardiovascular mortality and between the BMI and cardiovascular events are not entirely understood. In a meta-analysis of patients with chronic heart failure, the risks of a cardiovascular event and cardiovascular mortality were the highest in subjects with a low BMI and those who were overweight . In contWe analyzed a sample cohort database based on Korean National Health Insurance data to investigate the relationships between the BMI and all-cause or cardiovascular mortality, and between the BMI and cardiovascular events.We used the National Sample Cohort (NSC) database released from the Korean National Health Insurance Service , which is available to researchers through a simple application process and ethics approval. The NHIS constructed this cohort of 1,025,340 subjects  from all beneficiaries of the Korean National Health Insurance Program and the Medical Aid Program in 2002 . The cohThe NSC database consists of several datasets: a dataset that contains socio-demographic information  of the beneficiaries from the National Health Insurance and the National Medical Aid Programs; a dataset that contains medical claims, including information on the diagnosis  codes), admission, and treatment ; and a dataset that contains the cohort members of the National Health Screening Program. The National Health Screening Program in Korea provides biennial  health examinations for all medical insurance subscribers (at any age), other beneficiaries aged ≥40 years , and those between the ages of 44 and 66 years. This program includes a regular blood test, a chest X-ray examination, a physical examination, and a questionnaire on medical history. Approximately 70% of those eligible for a national health examination utilized these additional features. Data from the national death registration database of the Korea National Statistical Office  were linked with the NHIS NSC database.This cohort was followed up until the study ended  or until the member was disqualified from the NHIS due to death or emigration. The July 2014 initial release of the NSC database included datasets from 2002 to 2010, and the eligibility and socio-demographic information, the medical claims dataset, the National Health Screening dataset, and the national death registration database were added and updated annually. Researchers can use the NSC data with an Institutional Review Board (IRB) approval for the study proposal and a charge for the DB through the application procedure of the NHIS. In this study, we used the 2002–2013 NHIS NSC database, and the cohort was followed until 31 December 2013.The NHIS did not obtain informed consent individually because these data were not collected for study. The patient records of the NSC were anonymous and de-identified before the data were released by the NHIS. This study was approved by the IRB of Ewha Womans University Mokdong Hospital (IRB number: EUMC 2017-04-004).We analyzed adults from the cohort members of the 2002–2013 NSC dataset who were more than 30 years old , and who had taken the National Health examination at least once  between 2002 and 2012. The follow-up period began with their first health examination, and the information from this examination  was used as the baseline of each subject. Among them, 86,830 were excluded due to a medical history of stroke, ischemic heart disease (IHD), cancer, or chronic obstructive pulmonary disease; this information was obtained from the self-reported patient questionnaire administered during the first health examination and from the medical claim data  between 2002 and the year of the health examination. We also excluded subjects who lacked measurements for height, weight, blood pressure and fasting glucose level (875) as well as those who died during the year of the health examination . In total, 415,796 subjects were included in our analysis.2). Age was categorized into 10-year groups. Health behaviors, such as physical activity level, alcohol consumption, and smoking status, were explored in the questionnaire. Current smoking status was classified into smoking or not (non/ex-smoker). Alcohol consumption status was classified as heavy drinking  or non-heavy drinking ; average daily alcohol consumption was based on data regarding the frequency of alcohol consumption and the average amount of alcohol consumed during each drinking occasion. Physical activity level was grouped into none, 1−2, and ≥3 times/week. In 2009, the assessment of physical activity changed from one question on the weekly frequency of exercise to three questions on the number of days per week of physical activity according to intensity ; the total number of days across all three intensities was considered as the frequency of physical activity. Missing values were assigned to a separate category for each health behavior. Income was divided into quintiles, and the lowest income group included the beneficiaries of the National Medical Aid program. The family history of cardiovascular disease was based on the self-reported questionnaire responses for family history of stroke and heart disease.The BMI was calculated as body weight in kilograms divided by height in meters squared; these values were measured during the health examinations. The BMI was divided into six categories  between 2002 and the year of the health examination, and the health examination measurements . Total cholesterol was classified into three groups .The outcomes of interest were time-to-death, time-to-CVD death, and time-to-CVD incidence. We defined CVD incidence as the first admission with a diagnosis of CVD or CVD death. CVD was identified as ICD-10 codes I21-I25 and I60-I69 for the cause of death or a diagnosis reported in the medical claim data and was categorized into the following groups: IHD (ICD codes I20–I25), ischemic stroke (ICD codes I63), and hemorrhagic stroke (ICD codes I60-I62).2 as the reference for the all-cause mortality HRs and a BMI 21 kg/m2 as the reference for the CVD mortality HR. Then, we examined the same associations with a categorical BMI using Cox proportional hazards models, adjusting for age, smoking, and alcohol consumption status, levels of physical activity and income, and a family history of CVD. As a reference, we used the 25−27.4 kg/m2 BMI group for the mortality analysis and the 22.5−24.9 kg/m2 BMI group for the incidence analysis.The characteristics of the subjects are presented as the frequency and proportion for categorical variables. We first examined the associations between the continuous BMI and the all-cause and CVD mortality using age-adjusted Cox proportional hazards models, where the BMI was restricted to a three-knot cubic spline (using the lowest Bayesian information criterion (BIC)), and by reporting the hazard ratios (HRs). After manual inspection of the plots, we set a BMI of 25 kg/mWe also assessed the associations between BMI and all-cause and CVD mortality according to age, smoking, HTN, and DM status, and we examined the interactions between the BMI and HTN and between the BMI and DM status. In addition, we conducted a sensitivity analysis to avoid the possibility of reverse causality, in which we excluded subjects who died within less than 3 years after the baseline measurements  and additionally excluded subjects who were diagnosed with the corresponding diseases . For overall mortality, we also conducted a sensitivity analysis excluding subjects who died within less than five years after the baseline examination. In all sensitivity analysis, we used the variable of smoking status classified into never smoker, ex-smoker, and current smoker.If two BMI groups in the study were compared with a total number of subjects of fifty thousand and the ratio of the number of the subject of 4, and the proportions of subjects having the events in the control group is 2%  or 5% (for CVD events) during study period, the two-sided tests of whether the hazard ratio is one achieve 81% or 99% power at a 0.05 significance level when the hazard ratio is actually 1.25.P values were generated using two-tailed tests, and P values <0.05 were considered statistically significant. All statistical analyses were performed using SAS , STATA , and Prism  software.All analyses were conducted separately for each sex. The 2 for men and 23.2 ± 3.3 kg/m2 for women. The subjects aged <50 years and ≥60 years comprised 71% and 12% of all men, respectively, and 63% and 17% of all women, respectively. A total of 48.1% of men and 3.8% of women were current smokers, and 6.4% of men and 7.1% of women had a family history of CVD.A total of 415,796 subjects were included in our analysis, and the median follow-up duration was 7.0 years (interquartile range: 4–11 years). During the study period, 11,058 subjects  died, among whom 1,482 subjects (882 men and 600 women) died from CVD. The subject demographics are shown in 2. The risk of a CVD event increased nearly linearly across the entire range of BMI values in women and BMI values greater than 22.5 kg/m2 in men.2 BMI group. The highest mortality rate was observed in the < 20 kg/m2 BMI group , and this relationship was more prominent in men  and HTN status (among men). However, the effects of BMI values <20.0 kg/m2 and 20.0–22.4 kg/m2 on overall mortality were significantly stronger among men with DM than among men without DM (Pinteraction = 0.020). Among women with HTN and DM, those with a BMI ranging from 27.5 to 29.9 kg/m2 showed HRs that were not different those with a BMI ranging from 25 to 27.4 kg/m2, whereas among women without HTN and DM, those with a BMI ranging from 27.5 to 29.9 kg/m2 displayed a higher risk of overall mortality than those with a BMI ranging from 25 to 27.4 kg/m2 (Pinteraction = 0.015 for HTN and <0.001 for DM)  . These p for DM) .2 BMI group in men and 27.5–29.9 kg/m2 BMI group in women. Male subjects with a BMI < 22.5 kg/m2 and female subjects with a BMI < 20 kg/m2 showed significantly higher CVD mortality HRs than their counterparts with a BMI of 25–27.4 kg/m2; the highest CVD mortality was observed in the < 20 kg/m2 BMI group . IHD and ischemic stroke mortality were not significantly different according to BMI category, and the relationships were similar to those for CVD mortality. For women, hemorrhagic stroke mortality was higher among those with a BMI < 22.5 kg/m2 than among those with a BMI from 25–27.4 kg/m2. In subgroup analyses excluding subjects who died within less than 3 years after baseline examination, the patterns of the association between BMI and CVD mortality were similar, although the magnitude of the HRs reduced (and not significant) among men and increased among women . The associations between BMI and an IHD event and an ischemic stroke event were similar to the association between BMI and a CVD event. For hemorrhagic stroke events, the increase in risk was less evident; for men with a BMI < 20 kg/m2, the increase in the risk of a hemorrhagic stroke event was similar to the risk of hemorrhagic stroke mortality. These patterns of the association between BMI and CVD event were maintained in subgroup analyses excluding subjects who died within less than 3 years after baseline examination    Tables.2), and highest mortality in BMI values <20 kg/m2 in general Korean population. An increased risk of death was found in BMI <22.5 kg/m2 in men and BMI<20 kg/m2 in women for overall CVD, compared with those with a BMI of 25–27.4 kg/m2. On the other hand, the risk for cardiovascular events presented a linear pattern, with the lowest risk in normal weight subjects. The smoking status did not affect the relationship between BMI and mortality or CVD event, and the association was maintained in subgroup analyses excluding those who died within less than 3 or 5 years after recruitment. This suggested that the BMI was related to the overall and CVD mortality/event.In our study of more than 400,000 participants, we observed that BMI showed a U-shaped association with overall mortality with the lowest mortality in slightly obese subjects  [2 is an important predictor of both overall mortality and the combination of cardiovascular morbidity plus all-cause mortality [2 BMI group; these subjects are slightly obese according to the Asian obesity criteria of the World Health Organization [2 [We used the NSC database from the NHIS. The Korean universal health insurance system was initiated in 1963 for all citizens, and universal healthcare coverage was achieved in 1989. We used the sample cohort of 1,025,340 participants released by the NHIS, so it represents the total Korean population . Obesitylity [2) . In a Sportality . In anotnization . In a pr2, with the lowest risk observed among those with a BMI ranging from 20 to 22.5 kg/m2 [2 displayed the lowest CVD mortality rate, whereas women with a BMI ranging from 27.5 to 29.9 kg/m2 displayed the lowest mortality rate. A low BMI may indicate a low level of circulating total cholesterol or triglycerides, which is associated with an increased risk of hemorrhagic stroke according to previous studies [We found that CVD mortality rate showed a U-shaped association with BMI and that the lowest BMI group showed the highest CVD mortality rate. Several meta-analyses, mostly based on Western populations, evaluated the association between BMI and CVD mortality , 17, 18..5 kg/m2 ). Moreov2 and men with a BMI < 20 kg/m2 showed increased risk. This result is consistent with those of previous studies that showed that the BMI is related to ischemic stroke risk [CVD is a common cause of death worldwide, and the rate of CVD deaths will likely continue to increase . Obesityoke risk , 26. Theoke risk , 28 or aoke risk  between oke risk , 31.2, and 4.3% and 11.6% with a BMI of ≥30 kg/m2. Those with a low BMI were at low risk of CVD, but at high risk of dying once, they experience a CVD event. Those with a high BMI were at higher risk of a CVD event, but at lower risk of dying should they experience one. These results were similar to the phenomenon called the obesity paradox, in which patients with a higher BMI have better survival compared to those with a lower BMI [Interestingly, the associations between BMI and mortality and cardiovascular events show different patterns. The BMI showed a U-shaped association with overall mortality and cardiovascular mortality, but we observed that BMI was positively associated with the risk of CVD throughout the range. This linear pattern was consistent with the studies using nonfatal CVD or combined fatal and nonfatal CVD as an outcome , 32–34. ower BMI –37. Bettower BMI –39. A loower BMI –42. In aAlso of note is that subjects with DM displayed an inverse correlation between mortality rate and BMI, whereby lower BMI values were associated with higher mortality rates. This finding indicates that DM modifies the BMI-mortality relationship and may be associated with different effects between the BMI and mortality rate than those observed for the general population.Our study has several strengths. First, the NHIS database contains representative samples of population-based data, which are large-scale and stable. We were able to obtain nationwide health insurance data, including questionnaires, disease states, and laboratory and anthropometric data, that was generated by a public institution. Second, low body weight caused by illness could distort the association between underweight status and health due to reverse causation. However, to reduce this type of bias, we excluded subjects with a history of stroke, ischemic heart disease, cancers, and chronic obstructive pulmonary disease, and we analyzed the mortality data using several models that adjusted for multiple CVD risk factors. Additionally, the sensitivity analysis, which excluded subjects diagnosed with CVD or cancer as well as those who died during the first 3 years of follow-up, showed similar patterns.However, the NHIS database also has limitations. Although the cohort consisted of more than one million participants with the national representativeness, the number of deaths and the resultant power of the study might not have been enough to explore the association between the cause-specific mortality and each BMI category, particularly in subgroup analyses according to smoking or chronic disease status. The disease codes listed may not reflect the real disease status of the subjects because the codes are used for insurance claim purposes. This is an inherent limitation of the use of insurance databases. Additionally, there could be self-referral and selection bias, as the subjects who are more interested in their health are more likely to participate in routine health examinations, although this bias might not be large because NHIS health examinations are mandatory in Korea. A lack of evaluation of some main CVD risk factors such as LDL-cholesterol, HDL-cholesterol, serum uric acid and glomerular filtration rate in the national health examination was also another limitation, but it might not have much biased the association between BMI and CVD mortality/incidence because most of these metabolic and biologic factors were considered as mediating factors rather than as confounding factors. Moreover, we used the BMI as a marker of obesity; however, the BMI may not represent a subject’s obesity status accurately because it cannot distinguish between relative muscle and fat composition.2 or lower than 22.5 kg/m2 in a general Korean population. Additionally, we found an increased risk of CVD among subjects in the overweight and obese BMI groups. Therefore, health care professionals should provide personalized treatment plans to help subjects maintain an optimal BMI. Moreover, further research on body composition in relation to health outcomes is needed.In this study, we observed increased overall mortality risk among subjects with a BMI greater than 30.0 kg/mS1 TableAll HRs were adjusted for age, behavior, income, and family history of cardiovascular disease. Ex-smoker group among women was not presented due to the small number. BMI, body mass index; HTN, hypertension; DM, diabetes mellitus; HR, hazard ratio.(DOCX)Click here for additional data file.S2 TableAll HRs were adjusted for age, behavior, income, and family history of cardiovascular disease. Ex-smoker group among women was not presented due to the small number. BMI, body mass index; HTN, hypertension; DM, diabetes mellitus; HR, hazard ratio.(DOCX)Click here for additional data file.S3 TableAll HRs were adjusted for age, behavior, income, and family history of cardiovascular disease. BMI, body mass index; CVD, cardiovascular disease; HR, hazard rati.(DOCX)Click here for additional data file.S4 TableAll HRs were adjusted for age, behavior, income, and family history of cardiovascular disease. BMI, body mass index; CVD, cardiovascular disease; HR, hazard ratio.(DOCX)Click here for additional data file.S5 TableAll HRs were adjusted for age, behavior, income, and family history of cardiovascular disease. Ex-smoker group among women was not presented due to the small number. BMI, body mass index; HTN, hypertension; DM, diabetes mellitus; HR, hazard ratio.(DOCX)Click here for additional data file.S6 TableAll HRs were adjusted for age, behavior, income, and family history of cardiovascular disease. Ex-smoker group among women was not presented due to the small number. BMI, body mass index; HTN, hypertension; DM, diabetes mellitus; HR, hazard ratio.(DOCX)Click here for additional data file.S7 TableAll HRs were adjusted for age, behavior, income, and family history of cardiovascular disease. Ex-smoker group among women was not presented due to the small number. BMI, body mass index; HTN, hypertension; DM, diabetes mellitus; HR, hazard ratio.(DOCX)Click here for additional data file.S8 TableAll HRs were adjusted for age, behavior, income, and family history of cardiovascular disease. Ex-smoker group among women was not presented due to the small number. BMI, body mass index; HTN, hypertension; DM, diabetes mellitus; HR, hazard ratio.(DOCX)Click here for additional data file.S1 FigHRs were adjusted for gender, health behaviors , income, and family history of CVD. BMI, body mass index; CVD, cardiovascular disease; DM, diabetes mellitus; HR, hazard ratio; HTN, hypertension.(DOCX)Click here for additional data file.