Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma spp.) or environmental . Improving sanitation such as enhanced milking hygiene, implementation of post-milking teat disinfection, maintenance of milking machines are general measures to prevent new cases of mastitis, but treatment of active mastitis infection is dependant mainly on antibiotics. However, the extensive use of antibiotics increased concerns about emergence of antibiotic-resistant pathogens and that led the dairy industries to reduce the use of antibiotics. Therefore, alternative therapies for prevention and treatment of bovine mastitis, particularly natural products from plants and animals, have been sought. This review provides an overview of bovine mastitis in the aspects of risk factors, control and treatments, and emerging therapeutic alternatives in the control of bovine mastitis.Bovine mastitis, an inflammation of the mammary gland, is the most common disease of dairy cattle causing economic losses due to reduced yield and poor quality of milk. The etiological agents include a variety of gram-positive and gram-negative bacteria, and can be either contagious (e.g.,  Bovine mastitis is an inflammatory response of the udder tissue in the mammary gland caused due to physical trauma or microorganism infections. It is considered the most common disease leading to economic loss in dairy industries due to reduced yield and poor quality of milk . On an aBovine mastitis can be classified into 3 classes based on the degree of inflammation, namely clinical, sub-clinical, and chronic mastitis. A clinical bovine mastitis is evident and easily detected by visible abnormalities, such as red and swollen udder, and fever in dairy cow. The milk of the cow appears watery with presence of flakes and clots . ClinicaThis review article provides an overview of risk factors that are known to be associated with the incidence and severity of bovine mastitis. In spite of the fact that the use antibiotic remains as the main treatment strategy for bovine mastitis, concerns about emergence of antibiotic-resistance pathogens are continuously raising. In this regard, this review will highlight some therapeutic alternatives to antibiotic that may control bovine mastitis. The recent publications about the natural compounds derived from plants, animals, and bacteria will be discussed and their mechanisms of action also will be described in this review.There are several risk factors known to be associated with the incidence of bovine mastitis that play significant role, including pathogen, host, and environmental factors. All these factors were taken into consideration in the mastitis control programs .Staphylococcus aureus and Streptococcus agalactiae, and less common species like Mycoplama bovis and Corynebacterium, live on the cow’s udder and teat skin, colonizing and growing into the teat canal [Bacterial intra-mammary infection (IMI) is considered to be the main cause of bovine mastitis. Many bacterial species have been identified as causative agents for bovine mastitis. These bacterial infections can be classified into 2 types based on the bacterial origin—contagious and environmental . Contagiat canal . These aat canal . Contagiat canal .Escherichia coli (E. coli) or Strep. uberis invade and multiply in the cow’s udder, induce a host immune response and are rapidly eliminated [Streptococcus spp. (e.g. Strep. uberis), coliforms species , Pseudomonas spp., etc [Unlike contagious pathogens, environmental pathogens do not usually live on the cow’s udder and teat skin; instead, they exist in the bedding and housing of the herd. They are best described as opportunistic pathogens, looking for the chance to cause an infection. For example, they can enter the teat during milking owing to the liner slippage, or when cow’s natural immunity is weak, causing clinical mastitis. Environmental pathogens such as iminated . A wide pp., etc . Controlpp., etc .Staph. aureus is the most prevailing gram-positive pathogen known to be associated with various forms of clinical and sub-clinical mastitis [Staph. aureus is chronically infected mammary gland, therefore maintaining hygiene of udder and milking can protect healthy cow from infected cow, thereby reducing the infection [Staph. aureus does not trigger an immune response in cow as strong as E. coli or endotoxin, therefore the infection of Staph. aureus is always milder, leading to chronic mastitis that lasts for a few months [Staph. aureus do not cause abnormalities or fatality; however, it produces degradative enzymes and toxins that irreversibly damage the milking tissue, ultimately decreasing milk production [mastitis . The funnfection . Staph. w months . Staph. oduction .Staph. aureus infections is done by the use of antibiotics. However, Rainard et al [Staph. aureus are known as methicillin-resistant Staph. aureus (MRSA), which have a mecA gene conferring the resistance [Staph. aureus to produce biofilm and adapt to host environment makes it an even harder target for treatment of such infection [Treatment of rd et al  demonstrsistance . Besidesnfection ,21.Staph. aureus produces an exopolysaccharide called glycocalyx. It helps Staph. aureus to adhere to mammary epithelial cells and acquire nutrient, allowing it to survive in high shear environment, as well as protect it from environmental stress such as antibiotics, disinfectants, and evade phagocytosis [Staph. aureus are necessary [Biofilms are cluster of cells  enclosed in a self-produced matrix , adherent to biotic or abiotic surfaces . Biofilmocytosis ,25. Thisocytosis . As a reecessary .Strep. agalactiae is a gram-positive pathogen causing contagious mastitis. It can be found in bovine gastrointestinal tract as well as in the environment of dairy cows. It can be transmitted via milking machine and through oro-fecal route, particularly through contaminated drinking water; therefore, a recent study showed that maintaining udder and milking sanitary are not enough to control Strep. agalactiae infection, but fecal and environment management should also be taken into account [Strep. agalactiae causes sub-clinical mastitis with high SCC and low milk production even though no abnormalities were shown in milk [ account . Strep.  in milk . It can  in milk .Mycoplasma spp. is less common than Staph. aureus and Strep. agalactiae infection. However, it is highly severe and damage secretory tissues, and induce gland and lymphatic nodule fibrosis and abscesses [Contagious mastitis caused by bscesses . Outbreabscesses . The onlbscesses .E. coli is the most frequently found gram-negative pathogen. It invades the udder through teat, proliferate and initiate inflammatory response in dairy cow. It can be found in the environment surrounding dairy cow, such as bedding of the herd, especially in a wet condition [E. coli is usually clinical and transient. Symptoms are varied, ranging from mild with only local signs (red and swollen udder) to severe with systemic signs (fever). Severe clinical mastitis caused by E. coli can cause irreversible tissue damage in the mammary gland, complete loss of milk production, sometimes even leading to the death of dairy cow.ondition . MastitiE. coli rapidly induce an inflammatory response in the host. The virulence factor best known to trigger the inflammatory response is the endotoxin, which is found on the outer membrane of E. coli, known as lipopolysaccharide (LPS). The binding of LPS to toll-like receptor (TLR4) in association with other molecules, such as LPS-binding protein and cluster of differentiation 14 induce a series of signaling pathways [E. coli mastitis is mainly determined by the host factor rather than by E. coli pathogenicity [pathways . TLR4 enpathways ,34. NF-κpathways . Pro-infpathways ,36. The genicity .E. coli was classified as an opportunistic pathogen with different virulence factors, since its pathogenicity is not only mediated by single and specific virulence factor [E. coli can persist in the mammary gland, causing recurrent mastitis infections that are hard to treat, possibly due to the ability to produce biofilm at different levels [Nevertheless, e factor . In facte factor . Besidest levels ,37.Enterococcus faecalis is the predominant Enterococcus spp., followed by Ent. faecium. They are environmental gram-negative pathogens present in the organic bedding material of the herd. Pathogenesis of Ent. faecalis was reported related to the biofilm formation [Ent. faecalis and Ent. faecium were reported to be resistant to several antibiotics such as lincomycin, tetracycline, kanamycin, streptomycin, quinupristin/dalfopristin (Synercid), erythromycin, chloramphenicol, and tylosin owing to the presence of biofilm [ormation . In addi biofilm . This le biofilm .Staphylococcus (CNS), for example Staph. simulans, Staph. chromogens, Staph. hyicus, and Staph. epidermis, represents an emerging mastitis pathogen that has been isolated in many countries. The infections caused by CNS are relatively mild, usually remain sub-clinical but can be persistent, and are associated with an elevated SCC and decreased milk quality [Staph. aureus, reports show that their persistence in the udder has no relation with the ability of biofilm production [Staph. aureus [Coagulase-negative  quality . Howeveroduction . They ca. aureus .Strep. uberis is an environmental pathogen that causes recurrent mastitis, associated with clinical and sub-clinical infections [Strep. uberis to persist under environmental stress and resist antibiotic treatment [fections . It was reatment ,44. It hreatment .Genetic factors and dairy cow breeding have an effect on susceptibility or resistance to mastitis. Pure breed or cross breed of high-yielding cattle, particularly, Holstein-Friesian cattle, appear to be more genetically vulnerable to mastitis than are breeds giving medium yield . For insin vitro activity of leukocytes in milk hence increase the occurrence of IMIs [The structure of the udder also affects the susceptibility to the infection. Cattle with large funnel-shaped teats or pendular-shaped udder and blind quarters after calving are at greater risk of sub-clinical mastitis . Other t of IMIs .Another factor influencing infections is age. Older cow are more susceptible to infections, most probably because of the wider or permanently partially-open teat canal as a results of frequent milking . FurtherThe period between 3 weeks before and after parturition is defined as transition period, also known as periparturient period. Dairy cows are at a higher risk to acquire diseases like mastitis during this period . ResearcDuring lactation, there is a higher demand of energy and nutrient for the synthesis of colostrum and milk by the dairy cattle. So, when the feed intake does not meet the lactation demands, cattle exhibit negative energy balance . NegativEnvironmental conditions and management practices of the herds have decisive effects on animal health and welfare. Keeping the herd clean and comfortable can reduce the incidence and severity of mastitis . High stFive-point plan introduced by National Institute for Research in Dairying (NIRD) since 1960s is effective in controlling contagious mastitis pathogens . The fivThe main strategy to treat mastitis is by the use of antibiotics, such as penicillin, ampicillin, tetracyclin, gentamycin, etc., which can be given by intra-mammary infusion, intramuscular or intravenous injections . The DCTApart from this, any mastitis case detected during lactation is accompanied by a great concern of antibiotic residues in milk. When cow is detected with an active mastitis infection, the first thing to do is to cull the sick cow and milk out the cow completely to remove bacteria, milk clots, debris, and also toxins that might be released by the bacteria. Intra-mammary infusion of antibiotic is then applied for it to reach the udder as well as systematically into the blood circulation . In someDespite the cost, the overuse and misuse of antibiotics in treating bovine mastitis has brought some problems to the dairy industry. In addition, the presence of antibiotic residues in milk is also of concern. In general, the milk obtained during the antibiotic treatment followed by a waiting period has to be discarded since it cannot be consumed by the consumer due to the risk of allergies and drug resistance caused by antibiotic residues . Thus, hStaph. aureus, Strep. agalactiae, and E. coli. Vaccines targeting Staph. aureus and Strep. agalactiae are made up of either the whole organism  or subunits ; while for E. coli, the mutant core antigen J5 was used widely [Staph. aureus was studied in few reports. Schukken et al [Vaccinating cows can be deemed as a preventive mastitis treatment in herds. Most vaccines are designed to target d widely , 70. Howen et al  demonstren et al  reporteden et al ,74. Thesen et al .As mentioned before, mastitis is caused by a number of different bacterial pathogens; therefore, the lack of efficacy of vaccines might also be due to the multi-etiological nature of bovine mastitis. Not only the site of infection in the mammary gland varies among different bacterial strains, but their virulence characteristics and immunogenic capabilities can also be different . Hence, As stated above, even though the use of antibiotics remains as the main treatment strategy, but its effectiveness is limited, not to mention the development of antibiotic-resistant strains of pathogen has become a critical challenge in antibiotic treatment ,78. FurtIn vivo mastitis studies were usually performed with BALB/c female mice, while in vitro anti-inflammation assays were usually carried out in primary bovine mammary epithelial cells (bMEC) or immortalized bMEC lines such as MAC-T and BME-UV1. The plant extracts along with their mechanisms of action are summarized in Plants have served as a valuable source for ingredients in traditional medicine therefore they are gaining interests of researchers in treating bovine mastitis. As compared to antibiotics, plant-derived compounds have an advantage of not inducing resistance even after prolonged exposure. Another advantage of plant-derived compounds is their low toxicity . VariousScutellaria baicalensis and Scutellaria lateriflora, was claimed to attenuate inflammatory response by suppressing TLR4 mediated NF-κB and MAPK signaling pathways in LPS-induced mastitis in mice [E. coli strains isolated from mastitis milk samples with MIC of 4 mg/mL. Although the antibacterial activity of baicalin is low, it affects the drug resistance genes of E. coli, indirectly enhancing the sensitivity of E. coli to antimicrobial agents such as ampicillin, penicillin, streptomycin, ciprofloxacin. Before this, baicalin was shown to attenuate inflammation and apoptosis induced by Staph. aureus in mammary gland tissues of mouse model by regulating expression of pro-inflammatory cytokines and TLR2-related apoptotic factors [Baicalein is a flavone extracted from  in mice . The bin in mice  showed t factors ,86. Apar factors  also demStaph. aureus internalization in bMEC in a dose-dependent manner. It was suggested that the reduction of Staph. aureus internalization was related to the inhibition of NF-κB nuclear translocation; however, this was not correlated with the ability of thymol to decrease the antimicrobial peptide gene expression such as tracheal antimicrobial peptide and β-defensin.Thymol, a monoterpene phenol isolated from thyme, oregano, and tangerine peel, has shown to have an anti-inflammatory effect on mouse mammary gland. It also reduces the expression of pro-inflammatory cytokines (TNF-α and IL-6) and inflammation mediated proteins iNOS and COX-2 in a dose-dependent manner, by down-regulating the activation of MAPK and NF-κB signaling pathway . BesidesHO-1), cysteine/glutamate transporter, thioredoxin reductase 1 (TXNRD1), and NAD(P)H quinone dehydrogenase 1, which were up-regulated by activation of nuclear factor erythroid 2. In an in vivo study conducted by Zhang et al [Resveratrol is a natural polyphenol that is present in various plant species, such as grapevines, berries, and peanuts. It has beneficial effects in protecting MAC-T from oxidative cell damage caused due to hydrogen peroxide . Resverang et al , resveraStaph. aureus in a mouse model when compared with normal curcumin [Curcumin, the major compound of turmeric, was claimed to be one of the best potential therapeutic agent against bovine mastitis treatment . Fu et acurcumin . In anotcurcumin .Staph. aureus, Staph. chromogenes, Staph. siuri, Staph. warneri, Staph. xylosus, and E. coli) and found out that 3 of the essential oil Satureja montana L., Thymus vulgaris L. ct. thymol, and Origanum majorana L. were able to inhibit the tested pathogens. The study also found out that thymol, carvacrol and p-cymene are the most abundant components of these essential oils. Antimicrobial tests were carried out using both, pure component mixtures, as well as combined mixture of essential oils. In fact, the mixture of essential oils and mixture of pure components exhibited stronger inhibitory activity better than the single essential oil, suggesting there is synergistic effect between the mixtures. Fratini et al [Origarum vulgare and Leptospermum scoparium may be an effective alternative to staphylococcal infections owing to their synergistic effect. In addition, Cho and his co-workers also found out that treatment using oregano essential oil (OEO) can improve physical condition of the udder in tested cows comparable to that by gentamycin. Not only SCC and the number of WBC were significantly decreased, but Staph. aureus and E. coli were not detected as well. This finding suggested that OEO might be an alternative to antibiotics in controlling subclinical bovine mastitis [Despite having an effect on cell’s physiology, plant-derived compounds, especially essential oils, are reported to directly inhibit or kill mastitis pathogens . Fratinini et al  further mastitis . Howevermastitis .Staph. aureus and MAC-T cells. Growth of Staph. aureus was inhibited in a dose- and time-dependent manner. However only 0.05% CDO was found have a modest effect on the biofilm formation of Staph. aureus. When using MAC-T cells as an in vitro model of bovine mammary gland, 0.1% and 0.05% CDO managed to totally inhibit the adhesion and invasion of Staph. aureus into MAC-T cells. It was found out that major components of CDO, citral, and linalool are responsible for inhibition, owing to their abilities to alter the expression of Staph. aureus virulence genes [Terpeneless, cold-pressed Valencia orange oil was previ ously reported to have an antimicrobial effect on MRSA . Federmace genes . On the ce genes .Use of animal-derived compounds in treating bovine mastitis has been concentrated on bee products recently. Bee venom, containing the active component melittin, was administered to LPS-induced MAC-T cells to study its anti-inflammatory effect . AuthorsE. coli, and Staph. aureus. Besides, Chinese propolis also enhanced the mRNA expression of antioxidant gene HO-1, TXNRD1, and glutamate-cysteine ligase modifier subunit in mastitis infected cells, indicating the anti-oxidative effects of Chinese propolis.Propolis, a resinous substance produced by honey bee, has also been studied for its anti-inflammatory effect on MAC-T cells . Pre-treE. coli, Pseudomonas aeruginosa, Strep. agalactiae, and Staph. aureus, attributable especially to its iron-chelating ability, which can inhibit biofilm production through iron sequestration [On the contrary, immunomodulators naturally produced by mammals, such as lactoferrin, were preferred as potential non-antibiotic antimicrobial agents for treatment and prevention of bovine mastitis . Lactofestration .Lactobacillus and Bifidobacterium, have shown to have an antibacterial activity on tested bovine mastitis isolates [Lactococcus lactis subp. lactis CRL 1655 and L. perolens CRL 1724 isolated from bovine milk can inhibit bovine mastitis pathogens. These species were able to adhere to teat canal, therefore hypothesized to have a role in prevention of bovine mastitis during dry period [E. coli. In addition, both L. brevis 1595 and 1597 and L. plantarum 1610 showed high colonization capacities towards bMEC, suggesting they can be good candidates to compete with pathogens in mammary gland colonization [Other than bee products that are directly obtained from bee itself, lactic acid bacteria (LAB) found in the honey have been a new source of antibacterial agent . LAB plaisolates . In facty period . Apart fnization .L. lactis. Nisin form a complex with the cell wall, thereby inhibiting cell wall biosynthesis. The complex then aggregates and further incorporates into the cell wall, finally forming a pore in the bacterial membrane [Staphylococci were reported to have nisin resistance, therefore, discovery of new bacteriocins, alone or in combination with nisin, are highly desirable [Staph. aureus and Strep. pseudintermedius than wild-type and antibiotics combination. Besides, combination of nisin and dioctadecyldimethylammonium bromide nanoparticles increased the susceptibility of Staphylococci to nisin [Staph. simulans, either treated alone or in combination with nisin, can inhibit biofilm-forming Staph. aureus [Bacteriocins, antimicrobial peptides produced by bacteria, have emerged as potential alternative for bovine mastitis , 111. Onmembrane . Nisin imembrane , howeveresirable . Field eesirable  reportedto nisin . Another. aureus . Taken tStaph. aureus strain and tested on 36 Staph. aureus subclinical mastitis strains. They found out that all the tested strains were susceptible to all phage lysates. Other than Staph. aureus, bacteriophage was reported targeting E. coli too. Porter et al [E. coli. Bacteriophage cocktail had same effect as ceftiofur (10 μg/mL) in inhibiting E. coli growth. It also significantly reduced adhesion and invasion of E. coli. In addition, combination of bacteriophage with a non-antibiotic bismuth-based intra-mammary teat sealant, E. coli growth was inhibited, therefore, phage cocktail was suggested to have a potential to control E. coli infections in farm [Staph. aureus mastitis quarters showed significant reductions in pathogen levels and SCC [Bacteriophage are viruses that specifically infect bacteria and are harmless to humans, animals, and plants; thus, bacteriophage and their derivatives  are being deemed as valuable antimicrobial alternatives with a potential to reduce the current use of antibiotics in agri-food production, increasing animal productivity and providing environmental protection . Varela-er et al  separate in farm . Moreove in farm . For ins and SCC .Staphylococcus spp. causing bovine mastitis [Chitosan is a natural polysaccharide derived from chitin, proven to have broad spectrum of antimicrobial activity against fungi and bacteria. It was majorly reported to inhibit growth and biofilm formation of mastitis ,122. Chimastitis . Intra-mmastitis .In conclusion, effective mastitis control programs rely more on prevention rather than treatment. Currently, antibiotic treatment is still an established component in mastitis control programs. Antibiotics are often coupled together with others therapies; yet the effectiveness is still not satisfying. Therefore, searching for new therapeutic alternatives is necessary. A wide variety of natural products derived from plants, animals, and bacteria were investigated and reported to have potential in controlling bovine mastitis. Field studies should be considered to reassure the outcome of the alternative therapies before commercial applications.


Allergic rhinitis (AR) and diabetes mellitus (DM) share a common cause in inflammation; however, potential relationships between them are not clear. Therefore, we aimed to explore the associations between AR and DM. In this cross-sectional study, data were extracted from the Korean National Health and Nutrition Examination Survey 2007–2018 and weighted based on sociodemographic characters. AR and DM were defined by diagnoses thereof. Since self-reporting was not perfect, in order to complement the validity, we additionally performed sensitivity analysis by defining DM according to HbA1c and medication history. After excluding invalid data, the number of final participants was 29,246 . We calculated the odds ratio (OR) of newly self-reported DM among AR patients without DM history by multivariable logistic regression adjusted for potential confounders. A reverse association was also assessed. Patients with AR showed lower ORs for DM than those without AR . Similarly, DM patients showed lower ORs for AR than patients without DM . The same results were obtained in sensitivity analysis defining DM according to HbA1c levels or DM treatment and in stratification analysis for age, residency, comorbidity, BMI, and menopause. In conclusion, we discovered that AR and DM show mutual inverse associations, regardless of sex, in individuals aged 30 years and older. AR, with symptoms of sneezing, rhinorrhea, and nasal congestion, has an immense effect on one’s life. Research has shown that AR is mediated by type 1 allergic reactions related to helper T cell-1 (Th 1)2. Since AR is related to inflammation, several studies have explored relationships between AR and other diseases related to immune reactions, including cancer3, cardiovascular disease4, stroke5, and depressive disorder6.Allergic rhinitis (AR) affects 16.7% of Korean adults according to Korean Health Statistics 20187. In fact, some studies on the pathophysiology of DM have shown that DM is induced by insulin resistance in association with low-grade chronic inflammation9. Since the pathophysiologies of both AR and DM are related to inflammation, it would be reasonable to infer potential correlations between AR and DM, similar to cancer, cardiovascular disease, stroke, and depressive disorder, and documenting an association between AR and DM could provide novel perspectives on both diseases.Type 2 diabetes mellitus (DM) should be monitored and controlled carefully because it could cause complications such as hyperosmolar hyperglycemic state, blindness, neuropathy, and chronic renal failure11. Hashimoto et al. found that subjects with high levels of FPG had a low prevalence of rhinitis. Hwang et al. suggested that subjects with metabolic syndrome had lower odds of AR than those without. Both studies only considered the effects of DM and its related conditions on AR occurrence, and not vice versa. In addition, they only defined exposure as DM and its related conditions, and not the DM itself, and Hashimoto et al. only included male participants. Unlike previous studies, we explored the bi-directional association of AR with DM itself, not DM-like conditions.Previous studies have explored the associations between AR and patients with DM and its related conditions, such as metabolic syndrome and high-fasting plasma glucose (FPG)Table 12. Among the final study participants , 1703 participants were excluded due to missing values of HbA1c. With this definition, likewise, the results showed that DM risk was also reduced in AR patients. The lower part of Table The upper part of the table shows the association between AR and DM based on the number of participants whose DM was not diagnosed before AR , and menopause also showed that significant outcomes. Thus, even in stratification analysis, DM occurrence in AR patients was lower than non-AR. Likewise, even in stratified groups, as shown in Supplementary Table 4, AR occurrence in DM patients was lower than non-DM.In stratified analysis, likewise un-stratified analysis of this study, we also adjusted all covariates including age, family income, education status, marital status, smoking, drinking frequency, sleep deprivation, dyslipidemia, BMI, and the number of comorbidities. Age was divided into three groups: 30–46, 47–61, and ≥ 62. As shown in Table Our results showed that the prevalence of DM is significantly lower in AR patients and that of AR is significantly lower in DM patients among both men and women. In other words, AR patients had a lower likelihood of DM occurrence than patients without AR. Likewise, DM patients had a lower likelihood of AR occurrence than those without DM. To confirm the robustness of our findings, we performed sensitivity analysis by defining DM according to HbA1c or diabetes treatment. Furthermore, the results of stratification analysis by age, residency, comorbidity, BMI, and menopause were highly comparable. Thus, it is reasonable to suggest that AR inhibits the occurrence of DM and that DM inhibits AR occurrence regardless of sex.11. In Hashimoto et al., involving 10,093 male participants, they calculated the ORs of rhinitis in five groups divided by FPG level. They found that subjects with high levels of FPG had a low prevalence of rhinitis. Hwang et al. suggested that among 30,590 subjects, the ORs of AR were lower in participants with metabolic syndrome or impaired fasting glucose than in the healthy population. They, however, only focused on the effect of DM-like conditions on AR, not actual DM. High FPG does not mean diabetes, because plasma glucose could be increased by many other factors and may also be masked by therapeutic drugs for diabetes. Metabolic syndrome, likewise, is not same as diabetes. In other words, both studies only considered the effects of DM-like conditions on AR occurrence and not vice versa. Thus, we used actual DM as a variable and elucidated the bidirectional inhibitory associations between AR and DM.Several previous studies have explored the association between AR and DM-like conditions, such as metabolic syndrome, impaired fasting glucose, and high FPG14, when evaluating the associations between AR and DM. Since causal relationships could not be confirmed in this study due to cross-sectional design, we considered the sequence of diagnoses of AR and DM to enhance the validity of the association. Furthermore, strict exclusion criteria, consideration of many confounders, and stratification analysis also contributed to the validity.Although this study was cross-sectional in its design, we considered the temporality of the diagnosis, considering the Bradford-Hill criteria14. In other words, when a result can be explained by biologically well-known mechanisms, their association would be much more rigorous than a simple association. Our results might possibly be explained by the pathophysiology of helper T (Th) cell polarization, where Th1 and Th2 cells counteract each other. When immunologic stimulation is generated, premature Th cells would be differentiated into Th1, 2, and 17 cells15. Since the number of premature T cells is limited, domination of one type of Th cell suppresses the activation of the other type of Th cells by cytokine reactions. This phenomenon is called Th polarization16. In light of this theory, it is well known that AR is mediated by Th 2 domination2, and according to a recent review by Kartika et al., type 2 DM is induced by Th 1 domination8. With Th polarization, AR and type 2 DM downregulate reciprocally. Thus, our conclusion could satisfy the plausibility of the Bradford-Hill criteria, which reinforced the validity of the association we presented.Plausibility is also an important factor for confirmation of a causal relationship based on the Bradford-Hill criteriaAs mentioned above, the strengths of this study were the novelty of the topic and thorough evaluation of the associations, considering temporality. In addition, the phenomenon in the study might possibly be explained biologically. Thus, we consider the association between AR and DM to be meaningful.17. To address this issue, we considered temporal sequence of AR and DM diagnoses in the analysis. However, consideration of temporality could not fully ensure the causality, for example, we could utilize the age by year unit, which may not distinguish the overlap perfectly. Additionally, the measurement of the age of diagnosis of each disease may not be as same as the age of biological onset. Therefore, subjects, as an example, whose AR diagnosis was slightly prior to DM, might have been the patient who originally had DM prior to AR. To investigate this issue, we conducted a sensitivity analysis, excluding the gap under 1 year between both diseases (Supplementary Table 2), and the result was similar with the original outcomes.There are several limitations in this study. First of all, this was a cross-sectional study that could not reveal causation and could represent only association18. We also conducted the sensitivity analysis, comparing self-reported and clinically identified DM, showed the similar results (Supplementary Table 3). However, in the sensitivity analysis, 1703 participants were excluded due to missing value of HbA1c, who showed partially different general characteristics compared to included participants (Supplementary Table 5). Specifically, excluded participants were more likely to have low social economic status, be unmarried or obese, and have more comorbidities than included participants (result not shown). Therefore, it was possible that our results might have overestimated the true association regarding AR and DM.Second, the main exposure and outcome of this study were defined by self-reported questionnaires, which could lead to measurement bias, including recall bias based on a recall. Participants also could respond incorrectly, especially in the case of AR, which could lead to underestimation. In contrast, the agreement between self-reported and measured data for diabetes was relatively high (k = 0.82) in a previous validation study of KNHANESThirdly, since KNHANES did not discriminate detail DM types, so we could not use data of pure type 2 diabetes (T2D). To reduce heterogeneity of DM data and make more homogenous toward T2D, we restricted participant’s age over 30, but it was not exact T2D. Further studies with well discriminated data of DM types would be needed to discover exact association between T2D and AR.19. In the same manner, application of Th polarization in DM treatment could also be possible. With this study as the starting point, we can expect novel effective regimens of treatment for DM to be developed in the future.To our knowledge, this study is the first to demonstrate that AR and DM have mutual inverse association. Since we considered the temporality of the diagnoses and the plausibility of the phenomenon might be explained by Th polarization, the association between AR and DM appears to be significant. This clinical phenomenon could be used as the basis for additional research on AR and DM. For example, in further studies, it might be possible to show that Th polarization modulation can be exploited to treat DM and AR. In fact, immunotherapy based on Th polarization is already being executed for AR treatmentIn conclusion, we found that AR and DM have mutual inverse associations in both men and women aged over 30 years. Th polarization could be one of a possible explanation for these findings. Our results could provide a novel perspective on the mutuality between AR and DM and open new pathways to additional research on AR and DM.Data was extracted from a cross-sectional study, Korean National Health and Nutrition Examination Survey (KNHANES) 2007–2018 which was a nationwide population-based self-questionnaire survey conducted by the Korean Ministry of Health and Welfare. Participants were recruited by a two-stage stratified cluster sampling. Sampling frame was the latest population and housing census, conducted by Statistics Korea and supplemented by the data of public housing price conducted by Ministry of Land, Infrastructure and Transport, Korea. Stratification variables were administrative district, sex age, type of housing, and area of living space. Trained interviewers performed the interviews using structured questionnaires to obtain information including sociodemographic factors, health-related factors, lifestyle factors, the use of medical services and female reproductive factors. Data on lifestyle were recorded by self-reported questionnaire. HbA1c levels were measured via high performance liquid chromatography . All data of the participants was weighted based on a sociodemographic character and significance because KNHANES data represented total population of Korea. To be more specific, the Rao-scott chi‐square test for complex samples was used to compare the differences in the exposure group (PROC SURVEYFREQ) and the complex sample multiple logistic regression (PROC SURVEYLOGISTIC) was used to determine the bi-directional association between AR and DM.Among KNHANES 2007-2018, total number of study participants are 97,622. To determine the association between AR and DM, we excluded the data of 68,376 participants. First of all, from the 2010–2012 data, 25,534 participants were excluded due to absence of assessment of AR. Secondly, among participants aged under 30 years, 23,366 subjects were excluded to increase the homogeneity of the DM group, as near to type 2 DM. Lastly, patients with missing value on AR and DM were excluded . Subsequently, 29,246 participants , aged over 30 years, were included  or with current DM treatment was defined as DM patients. (Glycated hemoglobin (HbA1c) is a form of hemoglobin bonded to a sugar and the target analyze of a blood test for diagnosing and monitoring DM.) The reason why we conducted the main analysis by defining DM through self-reporting, rather than HbA1c, was based on our main goal to evaluate the temporal sequence between DM and AR. In this study, to clarify the temporal sequence of each association between AR → DM and DM → AR, we utilized the data regarding the age of diagnosis for both DM and AR. HbA1c was not proper for comparing temporality, since it only reflected the state at the latest measurement. Therefore, we chose self-reported morbidities as the main exposure and outcome measurements. For the AR → DM association evaluation, we additionally conducted sensitivity analysis by defining DM based on HbA1c and medication history.2) was divided into two categories, underweight/normal (under 23.0 kg/m2) and overweight/obese (above 23.0 kg/m2), according to the WHO-WPRO  guideline; normal range was 18.5 to 2320. Overweight and underweight were over 23.5 and under 18.5 respectively. Our database included a small population of underweight group, which could lack statistical power due to the small sample; therefore, we did not divide the under-23 group into two categories. We defined sleep disturbance as a few days’ experience of waking from sleep and difficulty going to sleep with lethargy in the daytime. “Sleep deprivation” was based on the weekly frequency of sleep disturbance: not at all, a few days, more than a week, and every day. The “number of comorbidities” was the sum of comorbidities assessed in the KNHANES, including liver cirrhosis, hepatitis C, hepatitis B, chronic renal failure, macular degeneration, glaucoma, cataract, otitis media, sinusitis, atopic dermatitis, depressive disorder, cancer , benign thyroid disease, asthma, tuberculosis, arthritis, myocardial ischemia or angina, stroke, and hypertension. Lastly, “residence” was divided into urban and rural: urban areas included Seoul, other metropolitan cities, and areas whose administrative district was larger than “dong” in Gyeonggi province; rural included all non-urban areas. All covariates were coded as categorical variables. In case of missing values in these variables, we coded them as a separate category; this allowed us to include the information on exposure and outcome variables from the entire individual in the major statistical analysis without omitting the individuals with any missing values present in the covariates. .Since the goal of this study was to assess the risk of DM in AR patients and vice versa, we considered several confounders known to affect both AR and DM occurrence, such as age, family income, education, marital status, ever smoking status, drinking frequency, BMI, sleep deprivation, dyslipidemia, the number of comorbidities, residence, and menopause. “Family income” was defined by monthly earnings, and it was divided into four groups: income under 150, 150–316, 316–518, and over 518 Korean-won (unit). “Education level” was assigned to seven categories, including pre-school, graduating from elementary school, middle school, high school, college, university, graduate school. “Marital status” was defined by four categories as follows: married and living with spouse, married but not living together or divorced, bereaved, and unmarried. “Ever smoking” was divided into three categories, including never, quit now, and still smoking. “Drinking frequency” was assigned to three categories, including less than once per month, two to four times per month, and more than two times per week. BMI , sleep deprivation, dyslipidemia, and the number of comorbidities. Stratified analysis was also performed by age group, residence, presence of comorbidity, BMI group, and menopause (-in women only). All analyses were performed using the statistical software package SAS version 9.4 , and weight distribution of the population was considered. Since both AR and DM had sex difference in many aspects of the diseases22, to evaluate precisely, all analyzing process had done in each sex, respectively.Multivariable logistic regression models were used to determine the associations between AR and DM Figs.  and 3. WSupplementary Information


Based on the androgen receptor (AR) expression, triple-negative breast cancer (TNBC) can be subdivided into AR-positive TNBC and AR-negative TNBC, also known as quadruple-negative breast cancer (QNBC). QNBC characterization and treatment is fraught with many challenges. In QNBC, there is a greater paucity of prognostic biomarkers and therapeutic targets than AR-positive TNBC. Although the prognostic role of AR in TNBC remains controversial, many studies revealed that a lack of AR expression confers a more aggressive disease course. Literature characterizing QNBC tumor biology and uncovering novel biomarkers for improved management of the disease remains scarce. In this comprehensive review, we summarize the current QNBC landscape and propose avenues for future research, suggesting potential biomarkers and therapeutic strategies that warrant investigation. Triple-negative breast cancer (TNBC) is defined by the lack of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression . CompareThe stark inter-patient and intra-tumoral heterogeneity render TNBC challenging to cure , 7. BaseAn updated version by Lehmann and colleagues classifies TNBC into four distinct molecular subtypes including two basal-like subtypes, i.e., (BL1 and BL2), mesenchymal (M), and luminal androgen receptor (LAR) . RetrospAlthough TNBCs exhibit greater chemosensitivity compared to non-TNBCs, many TNBC patients diagnosed with advanced-stage disease relapse following treatment with conventional anthracycline/taxane-based chemotherapy –5. HenceAndrogen receptor (AR) is expressed in approximately 10–43% of TNBCs depending on the AR positivity cutoff used; therefore, it has emerged as a promising therapeutic target for TNBC patients . AR antaAR is a transcription factor (TF) belonging to the nuclear steroid hormone receptor family. AR-mediated signaling plays a critical role in the development of breast tissue . There iThere have been conflicting reports regarding AR expression levels in TNBC, ranging from 7 to 75% –36. In ccis-regulatory DNA sequences [Additionally, AR splice variants (AR-Vs), which are produced due to structural rearrangement or alternative splicing of the AR transcript, have been suggested to underlie the variability in findings regarding the prognostic role of AR in TNBC . These vequences . AR-V7 eequences , 59. LimAR can drive tumor growth even in TNBC subtypes expressing low levels of AR. Although the precise mechanisms remain unclear, the AR-positive tumor cell subpopulation can support the growth of a cancer stem cell (CSC)-like cells, which promote chemotherapy resistance and tumor recurrence. Thus, AR-targeting agents may still be beneficial for TNBC patients exhibiting low (≤ 1%) AR expression . CompareAugmenting the complexity of the subject further, prognostic implications of AR expression appear to vary by ancestry. A recent AR assessment study  found thIn contrast to ER, PR, and HER2 testing, which is standard clinical practice, AR testing has not been standardized due to the lack of consensus regarding its prognostic value. However, given expanding data revealing that QNBC tumors have a more aggressive disease course and worse outcomes than AR-positive tumors, standardizing AR testing is critical. QNBC has a distinct molecular profile and should be considered a separate entity from TNBC. TNBC heterogeneity is associated with AR expression, suggesting that QNBC is a distinct, clinically relevant subtype . DiffereAlthough the prognostic value of AR is ambiguous, there is better agreement on its predictive value. A study by Masuda et al.  demonstrProtein expression analysis, gene copy number analysis, and gene sequencing have yielded promising therapeutic targets for TNBC Fig. . A recenE2F1, PDK1, CCL2, CEBPB, NFKBIL2, TGFB3, IL12RB2, IL2RA, and SOS1  and carbonic anhydrase IX (CAIX), are highly upregulated in TNBCs compared to non-TNBCs , 121. MoO-Bn-InsP5 , GSK2334470 (GlaxoSmithKline), OXIDs (S. Rapposelli Laboratory), and MP7 (Merck) are some of the PDK1 inhibitors under investigation [One of the differentially expressed genes in QNBC identified by Davis et al. is 3-phosphoinositide-dependent protein kinase-1 (PDK1), a downstream effector of PI3K . The roltigation .The chemokine CCL2 is involved in tumor development and progression by promoting the migration and infiltration of monocytes and tumor-associated macrophages (TAMs) . CCL2 ovShi et al.  identifiThe CCAAT enhancer-binding protein beta (CEBPB) is a TF regulating the expression of genes involved in inflammatory responses. High CEBPB has been correlated with expression of the chromosome 19 miRNA cluster (C19MC), the expression of which has been linked to TNBC . Due to From analyses in TCGA datasets, Peluffo G et al.  identifiAs part of the SCF-SKP2 ubiquitin ligase complex, S-phase kinase-associated protein 2 (SKP2) is involved in the degradation of p21, p27 (a CDK inhibitor), and p57, among other proteins. SKP2 is a known oncoprotein being involved in DNA replication during the S-phase of the cell cycle . SKP2 waQNBC is an aggressive and poorly understood form of BC. This review presents recent findings that aimed to improve our understanding of QNBC tumor biology required to identify prognostic markers and therapeutic targets. Studies assessing the differential expression of biomarkers between QNBCs and TNBCs may yield novel actionable targets unique to QNBC. These discoveries may also benefit TNBC patients of African ancestry, who are predominantly diagnosed with a QNBC phenotype. Cumulative evidence suggests that QNBC is highly proliferative and immunogenic, rendering it an ideal candidate for cytotoxic chemotherapy and immunotherapy. However, QNBCs have worse clinical outcomes even after treatment with adjuvant chemotherapy. Besides, acquired resistance to taxanes is also commonly observed. Thus, combining these approaches with agents targeting QNBC biomarkers may enhance treatment response and improve prognosis.A vast majority of these markers can be evaluated through clinically applicable methods such as immunohistochemistry (IHC). However, the successful characterization of QNBC patients and tailored clinical decision-making required the establishment of robust AR expression cutoffs. Although nuclear staining indicates active AR signaling, there is currently no standard scoring method for nuclear AR IHC staining. Often, CAP/ASCO staining guidelines for ER and PR are applied for AR since they stain similarly; however, there is no consensus on the threshold value for AR status assessment. One way to overcome this is to measure AR levels using a more reliable androgen-driven gene signature. Determining prior treatment, clinical outcomes, and key AR-dependent proteins, which may differ according to BC subtypes, will shed more light on the mechanistic action and prognostic value of AR in TNBC required for its implementation into routine clinical testing.It is imperative to treat QNBC as a unique disease and thoroughly investigate its biology. Examining the role of epidemiological and non-biological factors in QNBC is equally important in gaining a holistic understanding of its etiology and uncovering novel modifiable risk factors.


Olean-12-en-27-oic acids possess a variety of pharmacological effects. However, their effects and underlying mechanisms on osteoclastogenesis remain unclear. This study aimed to investigate the anti-osteoclastogenic effects of five olean-12-en-27-oic acid derivatives including 3α,23-isopropylidenedioxyolean-12-en-27-oic acid (AR-1), 3-oxoolean-12-en-27-oic acid (AR-2), 3α-hydroxyolean-12-en-27-oic acid (AR-3), 23-hydroxy-3-oxoolean-12-en-27-oic acid (AR-4), and aceriphyllic acid A (AR-5). Among the five olean-12-en-27-oic acid derivatives, 3-hydroxyolean-12-en-27-oic acid derivatives, AR-3 and AR-5, significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced mature osteoclast formation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, F–actin ring formation, and mineral resorption activity. AR-3 and AR-5 decreased RANKL-induced expression levels of osteoclast-specific marker genes such as c-Src, TRAP, and cathepsin K (CtsK) as well as c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Mice treated with either AR-3 or AR-5 showed significant protection of the mice from lipopolysaccharide (LPS)-induced bone destruction and osteoclast formation. In particular, AR-5 suppressed RANKL-induced phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). The results suggest that AR-3 and AR-5 attenuate osteoclast formation in vitro and in vivo by suppressing RANKL-mediated MAPKs and NFATc1 signaling pathways and could potentially be lead compounds for the prevention or treatment of osteolytic bone diseases. Bone maintains its structure and function through coordinating the activity of osteoclasts and osteoblasts, a process known as bone remodeling ,2. OsteoThe differentiation of osteoclast is regulated by two crucial cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) ,10,11,12Aceriphyllum rossi Engler (Saxifragaceae) suppressed RANKL-induced osteoclast formation. 3-Hydroxyolean-12-en-28-oic acid derivatives including oleanolic acid and maslinic acid, exert anti-osteoclastogenic effects in vitro and in vivo [Natural products have been utilized as an excellent source for new drug discovery . Natural in vivo ,24. HoweA. rossi in RAW264.7 cells . Stimulation of mouse BMMs with M-CSF and RANKL induced osteoclast differentiation from the precursors. However, the treatment of BMMs with AR-3 or AR-5 suppressed the formation of TRAP-positive multinucleated osteoclasts in a concentration-dependent manner A,B. The was used . Quercet± 0.3 µM . We furt± 0.3 µM C.Next, we determined the effects of AR-3 and AR-5 on the cell viability of BMMs to determine the possibility that AR-3 and AR-5 inhibited RANKL-induced osteoclastogenesis due to the potentially cytotoxic effects of these compounds on BMMs. However, these compounds did not decrease the cell viability of BMMs, but rather increased the cell viability of BMMs at concentrations utilized in this study D. CollecWe next determined whether AR-3 and AR-5 could inhibit filamentous actin-ring formation, which is the most obvious characteristic of mature osteoclasts . StimulaThe effects of AR-3 and AR-5 on the suppression of osteoclastogenesis in vivo was assessed using an inflammation-induced bone loss mouse model. The mice were treated with LPS in the presence and absence of AR-3 or AR-5, respectively. After nine days, the femurs were examined by micro-CT. A three-dimensional analysis revealed that the LPS administration caused a marked trabecular bone loss in the femurs. However, oral administration of either AR-3 or AR-5 considerably offered protection from LPS-induced bone loss A. With rTRAP, MMP-9, CtsK, and DC-STAMP in a concentration-dependent manner during RANKL-induced osteoclastogenesis . Antibodies against anti-β-actin, anti-CtsK, and anti-c-Src were obtained from Santa Cruz Biotechnology . Alexa Fluor 488 goat anti-rabbit secondary antibody and FITC (fluorescein isothiocyanate)-conjugated phalloidin were purchased from Invitrogen . M-CSF was obtained from Prospec . RANKL was obtained from R&D systems . DAPI  and quercetin were obtained from Sigma-Aldrich . AR-1, AR-2, AR-3, AR-4, and AR-5 were isolated from eviously ,46,47, a2. Floating cells were cultured for three days with M-CSF (30 ng/mL), and then adherent bone marrow-derived macrophages (BMMs) were used for further experimentation.RAW264.7 cells were cultured in DMEM  supplemented with 10% heat-inactivated FBS  and penicillin (100 units/mL)-streptomycin (100 μg/mL). Bone marrow cells were isolated from femurs and tibias of six week-old male ICR mice  as described previously . BrieflyThe in vitro osteoclastogenesis assay was performed as described previously . Briefly5 cell/mL) were seeded onto 96-well plates and treated with the test compounds in the presence of 30 ng/mL M-CSF. After incubating for 72 h, MTT (0.5 mg/mL) was added and further incubated for 3 h. Insoluble formazan products were dissolved in dimethyl sulfoxide and a microplate reader was used to measure the absorbance at 540 nm.Cytotoxic effects of the test compounds on BMMs was assessed using the 3--2,5-diphenyl tetrazolium bromide (MTT)-based assay. Briefly, BMMs  and stimulated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of different concentrations of AR-3 and AR-5. The compound and culture medium were replaced after every two days. After seven days, the cells were completely removed from wells and the plate was thoroughly washed using distilled water. Resorption pits were captured using a model H550L microscope  and quantified with ImageJ software (Java 1.6.0_20 (64 bit); NIH, Bethesda, MD, USA).Bone resorption assay was performed as previously described . Briefly6 cell/mL) were seeded onto cover glass and stimulated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence of different concentrations of AR-3 and AR-5 for seven days. The compound and culture medium were replaced every two days. Cells were permeabilized, stained with FITC-phalloidin, and then mounted. Images were captured using an inverted laser scanning confocal microscope  equipped with an external argon laser, HeNe laser Green, and HeNe laser Red. Images were captured at colony midsection using a UPLSAPO 60X NA1.35 oil immersion objective (OLYMPUS).BMMs  and RANKL (100 ng/mL) in the presence of different concentrations of AR-5. After five hours of incubation, cells were fixed and the wound edge closure was measured with a microscope and the number of migrated cells was standardized to the vehicle treated control.For the preparation of whole cell lysates, the cells were lysed in a lysis buffer  in the presence of a protease inhibitor cocktail (Sigma-Aldrich). The lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by western blot analysis. Western blots were incubated overnight with the indicated primary antibodies (1:1000 dilution), and anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (1:2000 dilution) were used to visualize signals using an enhanced chemiluminescence system . The band intensity was quantified using ImageJ software .Cells were rinsed once in PBS, fixed in fresh 4% paraformaldehyde for 20 min at room temperature, then permeabilized in 0.5% TritonX-100. Nonspecific sites were blocked by incubating with PBS containing 1% goat serum before incubating the cells with an antibody against anti-NFATc1 (1:200 dilution). After washing four times with PBS, the cells were incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:250 dilution) for 4 h at room temperature, washed, stained with DAPI, and mounted. Confocal images were acquired using an OLYMPUS FV1000 inverted laser scanning confocal microscope equipped with an external argon laser, HeNe laser Green, and HeNe laser Red. Images were captured at colony midsection using a UPLSAPO 60X NA1.35 oil immersion objective (OLYMPUS).CtsK, 5′-GAC ACC CAG TGG GAG CTA TG-3′ (sense) and 5′-AGA GGC CTC CAG GTT ATG GG-3′ (antisense); TRAP, 5′-ACT TGC GAC CAT TGT TAG CC-3′ (sense) and 5′-TTC GTT GAT GTC GCA CAG AG-3′ (antisense); MMP-9, 5′-TGG GCA AGC AGT ACT CTT CC-3′ (sense) and 5′-AAC AGG CTG TAC CCT TGG TC-3′ (antisence); β-actin, 5′-GGG AAA TCG TGC GTG ACA TCA AAG-3′ (sense), and 5′-AAC CGC TCC TTG CCA ATA GT-3′ (antisense). The primers for DC-STAMP were purchased from Origene . PCR conditions were: 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s. All reactions were performed in triplicate and β-actin was utilized as an internal control. The 2−ΔΔCq method was used to determine the relative gene expression.The cells were collected and total RNA was isolated using an RNeasy Mini Kit according to the manufacturer’s protocol . First-strand cDNA was prepared from 1 μg of the total RNA. Real-time qPCR analysis was performed using TOPreal qPCR 2X PreMIX  and a Rotor-Gene Q real-time PCR cycler (Qiagen). The primers used included: n = 4/group). The mice were orally administrated with AR-3 , AR-5 , or the control vehicle (corn oil) for 1 h before the first administration of LPS (5 mg/kg) via intraperitoneal injection and then every other day for eight days. LPS was intraperitoneally injected on days 2 and 6. The mice were dissected on day 9. The femurs of the mice were collected and fixed in 4% paraformaldehyde. Intact left femoral metaphysic regions of each mouse were evaluated by high-resolution micro-CT analysis using an NFR-Polaris-S160 apparatus  with a current of 90 μA, a source voltage of 45 kVp, and an isotropic resolution of 7 μm. Femoral scans were performed over 2 mm from the growth plate, with a total of 350 sections per scan. After three-dimensional reconstruction, trabecular number (Tb.N), bone mineral density (BMD), trabecular separation (Tb.Sp), and bone surface/bone volume (BS/BV) were examined with quantitative analyses using INFINITT-Xelis software . For histological analyses, the femurs were fixed in 4% paraformaldehyde (Sigma) for a day, decalcified in 12% EDTA for three weeks, and embedded in paraffin. Sections of 5 μm thickness were prepared and stained using TRAP in order to examine the osteoclast formation.All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University . Six week-old ICR-mice were randomly divided into control vehicle-treated, vehicle + LPS-treated, AR-3 + LPS-treated, and AR-5 + LPS-treated groups . Statistical significance was assessed by one-way analysis of variance and Tukey’s test in order to examine the differences between the two groups using SPSS version 14.0 .


In contrast, lnc-EPC1-4 overexpression inhibited cell proliferation and induced cell apoptosis, indicating that it functions as a tumor suppressor gene. Collectively, these findings show that lnc-FAM72D-3 and lnc-EPC1-4 play a novel role that might contribute to hepatocarcinogenesis and identify potential candidate biomarkers for HCC diagnosis.Long noncoding RNAs (lncRNAs), such as LINC00462, HOTAIR, and MALAT1, are significantly upregulated in hepatocellular carcinoma (HCC) tissues. However, lncRNA expression in the serum of HCC patients is still unclear. To identify candidate lncRNAs for HCC diagnosis, we purified exosomal total RNA from the serum of healthy volunteers (controls) and hepatitis, cirrhosis, and HCC patients. To assess the function of lncRNAs, small interfering RNAs and overexpression vectors were designed and cell viability and cell apoptosis assays conducted. The exosomes of the control group had a larger number of lncRNAs with a high amount of alternative splicing compared to hepatic disease patients. qPCR assays showed that lnc-FAM72D-3, lnc-GPR89B-15, lncZEB2-19, and lnc-EPC1-4 are differentially expressed in HCC. Furthermore, the expression level of lnc-EPC1-4 correlated with age. While the expression levels of lnc-GPR89B-15 and lnc-EPC1-4 correlated with serum alpha-fetoprotein level.  Liver cancer, also called hepatocellular carcinoma (HCC), is the fifth-most common cancer worldwide, with a mortality rate that increased by almost 3% per year from 2010 to 2014 [Long noncoding RNAs (lncRNAs) are non-protein-coding transcripts with a length of >200 nucleotides. lncRNAs participate in various biological processes, such as tumorigenesis, metastasis, and proliferation –8, so thCirculating exosomes are small 30–150 nm membrane vesicles that are released into extracellular environments, such as serum or plasma . They usThis study determined whether exosomal lncRNAs have functions in hepatic diseases and could potentially be used as diagnostic biomarkers. We purified exosomal lncRNAs from 45 healthy volunteers, 45 hepatitis patients, 45 cirrhosis patients, and 45 HCC patients. We conducted RNA sequencing (RNA-seq) to obtain expression profiles and performed correlative and functional analyses of candidate lncRNAs.To investigate exosomal lncRNAs involved in hepatic diseases, we collected serum from healthy volunteers and hepatitis, cirrhosis, and HCC patients. Exosomes were purified by ultracentrifugation, and their sizes were determined by NTA. As shown in To further evaluate potential differences between exosomes of healthy volunteers and hepatic disease patients, we measured the particle and protein concentrations of exosomes. The control and hepatitis groups had similar particle concentrations, but the particle concentration was 50% lower for the cirrhosis group and 20% higher for the HCC group . The hepTo further qualify differences between the exosomes of healthy volunteers and hepatic disease patients, we purified exosomal total RNA and conducted RNA-seq. RNAs were annotated, and lncRNAs were extracted from the data for further analysis. For all four groups, lncRNAs were mostly intronic. In the control group, we determined 946 relevant genes to be involved in lncRNA formation, with 1955 associated alternative splicing events . In contTo determine the potential relevance of differentially expressed lncRNAs from the hepatitis, cirrhosis, and HCC groups, we conducted KEGG and GO analysis. In KEGG pathway annotation, the pathways were mainly RNA transport and protein processing in the endoplasmic reticulum : Ras, RaTo further investigate the expression differences in exosomal lncRNAs of the four groups, we performed qPCR and examined the expression levels of nine lncRNAs. The expression levels of lnc-GPR89B-15 and lnc-ZEB2-19 verified by qPCR were consistent with RNA-seq results in hepatitis, cirrhosis, and HCC groups . lnc-FAMNext, to determine the relationship between exosomal expression and diseases, we compared the expression levels of these lncRNAs between the control group and hepatitis, cirrhosis, and HCC groups . As indihsa-miR-29b-1-5p, hsa-miR-511-5p, and SLCO1B1. To further assess the functional effects of lnc-FAM72D-3 expression modulation, we performed cell proliferation and apoptosis assays. HepG2 cells transfected with lnc-FAM72D-3 siRNA-1 or lnc-EPC1-4 siRNA-1 and MHCC-LM3 cells transfected with pcDNA3.1-FAM72D-3 or pcDNA3.1-EPC1-4 were evaluated by MTS and flow cytometry assays. When HepG2 cells were transfected with lnc-FAM72D-3 siRNA-1 for 48 h, ~50% of HepG2 cells were viable, which was low compared to the siRNA negative control (NC) group, indicating that lnc-FAM72D-3 siRNA-1 significantly inhibits HepG2 cells proliferation  [In this study, we examined exosomal lncRNAs that are differentially expressed in hepatic disease patients. Increased lnc-FAM72D-3 and decreased lnc-EPC1-4 levels are clearly correlated with HCC; these findings were verified by qPCR for a group of 180 healthy volunteers and hepatic disease patients. Functional validation with MTS and cell apoptosis assays showed that lnc-FAM72D-3 knockdown induces significant inhibition of cell proliferation and promotes cell apoptosis, while upregulation of lnc-EPC1-4 expression has the same effect. There are currently no reports on the exact underlying mechanism; however, we have elucidated new roles for them in HCC and other hepatic diseases. On the basis of our bioinformatic prediction of downstream targets of lnc-FAM72D-3 and lnc-EPC1-4, several candidate target genes and miRNAs have been shown to regulate cell proliferation and cell apoptosis. These included hsa-miR-511-5p, which we had demonstrated increases by pcDNA3.1-lnc-EPC1-4 transfection. Consistent with our findings,  (TRIB2) . TRIB2 i (TRIB2) –29. MiR- (TRIB2) . TherefoCDCA7, as a target gene of hsa-miR-29b-1-5p, is overexpressed in several malignancies [We also demonstrated that SLCO1B1 is targeted by lnc-EPC1-4. The sequential administration of sorafenib (front-line treatment) and regorafenib (second-line treatment) efficiently improves therapeutic efficacy in advanced HCC. Influx transporters  are highly involved in the metabolism and clearance of these chemotherapy agents in the liver, while downregulation of these influx transporters might inhibit cellular uptake, thereby discounting their clinical benefits –33. Hu egnancies , 35. lncOur results also demonstrated that lnc-FAM72D-3 siRNA-1 induces hsa-miR-5787 expression, while pcDNA3.1-lnc-FAM72D-3 inhibits hsa-miR-5787 expression. However, modulation of lnc-FAM72D-3 fails to affect eukaryotic translation initiation factor 5 (EIF5), which is a target of has-miR-5787 . EIF5 noIn summary, lnc-FAM72D-3 and lnc-EPC1-4 might be used as diagnostic and prognostic biomarkers for HCC. Future studies that characterize the expression of additional candidate targets of these lncRNAs might help us better define their mechanisms, which might ultimately lead to the development of new diagnostic and therapeutic tools for HCC.g for 20 h at 10°C. The supernatants were discarded, and pellets containing exosomes were resuspended in phosphate buffer solution (PBS). The size of the exosomes was validated by using nanoparticle tracking analysis , and the content of membrane proteins, such as CD63, CD9, and TSG101, was verified using western blotting.Whole blood was collected from 45 healthy volunteers (controls), 45 hepatitis patients, 45 cirrhosis patients, and 45 HCC patients. Serum was isolated from the whole blood and ultracentrifuged using a Beckman L8 ultracentrifuge  at 100,000×Exosomes, HepG2 cells, and MHCC-LM3 cells were lysed in 1% sodium dodecyl sulfate (SDS) lysis buffer. Protein concentrations were evaluated using the bicinchoninic acid (BCA) protein assay kit . Proteins were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. The nitrocellulose membranes were blocked with 5% nonfat milk in PBS for 1 h at room temperature and incubated overnight at 4°C with primary antibodies anti-CD63 , anti-TSG101 , anti-CD9 , and anti-calnexin . After several washes, the nitrocellulose membranes were incubated in a blocking buffer with a secondary antibody coupled to horseradish peroxidase (HRP) for 2 h at room temperature. The complexes were detected by electrochemiluminescence (ECL) .2 at 37°C. The cells were maintained in high-glucose Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum  and passaged every 3 days.HCC cell lines  were obtained from the American Type Culture Collection . The human normal liver L02 cell line and Huh-7, MHCC-LM3, and MHCC97-H cells were obtained from CELLCOOK  and cultured with 5% COFull-length lncRNAs were amplified by polymerase chain reaction (PCR) using Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific), and corresponding complementary DNAs (cDNAs) were subcloned into the NheI and KpnI sites of pcDNA3.1(+)  according to the manufacturer’s instructions. The vectors constructed were verified by Sanger sequencing. Small interfering RNAs (siRNAs) for candidate lncRNAs were designed and synthesized. The vectors and siRNAs were transfected into cell lines using lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The expression level of lncRNAs and their targeted miRNAs and messenger RNAs (mRNAs) were determined by real-time quantitative PCR (qPCR) .6/mL into 96-well plates (100 μL/well), treated with overexpression vectors or siRNAs, and examined after 24, 48, and 72 h. A 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) mixture  was added for 3 h, and the optical density of the cells was detected at a frequency of 490 nm.Cells were seeded at a density of 1 × 10Cells were resuspended in 1× binding buffer, and 5 μL of fluorochrome-conjugated annexin V and 5 μL of 7-aminoactinomycin D (7-AAD) staining solution (Thermo Fisher Scientific) were added. The cells were assessed using a fluorescence-activated cell sorting Calibur flow cytometer, and the percentage of the apoptotic cells was measured.q < 0.05 and fold change > 2.0).Total RNA was purified from exosomes from the isolated serum from 45 healthy volunteers (controls), 45 hepatitis patients, 45 cirrhosis patients, and 45 HCC patients using the RNeasy Mini Kit . RNA integrity was evaluated on the basis of the RNA integrity number (RIN) using Agilent Bioanalyzer 2100 . RNA was further purified using the RNA Clean XP Kit , and the DNA residue was removed using the RNase-free DNase Set (QIAGEN). RNA quality and concentration were determined using NanoDrop 2000 (Thermo Fisher Scientific). Ribosomal RNA (rRNA) was removed using the NEBNext rRNA Depletion kit . Then, 1 μg of total RNA was used for library preparation using the VAHTSTM mRNA-seq v2 library Prep Kit  according to the manufacturer’s instructions. RNA was fragmented and then double-stranded cDNA was synthesized. End-polishing was performed, and the cDNA fragments were ligated with adapters and then subjected to universal PCR amplification in order to obtain a sufficient library for sequencing. The library quality was assessed using Agilent Bioanalyzer 2100. RNA-seq was performed using Illumina Hiseq 4000 . The data were assembled and annotated with corresponding symbols for transcripts. Differentially expressed lncRNAs were screened using R software  according to the manufacturer’s instructions. PCR was performed using the GoTaq qPCR Master Mix (Promega Corporation). PCR amplification was performed using the ABI 7500 system . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control.t-tests, one-way analysis of variance (ANOVA), and two-way ANOVA when data corresponded to a normal distribution. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS Statistics 19  and GraphPad Prism . Comparisons were performed using independent This study was approved by the ethics committee of Third Affiliated Hospital of Sun Yat-sen University. The patients provided informed written consent.Supplementary FiguresSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4


This paper focuses on stereotype threat and its effects on sense of belonging in the German Physics Olympiad science competition. Participants completed questionnaires about sense of belonging, stereotype endorsement, interest, and self-concept in physics, as well as about value and success expectations of studying physics in college. Female participants who endorsed negative stereotypes about female talent for physics felt less sense of belonging to physics. This effect did not manifest for male participants. Sense of belonging to physics significantly predicted value and success expectations for studying physics in college beyond what is predicted by interest and self-concept in physics. These findings suggest that sense of belonging is influenced by stereotype threat, which was shown to cause gender differences in science. Nevertheless, sense of belonging could be included into the expectancy-value theory based on its predictive impact on value and success expectations of studying physics. Lower interest in science, technology, engineering, or mathematics (STEM) careers and often lower numbers of girls in science precipitated much research on gender differences in science, applying different rationales and theoretical approaches e.g., . DecliniOf the various approaches that try to explain the reasons for the gender gap, two figures very prominently. First is stereotype threat theory. Stereotype threat theory explains the behavior of stereotyped minorities including females in sciences based on negative stereotypes that affect behavior and performance of the minority group due to their social identification with the stereotyped group e.g., . The impThis study focuses on a sense of belonging as a central factor in girls’ physics motivation by connecting the theories of expectancy-value and stereotype threat in one model. The addressed research questions are:Are girls in a physics environment adversely affected by stereotype threat’s consequences, in this case analyzed as having lower sense of belonging due to prevalent science gender stereotypes?Does sense of belonging influence success expectations and value of studying physics?We chose to research these questions using the German Physics Olympiad as a prototypical physics environment.Humans are motivated by the need to form social attachments and feel belonging to others . This neThis importance of social connections can also be seen when analyzing the structure of sense of belonging. Belonging in school can be divided into academic belonging and social belonging , wherebyIntentions to continue in a field, choosing it in college or as a career and belonging are also closely connected. When belonging is felt not just within school in general, but to a certain domain, it influences students’ intention to remain in a domain through college e.g., . It is pA possible explanation of the connection of belonging and career choices can be found in school, where belonging also predicts value and success expectations for middle school subjects  and highIn sum, belonging is an essential part of educational settings. As college belonging alone was found to be one of the main determinants for completing college , belongiWomen and girls in science are faced with negative stereotypes about them, and these negative stereotypes threaten both their belonging to the group and confirmation of the stereotype that goes along with their gender identity see . Within Intervening into the potentially vicious cycle of stereotype threat is important , as formBeyond this, stereotype threat was also shown to reduce girls’ interest in pursuing STEM in school or college, long-term interest, and career aspirations. In sum, among other things, negative stereotypes about the abilities of women and girls in STEM cause a reduced sense of belonging e.g.,  and loweIn analyzing career aspirations in science and researching reasons for the gender gap, making a division between school and general interest seems reasonable, as it was found by The Physics Olympiad shows the typical male dominance and thereby one of the assumed stereotype threat consequences. Although they are interested in physics, girls do not continue along that path in physics, do not perform up to their full potential, and end up dropping out of the contest. Nevertheless, the phenomenon of stereotype threat has not yet been researched in this context. Also, the predominantly male environment might signal to women that they do not belong. This lack in sense of belonging, in return, could adversely influence their success and value beliefs according to the expectancy-value theory. Sense of belonging itself as a link between stereotype threat and the expectancy-value theory has not been investigated in the context of science competitions.Even though out-of-school programs seem less affected by the prevalent problem of declining science interests of students , the PhyHypothesis 1:We hypothesize that endorsing stereotypes about girls and women in physics negatively affects sense of belonging of the female Physics Olympiad participants, whereas the male participants’ sense of belonging should not be affected.The lens of stereotype threat theory e.g.,  was applHypothesis 2: We hypothesize that sense of belonging is impacting success expectations in and the value of choosing a career in physics for participants of the Physics Olympiad.Based on previous evidence on success expectations and career aspirations in STEM fields e.g., , regardiThe present study addressed first-round participants of the German Physics Olympiad, which is a national contest for non-university students under 20 years old. By means of achievement-based selection on written and experimental tests, the number of participants is reduced over four rounds, until only five participants are selected for the national International Physics Olympiad team. Participants in the contest have to solve tasks on their own (either at home or in school) in rounds 1 and 2. In these rounds, they do not interact directly with other competitors. In rounds 3 and 4, students meet and compete in interaction with other candidates.Stereotypes were not explicitly triggered to elicit stereotype threat effects. Instead, it was assessed whether students endorsed common stereotypes about women’s and girls’ talent for physics and science. The regular physics setting, which interested students encounter when pursuing physics, was thus not changed or adapted. It was thereby assessed if students were subject to stereotype threat in a regular physics setting.via e-mail, letters, and posters. Participation was voluntary and anonymous; it did not have any positive or negative effect on the participation or success in the Physics Olympiad. As an incentive, participation in the project was connected to a lottery that gave out several prizes .The current study was part of a larger project  which evaluates factors influencing participants’ success in the science Olympiads in Germany. For the current study, only the data of the first-round are included, which means that participants were participating alone and not interacting with other participants. All 931 first-round contestants of the German Physics Olympiad were informed and invited to participate in the study M = 16.25, SD = 1.04) and 174 indicated male gender ; 24 individuals did not indicate their gender. Participants did not differ from the overall sample regarding gender  or class level , but participants were significantly younger than the overall sample  and performed significantly better . The significant difference in age was not regarded as problematic, as students still not significantly differed in the other characteristics. Especially the nonsignificant results in class level, suggesting the same amount of physics education, and gender, suggesting that the gender ratio was maintained, lead to this conclusion. Therefore, it can be assumed that participants in the study are reasonably similar to the overall sample of participants in the Physics Olympiad.A total of 282 first-round contestants responded to the invitation (30.29% response rate) and constituted the sample for the current study. Of these, 84 participants indicated female gender ; negative affect focuses on the feelings one has when participating in the environment ; trust focuses on trusting the setting not to be biased ; desire to fade focuses on the wish of being active in the environment ; and finally acceptance focuses on the feeling of being seen as a member by other participants of the environment . The scale was used without further division into subscales for the analysis, similar to Sense of belonging was measured with a shortened 15-item version of Math Sense of Belonging Scale of 2  = 188.24, p < 0.001, CFI = 0.94, TLI = 0.92, RMSEA = 0.07 , and SRMR = 0.08. According to In order to gauge the structural validity of our adapted sense of belonging scale, a confirmatory factor analysis for sense of belonging was calculated with Mplus  were measured with four items each from an adapted and shortened scale from At the end of the questionnaire, stereotype endorsement was measured by four items adapted from Self-concept in physics as the competence one perceives as having in physics was measured by six items (rated from 1 “strongly disagree” to 4 “strongly agree”) from the German Program for International Student Assessment (PISA) questionnaire . Specifically, we first regressed sense of belonging on the dummy of gender:β0 denotes sense of belonging in the reference group , whereas the slope of gender β1 indicates the extent to which females differ in their sense of belonging from the reference group. The second step included stereotype endorsement as a further predictor:Here, the intercept β2 indicates the overall impact of stereotype endorsement on belonging for both males and females combined.Whereby, the slope z-standardized before multiplication):Finally, whether females are adversely influenced by gender science stereotypes was investigated by additionally including an interaction term of gender and stereotype endorsement  = 0.46, p = 0.650, Cohen’s d = 0.07]. Female participants reported significantly lower sense of belonging , success expectations , and self-concept  than male participants. Male and female participants did not differ in value  or interest .Mean values  in senser = −0.13, p = 0.031). Splitting the group by gender shows that females’ sense of belonging was significantly correlated with stereotype endorsement , whereas males’ was not .Correlations of the scales can be found in β = −0.19, p = 0.002). In the second step of the dummy regression (Model 2), stereotype endorsement was added as a predictor of sense of belonging. The results evidenced an overall negative effect of stereotype endorsement on sense of belonging in physics . The interaction term of gender and stereotype endorsement was introduced to the model in the third and last step of the dummy regression (Model 3). The model showed that this overall relation between stereotype endorsement and sense of belonging was due to the females in the sample, who had a significantly stronger relation  compared to no relation for the males  was calculated  to analyhe males . The resβ = 0.50, p < 0.001 for expectancy; β = 0.42, p < 0.001 for value; β = 0.25, p < 0.001) and value . Specifically, and in addition to sense of belonging (Model 1: R2 = 0.25), in Model 2, self-concept in physics  but not interest  significantly predicted success expectations (R2 = 0.42). The reverse was shown for value: additionally to belonging (Model 3: R2 = 0.18), in Model 4, interest  but not self-concept  significantly predicted value of choosing to study physics or take a job in physics (R2 = 0.38). These results are according to the hypothesis that sense of belonging predicts two important factors of the expectancy-value theory.Linear regressions separately for success expectations and value for choosing physics at university or a job in physics were calculated to assess Hypothesis 2, whether sense of belonging predicted students’ career expectations. Sense of belonging was significantly predicting both variables , showing that females had a lower self-concept in physics than their male counterparts. In the second step (Model 2), stereotype endorsement was included as a further predictor. Stereotype endorsement did not significantly predict self-concept , thus indicating that stereotype endorsement did not have an overall negative effect on self-concept. The interaction term of gender and stereotype endorsement was introduced to the dummy regression in the last step (model 3). It was significantly predicting self-concept . This shows that gender moderates between stereotype endorsement and self-concept. On the other hand, the moderation between stereotype endorsement and interest did not turn out to be moderated by gender .We added two further dummy regressions to analyze if gender also moderated between stereotype endorsement and interest or self-concept , thus po2  = 320.14, p < 0.001, CFI = 0.93, TLI = 0.91, RMSEA = 0.06 , and SRMR = 0.07. According to β = −0.17). Also, sense of belonging significantly predicts the value of choosing a career in physics (β = 0.26) additionally to interest (β = 0.44), as well as success expectations for choosing a career in physics (β = 0.58) additionally to success expectations (β = 0.20). The suggested model, which combines the impact of negative stereotypes and the expectancy-value theory, thus appears to fit within the Physics Olympiad context.Lastly, the previous results were used to adapt the hypothesized research model in Research and research-based interventions need to explore how to retain the numbers of young women once they have decided on a physics career path. In the present study, the Physics Olympiad competition served as a prototypical physics environment; females not only are participating in lower numbers but also drop out of the competition in disproportionally higher numbers than the male participants. This paper addressed this problem by introducing sense of belonging as a previously rarely considered variable and situating it within an expectancy-value framework: female participants were found to have lower sense of belonging than male participants, indicating that the differences in belonging are due to the male-dominated environment signaling lower belonging by activating negative gender science stereotypes. However, above and beyond domain-specific self-concept and interest, sense of belonging predicted expectations and value of studying physics at university, two factors that, according to the expectancy-value theory, are predicting career choices. The adapted research model was shown to fit well, suggesting that belonging should be considered as an important factor within science career choices with regard to gender and the consequences of stereotype threat.The benefits of sense of belonging might be stifled by the problematic stereotype threat effects. Although participating female students were a group of highly interested and talented students, they nevertheless had a lower sense of belonging to the physics community than their male counterparts. Further, although there was no significant gender difference in endorsing the gender science stereotypes, girls’ belonging was significantly reduced the more they endorsed the stereotypes, whereas male participants were unaffected by endorsing stereotypes. We interpret this gender differential effect of stereotype endorsement on sense of belonging as stereotype threat in the physics competition, of which only female participants are susceptible. Adding to previous research on the field already showing lower sense of belonging for women in physics e.g.,  and womeAccording to the expectancy-value model, success expectations and value are decisive for achievement-related choices, in this case choosing to study physics at the university level or choosing a job in physics. A rarely investigated variable in this process, beyond domain-specific self-concept and interest, is sense of belonging that connects the social environment to the individual’s need to belong to a group. The present study’s findings underscore the importance of sense of belonging in the school-to-university transition phase; sense of belonging to physics significantly predicted success expectations for studying physics at university and value of doing so. Sense of belonging had a predictive value above and beyond the expected influence of self-concept and interest e.g., . TherebySeeing these results with regard to stereotype threat, they appear to emphasize the problems for females in science even more. Although the girls participating in the Physics Olympiad are interested and see themselves as capable of competing in physics, they are negatively affected by stereotypes. The stereotypes affect them negatively not just in their belonging but also in their self-concept, thereby possibly lowering their career aspirations even more.Participation in this study was voluntary. Although there are only few differences regarding demographics between the German Physics Olympiad participants who partook in this study and those who did not, we cannot extend this conclusion to other motivational variables. However, even if only highly interested and engaged students participated in the assessment, this would not bias the interpretation of the present study’s findings that higher interest does not immunize against stereotype threat. We regard our findings as evidence that even among the highly interested students, females face adversity in physics, complementing previous research on stereotype threat. Further research should nevertheless look more closely into sense of belonging and note differences between school and extracurricular competitions as well as the differences in characteristics of students and participants in science competitions.Further, all variables were measured at one time as the present study employed a cross-sectional design. Although we were guided by theoretical assumptions of underlying mechanisms, we strictly speaking cannot draw causal inferences. In the future, more research should focus on longitudinal or experimental designs to corroborate present findings pertaining to causal relations.Finally, individuals suffering from stereotype threat due to negative stereotypes are usually inferred indirectly in previous studies, but not assessed by self-report measures. In our study, we inferred the existence of the stereotype threat effect by explicitly measuring stereotype endorsement, as previous research has shown that higher stereotype endorsement led to higher stereotype threat susceptibility see . HoweverThe present study findings provide further insight into the pernicious hold that gender science stereotypes have over women and girls in STEM; stereotype threat not only reduces interest in pursuing physics overall but also specifically reduces belonging of those girls with an interest beyond normal curricular physics education and who engage in physics activities in their extracurricular leisure time.From our findings, we draw two related implications:Based on the finding that even highly interested young women participating in an out-of-school physics competition were susceptible to stereotype threat, which consequently lowered their sense of belonging, implies that competitions such as the Physics Olympiad need to address this in two possible ways: first, by adapting their environment in such a way that the gender science stereotypes or gender itself is made less salient in the achievement situation. Previously, three possible ways for this have been shown: using role models with regard to stereotypes in the domain e.g., , reducinBased on the finding that sense of belonging predicted career-related success expectations and value beliefs above and beyond domain-specific self-concept and interest implies that the feeling of belonging to an environment, being a member of it, valued and accepted, is an overlooked social component to motivation and career-related choices. Future research should dedicate itself in further situating sense of belonging within the expectancy-value framework and determine which features of the environment or personal characteristics further an individual’s sense of belonging.In conclusion, this paper supports the idea that the expectancy-value theory and sense of belonging should be connected into one framework to systematically study and understand students’ physics career decisions. Belonging is a significant predictor of success expectations and value of studying physics at university both for females and males. Still, no matter how much they engage in physics in their extracurricular time, girls seem susceptible to stereotype threat and report lower sense of belonging to physics when they endorse negative stereotypes about females’ physics talent and ability. Therefore, promoting students’ interest by offering the opportunity to participate in out-of-school competitions such as the Physics Olympiad does nothing for a more equitable gender participation pattern in physics fields without further inclusion of gender-sensitive measures. To tap into the full potential of talented and interested young women in physics, the competition design and environment must counteract the pernicious effects of gender science stereotypes.The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.AL wrote the first draft. AL, MK, and UK contributed to the interpretation of statistical analyses and edited and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.