Approximately one-third of Japanese couples currently worry or previously worried about infertility. To develop strategies for the primary prevention of infertility as a population approach, it is important for the general population to be knowledgeable about fertility and infertility. The internet may contribute to the dissemination of information regarding infertility and fertility. However, few studies have examined online information about fertility.This study aimed to quantitatively examine online Japanese-language information about lifestyle factors associated with reduced fertility.We conducted online searches, using the 10 search terms with the highest numbers of searches that people hoping to conceive are likely to input in two major search engines in Japan (Google Japan and Yahoo! Japan). From the 2200 retrieved websites, 1181 duplicates and 500 websites unrelated to our objective were excluded, resulting in a final dataset of 519 websites. Coding guidelines were developed for the following lifestyle factors associated with reduced fertility: sexually transmitted diseases, psychological stress, cigarette smoking, alcohol use, nutrition and diet, physical activity and exercise, underweight, overweight and obesity, and environmental pollutants.P<.001). The association between reduced fertility and nutrition and diet was mentioned significantly more frequently by the media than by hospitals and clinics (P=.008). With regard to the sex of the target audience for the information, female-specific references to psychological stress, sexually transmitted diseases, nutrition and diet, underweight, physical activity and exercise, and overweight and obesity were significantly more frequent than were male-specific references to these lifestyle factors .In terms of the website author’s professional expertise, 69.6 % of the coding instances for the selected lifestyle factors were mentioned by hospitals, clinics, or the media, whereas only 1.7% were mentioned by laypersons. Psychological stress (20.1%) and sexually transmitted diseases (18.8%) were the most frequently mentioned lifestyle factors associated with reduced fertility. In contrast, cigarette smoking, alcohol use, nutrition and diet, physical activity and exercise, underweight, overweight and obesity, and environmental pollutants were mentioned relatively infrequently. The association between reduced fertility and sexually transmitted diseases was mentioned significantly more frequently by hospitals and clinics than by the media (Of the lifestyle factors known to be related to reduced fertility, cigarette smoking, alcohol use, and male-specific lifestyle factors are mentioned relatively infrequently in online information sources in Japan, and these factors should be discussed more in information published on websites. At the 1994 United Nations International Conference on Population and Development, reproductive health was defined as “a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity, in all matters relating to the reproductive system and to its functions and processes” , and repInfertility is defined as the failure to achieve conception following at least 12 months of unprotected sexual intercourse . InfertiNumerous factors may contribute to reduced fertility in men and women. In addition to genetic background and reproductive history, environmental factors and current lifestyle habits have been proposed as causes of male and female infertility . AccordiA recent study found that about 60% to 70% of the reproductive-aged population in Japan responded incorrectly to a question about the association between cigarette smoking and reduced fertility . SimilarIn terms of sex differences, several studies have shown that, compared with women, men are less knowledgeable regarding the associations between lifestyle factors and reduced fertility ,29. AlthCurrently, the internet is a preferred and common source of health information . About 7Previous studies regarding information on infertility and fertility in the media have examined (1) newspaper reports about assisted reproductive technology ; (2) infPeople who are diagnosed with infertility and those receiving infertility treatment may receive accurate information on fertility from health care professionals. However, no studies have examined what kinds of online content are accessible to the general population, although online information is especially important for people who do not visit medical institutions but are trying to conceive or hoping to have a child in the future. Thus, it is crucial to examine online information on the lifestyle factors associated with reduced fertility.The study aimed to (1) quantitatively examine the information on lifestyle factors associated with reduced fertility accessible to people hoping to have a child who seek information regarding fertility on the internet and (2) identify the characteristics of this information in terms of lifestyle factors, the webpage author’s professional expertise, and the sex of the target audience .We used a Japanese-language search string input into the two most popular search engines in Japan, Google Japan  and YahoA flow diagram depicting the search terms generation procedure is presented in As a next step, we entered these 6 keywords into Google Japan and Yahoo! Japan and listed the related keywords shown by the search engines . Related keywords are words that are frequently searched in combination with the main keywords . AdditioAt the end of this process, the final search was performed using the following keywords: “ninkatsu” (trying to conceive), “ninshinshitai” (hoping to get pregnant), “funin” (infertility), “bebimachi” (waiting for a baby), “funin genin” (infertility cause), “kodomohosii” (hoping to have a child), “akachanhosii” (hoping to have a baby), “ninshindekinai” (I cannot get pregnant), “shizenninshin” , and “huninsho genin” (infertility cause).The unit of analysis in this study was the webpages suggested by the search engines. Because websites differ considerably in size, coding entire websites could introduce biases based on size ,48. MoreThe first 100 results retrieved using each search engine were collected by the first author. Each time we input search keywords or changed search engines, the search history and cookies were cleared. We conducted online searches from October 21 to November 3, 2019. The flow diagram of the webpage selection is presented in There are no current clinical practice guidelines in Japan regarding lifestyle factors associated with reduced fertility. We created a list of lifestyle factors associated with reduced fertility by reviewing information from the Japan Society of Obstetrics and Gynecology , MinistrEach webpage was categorized according to the author’s professional expertise: hospitals and clinics, media organizations, public administration institutions, business and service entities, academic and medical societies, pharmaceutical and medical device firms, alternative medicine practitioners, or laypersons. Hospitals and clinics indicated that the content appeared on the website of a hospital or clinic, including blogs written by clinicians. Media organizations indicated that the content appeared on the website of a mass media organization such as a newspaper, magazine, or news site. Public administration institutions indicated that the content appeared on the website of a public organization such as the government, a municipality, public health care center, or specialized public consultation support center. Business and service entities indicated that the content appeared on the website of an enterprise such as a marriage support service company, bridal company, recruiting company, children’s goods retail business, cooking school, counseling organization, architectural firm, stationery company, or hospital search service company. Academic and medical societies indicated that the content appeared on the website of a medical society such as an association of physicians or medical institutes. Pharmaceutical and medical device firms indicated that the content appeared on the website of a pharmaceutical company, medical device firm, or pharmacy. Alternative medicine practitioners indicated that the content appeared on the website of a practitioner of alternative medicine such as osteopathy, herbal medicine Kampo, acupuncture, moxibustion, or yoga or on the website of a health food company. Laypersons meant that the content was written by persons who were currently trying or had previously tried to conceive, including patients.We created coding rules for the selected lifestyle factors. These coding guidelines are summarized in We analyzed the textual data on the websites retrieved using the search terms described above. First, we read the text carefully. Second, all data were coded on the selected lifestyle factors, author’s professional expertise, and sex of the target audience . Finally, data from all webpages were assembled and pooled in Microsoft Excel. When information on lifestyle factors was provided, the code of 1 was assigned. When no information on lifestyle factors was provided, we assigned the code of 0. The URL and title of each webpage were saved as a reference during the data analysis. Because multiple types of lifestyle factors could be listed on a single website, instead of calculating the number of webpages mentioning a particular lifestyle factor, we calculated the number of mentions (codes) for each selected lifestyle factor associated with reduced fertility.Approximately 20% of the final dataset  was evaluated by two independent, blinded raters (RY and EF) to examine interrater reliability. Using the coding guidelines created by the first author of this study (RY), EF was instructed on applying the coding system in a training session that lasted about 1 hour. In a pilot test phase, the two raters applied the coding system to 10 webpages randomly selected from the full sample. No problems were identified during this phase. After a formal reliability assessment phase was completed, the first author (RY) calculated the interrater reliability index.P<.05 for all comparisons. Analyses were performed using R for Windows version 3.5.1 .To assess interrater reliability, the Gwet agreement coefficient (AC1) statistic, which is less affected by prevalence compared with the Cohen kappa , was useThis study was granted an exemption from the requirement of ethics approval by the ethical review committee at the Graduate School of Medicine, The University of Tokyo, because we aimed to analyze online information, meaning that this study was not medical research involving human subjects, and because the authors had no conflicts of interest related to this study.The assignment of all codes ranged from 0 to 9 (mean 1.017) per page. The assignment of codes ranged from 0 to 8 (mean 1.058) for hospitals and clinics, from 0 to 8 (mean 0.974) for media organizations, from 0 to 9 (mean 1.147) for public administration institutions, from 0 to 6 (mean 1.029) for business and service entities, from 0 to 6 (mean 1.389) for academic and medical societies, from 0 to 7 (mean 2.105) for pharmaceutical and medical device firms, from 0 to 3 (mean 0.367) for alternative medicine practitioners, and from 0 to 3 (mean 0.391) for laypersons. Of the webpages retrieved, 0 codes were assigned to 60.7% (315/519). The number of webpages for which 0 codes were assigned was 53.4% (110/206) for hospitals and clinics, 66.2% (102/154) for media organizations, 68% (23/34) for public administration institutions, 60% (21/35) for business and service entities, 50% (9/18) for academic and medical societies, 53% (10/19) for pharmaceutical and medical device firms, 77% (23/30) for alternative medicine practitioners, and 74% (17/23) for laypersons.The distributions of the webpage author’s professional expertise are summarized in A review of the coding by the two independent, blinded raters on the randomly selected subsample of 100 webpages revealed that the interrater agreement was excellent. The two raters agreed on the assignment of codes in 98.00% (3528/3600) of coding instances. The Gwet AC1 statistic for the assignment of codes ranged from 0.969 to 1.000 (mean AC1 0.985) for the author’s professional expertise, from 0.957 to 0.992 (mean AC1 0.979) for lifestyle factors, and from 0.950 to 0.986 (mean AC1 0.978) for the sex of the target audience. For the data analysis in this study, we used the coding of the first author.Of the coding instances concerning lifestyle factors associated with reduced fertility, 20.1% (106/528) related to psychological stress and 18.8% (99/528) related to sexually transmitted diseases. Together, codes on these two lifestyle factors accounted for 38.9% (205/528) of all lifestyle factor-coding instances. Websites referring to the associations between reduced fertility and nutrition and diet, cigarette smoking, underweight, physical activity and exercise, overweight and obesity, alcohol use, and environmental pollutants were relatively rare.P<.001). In contrast, of the coding instances referring to the association between nutrition and diet and reduced fertility, 26% (18/68) were published by hospitals and clinics and 40% (27/68) were published by media organizations; the association between nutrition and diet and reduced fertility was significantly more frequently mentioned by the media than by hospitals and clinics (P=.008). The other lifestyle factors associated with reduced fertility did not show statistically significant differences between hospitals/clinics and media organizations.P=.002, sexually transmitted diseases: P<.001, nutrition and diet: P<.001, underweight: P<.001, physical activity and exercise: P<.001, overweight and obesity: P<.001).This study quantitatively examined online information on the lifestyle factors associated with reduced fertility. The main findings of the study confirm that a large proportion of the information on lifestyle factors associated with reduced fertility was disseminated by hospitals, clinics, and the media. Furthermore, the findings show that the frequencies of descriptions provided by different entities varied by the particular lifestyle factor examined.In terms of the professional expertise of the author publishing online information on lifestyle factors associated with reduced fertility, following the categorization of professional expertise described in the Methods section, our study demonstrated that, of the codes assigned for the examined lifestyle factors associated with reduced fertility, 41.2% 218/528) were published by hospitals and clinics, 28.4% (150/528) by media organizations, and 1.7% (9/528) by laypersons. This may be because of the strategy of search engine optimization used by hospitals and clinics to make their websites appear higher in user lists of search results generated by the search engines. This may also be because lifestyle factors associated with reduced fertility are discussed in the context of infertility examinations or treatments. The large number of references to lifestyle factors associated with reduced fertility by media organizations may be associated with the mass media coverage of the public debate  after th were pubPsychological stress and sexually transmitted diseases were the two most frequently mentioned lifestyle factors associated with reduced fertility. In contrast, the associations between reduced fertility and cigarette smoking, alcohol use, physical activity and exercise, underweight, and overweight and obesity were less frequently discussed. Our findings are consistent with those of a previous study showing that people lack a general understanding of fertility, including the associations between reduced fertility and cigarette smoking and overweight and obesity . In 2017Our research has revealed that associations between reduced fertility and both psychological stress and sexually transmitted diseases were more frequently discussed by hospitals and clinics than by the other types of webpage author. This may be because lifestyle factors associated with reduced fertility are discussed in the context of infertility distress regarding infertility examinations or treatments ,60. We aProviding treatment is the main role of hospitals and clinics, and these entities may use their websites as a tool for patient acquisition. However, for individuals seeking fertility information on the internet because they are trying to conceive, the content of hospital and clinic websites may be a major source of information on fertility. This idea is supported by the finding that hospital and clinic websites rank higher in lists of search results when users use web search engines. Hence, various kinds of information provided by hospitals and clinics may be beneficial for people who are trying to conceive. In previous work, scholars have recommended that, despite the pressures of a competitive environment, hospitals and clinics offering fertility treatment should present educational information in an ethically balanced manner . The samWe found that there were fewer mentions of lifestyle factors associated with reduced fertility for men than for women. This may be because (1) men are less likely to seek information on infertility , (2) malAs mentioned above, infertility may harm the self-esteem of people who are trying to conceive because of its latently stigmatizing nature . People This study has several limitations. First, we limited our examination to online information, which does not capture all circulating public messages. Television broadcasts and print newspapers and magazines may also be widely used sources of information about lifestyle factors associated with reduced fertility. Second, although a substantial number of websites n=519) were retrieved for this analysis, availability, accessibility, and time limitations made it unfeasible to analyze all relevant sites. Third, despite the fact that we selected search terms based on related words indicated by the selected search engines, these search terms may have reflected our own biases. For example, it is possible that search terms were relatively easy for women to use. However, because men were less likely to seek information regarding infertility , the web19 were rIn terms of the webpage author’s professional expertise, authors from hospitals, clinics, and the media relatively frequently mentioned lifestyle factors associated with reduced fertility, whereas laypersons mentioned them relatively rarely. Regarding the specific lifestyle factors associated with reduced fertility, psychological stress and sexually transmitted diseases were more frequently discussed compared with the other factors. The association between reduced fertility and sexually transmitted diseases was more frequently discussed by hospitals and clinics than by the media. Conversely, the association between reduced fertility and nutrition and diet was significantly more frequently mentioned by the media than by hospitals and clinics. With regard to the sex of the target audience, male lifestyle factors were less frequently discussed than were female lifestyle factors. The authors of fertility-related websites should more frequently mention information on lifestyle factors associated with reduced fertility overall, moving beyond only psychological stress and sexually transmitted diseases to also discuss nutrition and diet, cigarette smoking, underweight, physical activity and exercise, overweight and obesity, alcohol use, and environmental pollutants. These authors should also make an effort to provide specific information on men’s lifestyle factors.


Few studies have examined health literacy and fertility knowledge among women from low income, socio-culturally diverse communities presenting for fertility care in the United States. Our study sought to examine demographic predictors of fertility-related knowledge among infertile women from low and high-resource communities in two major metropolitan centers in the United States.Fertility Knowledge Assessments were administered to women presenting for fertility care at county medical centers serving low-resource, largely immigrant patients and to women from largely affluent populations presenting to comprehensive fertility centers in two cities. The influence of demographic predictors on fertility knowledge was examined through regression analysis.n = 70), the mean age was 32.8 ± 6.1 years vs 35.0 ± 5.0 years in the fee-for-service/high resource clinic . Among the LR patients, 74% were immigrants, 71% had an annual income <$25,000 and 52% had completed high school. Among HR patients, 36% were immigrants, 60% had an annual income >$100,000, and 95% had some college or above. On average, women from HR settings scored 3.0 points higher on the Fertility Knowledge Assessment than their LR counterparts (p < 0.001). Upon multivariate analysis, education level remained the sole independent factor associated with fertility knowledge assessment score (p < 0.001). Stratifying by resource level revealed that income was highly associated with fertility knowledge (p < 0.01) among high resource individuals even when adjusting for education level.A total of 143 women were included in our analysis. In the county hospital/low resource clinic (LR, Women from low resource, largely immigrant communities, seeking fertility care have greater disparities in fertility knowledge and lower health literacy compared to women from high resource clinical settings. Further studies are needed to understand these barriers and to develop targeted inventions to lower disparities and improve care for these vulnerable populations. Low health literacy has been extensively correlated with poor health outcomes and reduced use of health resources across a range of conditions including cancer, diabetes, and asthma . As an eInfertility is a disease of reproductive health that exerts a profound impact on an estimated 80 million people worldwide. The World Health Organization ranks infertility as the 5th highest generator of disability among the global population of all people under 60 years of age –16.In a large sample of infertile women of various ages and educational backgrounds across Canada, Daniluk et al. found that the majority of women lacked significant knowledge related to age-related fertility and ART options, treatment limitations, and their associated costs . VassardA systematic review of the available evidence on fertility awareness worldwide, including 71 studies published between 1994 and 2017 among 26 countries, found that overall fertility awareness is low to moderate among reproductive-aged women and that higher education correlated with greater fertility awareness . The majNo study to our knowledge has quantitatively and comparatively examined fertility knowledge among infertile women within the United States across socio-economic background strata, and specifically within largely immigrant communities –19.We sought to test the hypothesis that socio-economic and demographic factors  are associated with respondents’ fertility knowledge level. This, in turn, may serve as an indirect pathway to measure barriers to access to fertility care.We designed a multi-institutional cross-sectional study, using paired clinical sites at the University of California, San Francisco School of Medicine (UCSF), San Francisco, CA and Baylor College of Medicine (BCM), Houston, TX. All study procedures were reviewed and approved by the Institutional Review Board at each university.The low-resource (LR) locations comprised of infertility clinics operating within the infrastructure of ambulatory women’s health clinics at university-affiliated urban public teaching community hospitals: San Francisco General Hospital and Ben Taub Hospital in Houston. These clinics operate weekly and provide free and low cost medical care to a largely immigrant, culturally diverse, and indigent population. Medical students and OB/GYN residents are supervised by a board-certified Reproductive Endocrinology and Infertility (REI) specialist or a senior REI fellow.The high-resource (HR), fee-for-service sites included the UCSF Center for Reproductive Health (CHR) and the Texas Children’s Family Fertility Center at Texas Children’s Hospital Pavilion for Women. These comprehensive tertiary fertility care centers are largely self-pay and offer services including intrauterine insemination (IUI), in-vitro fertilization (IVF), fertility preservation programs, and ovum donation programs.Infertile women desiring conception presenting for initial care at study sites were eligible to participate. Infertility was defined as the inability to conceive after 12 months of regular unprotected intercourse for women < 35 years old or 6 months for women ≥35 years old. Women presenting for infertility consultation were potential candidates for enrollment. Pregnant or women actively undergoing infertility evaluation and treatment were ineligible. Patients who had received some form of infertility evaluation or treatment greater than 12 months ago were eligible.Women were recruited from February 2013 to February 2014. At all study sites, bilingual (Spanish-English) research coordinators identified new patients from the complete list of patients being seen at each clinic. Telephone-based certified medical interpreters were utilized to recruit, consent, and complete study forms for patients speaking other languages. Coordinators identified new patients, presented the study, obtained written informed consent, and ensured study forms were completed. Study forms included a Fertility Knowledge Assessment and Social-Medical Demographics form  included questions on basic infertility knowledge (anatomy and etiology of disease), prevalence, treatment, and questions pertaining to female and male infertility. Each response was recorded as a binary variable; correct answers were given a 1 point value and incorrect responses were given a 0 point value. The composite knowledge quiz score ranged 0–11 and percentage of questions correct was calculated for each subject. Internal consistency was measured with Cronbach’s alpha .. 2010 US median household income census data was utilized to categorize subjects as follows: less than $25,000, $25,000–$50,000, $50,000–$75,000, $75,000–$100,000 and greater than $100,000. Country of origin was re-grouped to region of origin and included the United States, Mexico/South & Central America, Europe, Asia, and Other. Primary languages reported included English only, Spanish only, Mandarin, Cantonese, Vietnamese, Arabic, Tagalog, Korean, Bilingual, and Other. Based on response data, primary language was re-categorized to English, Spanish, and Other. Ethnicity was initially reported as White/European American/ Caucasian, African-American/Black, Latino/Hispanic, Asian, Pacific Islander, Native American, American Indian, Alaskan Native or Indigenous, Mixed/Multi-ethnic, and Do not know. Ethnicity was re-grouped to White, Hispanic, Black, Asian, or Other upon collection of responses. Partner status was dichotomized to Married or Unmarried  and occupation to Employed and Unemployed.Demographic predictors including age, education, annual household income, region of origin, primary language, ethnicity, marital status, and occupation were determined through questionnaires. Medical demographics including parity, prior fertility treatment, number of ectopic pregnancies, therapeutic abortions, spontaneous abortions, and total prior pregnancies were additionally assessed. Education was dichotomized to Grade School/ High School, General Education Development diploma (GED) or Some College/Abovep > 0.2. Constant variance was observed, residuals were normally distributed, and removal of outliers did not significantly affect regression coefficients or standard errors. Collinearity was investigated by assessing the variance inflation factor (VIF) for the model. All included variables had a VIF < 10 (mean 3.68). An Oaxaca Decomposition analysis was performed on the model but did not shed additional light on these findings beyond results presented with stratifying by resource level. Coefficients, 95% confidence intervals (CI), and p values were utilized to describe the results of regression models. Statistical significance was set at a level of p < 0.2. Stata 10  was used for all analysis.Descriptive statistics were utilized to describe the study population. Missing variables were not replaced. Bivariate comparisons were made between variables and quiz scores with Pearson’s chi-square analysis for categorical variables and linear regression for continuous variables. Linear regression was performed to investigate the association between baseline fertility knowledge and each outcome measure. Multivariate linear regression was used to examine relationships between baseline fertility knowledge and resource level while controlling for other predictor variables. Initial covariates were chosen a priori based on clinical relevance, literature review, and expert advice. Some variables were removed from the model to be parsimonious for A total of 143 women were included in the analysis: 49% participants were seen at LR clinics and 51% were seen at HR clinics. The mean age of women across all sites was 33.9 ± 5.7 years. Across all sites, the majority were partnered , born in the United States (54%), employed (77%), attended some college or above (71%), and had an annual household income of less than $25,000 (37%)  women were from LR sites while 73 51%) were from HR sites  (64%), partnered (78%), White (41%), completed some college or above (95%), employed (96%), spoke English (68%), had an annual household income greater than $100,000 (60%), and had not received any form of fertility treatment (76%). In this group, 57% reported prior pregnancy. The mean fertility knowledge assessment score in this group was 8.04 ± 2.3 points out of 11.A one-way ANOVA demonstrated a statistically significant difference in fertility knowledge scores between patients from LR and HR settings. Further analysis Table  demonstrp > 0.2 for all levels) and educational level (p = 0.16) was not related to knowledge score. In the HR setting, household income was strongly associated with quiz score results  after holding all other variables constant including education.Upon multivariate analysis Table , the relInvoluntary childlessness often becomes a central and preoccupying issue in infertile patients’ lives; this can be compounded by lack of access to basic care and fertility treatment. Direct barriers to fertility care include cost of treatment, marital status, employment, and insurance type –22. ThesOur study is the first comparative evaluation of health literacy and influencing factors among women across different sociocultural and income backgrounds presenting for fertility care in metropolitan centers in the United States. The principal strength of our study is our ability to access socio-culturally, largely immigrant women seeking infertility care, a population that is challenging to access as there are relatively few public health centers that provide fertility services by specialized providers to this population. We recruited subjects in-person through infertility clinics based at public hospitals and thus were able to access populations that may have limited access to Internet and/or social media sites.Pioneering studies by Becker and Nachtigall conducted at San Francisco General Hospital (SFGH) showed that difficulty finding a physician, legal immigration status, and language limitations provide additional barriers in accessing fertility care , 26. MorThe data from prior studies of populations with higher socioeconomic status and health literacy identified several factors strongly associated with fertility knowledge including resource level, region of origin, ethnicity, primary language, education level, and parity , 31. TheIdentification and recognition of factors associated with lower health literacy are critical to develop targeted interventional strategies to improve fertility knowledge. For example, clinicians could develop additional Spanish language written or video resources to educate their patients about reproductive options as part of their initial visit, thereby focusing on primary language as a barrier to reproductive knowledge. Recently, Garcia et al. found that regardless of education level, tailored oral education significantly increased fertility knowledge in a group of oocyte donors . MoreoveThere are several limitations of this study. In the absence of a well-validated fertility knowledge instrument available at the inception of our study, we developed a tool to assess fertility knowledge. Of note, in 2017 Kudesia et al. developed the Fertility and Infertility Treatment Knowledge Score (FIT-KS), which is a short survey, utilized as a quick assessment of fertility knowledge . AlthougFurthermore, given the option for UCSF HR participants to mail or email completed study forms, this could have contributed to a selection bias to favor those who have access to computers; however, since in HR clinical settings, e-communication is one of the main routes of nurse-patient communication, we suggest that this bias was unlikely to contribute to skewed results. Future studies should examine sources of participants’ fertility knowledge and how prior pregnancy and infertility treatment impacts fertility health literacy. Fertility knowledge is essential for patients to access care and appropriate therapeutics. In no other population setting is this need likely greater than in the growing and diverse immigrant populations of the United States. Designing and implementing a fertility education program and tools for patients from these communities and assessing the impact of such educational efforts is an important next step.Women from low-resource, largely immigrant communities, seeking fertility care have greater disparities in fertility knowledge and lower health literacy. Further studies are needed to understand these barriers and to develop targeted inventions to lower disparities and improve care for these vulnerable populations.Additional file 1.


Hibiscus sabdariffa were spray-dried using maltodextrin (MD) and gum arabic (GA) as carrier agents. A Taguchi L8 experimental design with seven variables was implemented. Physicochemical properties in the encapsulates were evaluated by Ultraviolet-Visible  X-ray Diffraction (XRD), spectroscopy and gravimetric techniques. Treatments with aqueous extracts showed the highest concentration of total soluble polyphenols (TSP) 32.12–21.23 mg equivalent gallic acid (EAG)/g dry weight (DW), and antioxidant capacity (AOX) in the 2,2-azinobis-3-ethylbenzotiazoline-6-sulfonic acid (ABTS) assay. The best treatment for TSP and AOX was T4: 2.5% Hibiscusw/w, aqueous extract, decoction, extract-to-carrier ratio 1:1 (w/w), proportion to carriers (MD:GA) 80:20 (w/w), 10,000 rpm, 150 °C. The Taguchi L8 design is a tool that allows the use of multiple variables with a low number of treatments that indicate the drying conditions that give the best parameters, focusing mainly on TSP and AOX, also, it is a good alternative for the preservation and stability of the phenolic compoudns in Hibiscus.Aqueous and ethanolic extracts of  Hibiscus sabdariffa L. is a shrub that belongs to the Malvaceae family, is a good source of dietary fiber (DF) rich in non-starch polysaccharides, and phenolic compounds (PC) such as flavonoids , anthocyanins , and phenolic acids  . Fo. FoHibisnsidered  46]..HibiscusThe AOX in the aqueous extracts showed two-fold higher values than ethanolic extracts with a correlation with the results of TSP see . When thHibiscus, water, decoction, extract: carrier 1:1, carrier ratio 80:20, 10,000 rpm, 150 °C) 216.28 ± 5.24 mmol ET/g DW , the treatment with the highest value corresponds to T4  showed highest AOX with 271.95 ± 6.63 mmol ET/g DW value; according to the results (see Hibiscus (p.e. catechin and quercetin) which, given the position of the hydroxyl groups, are responsible for the high chelating activity and the radical quenching power . Pr. PrHibisults see , as wellng power .Hibiscus sabdariffa extracts encapsulates. The T4 treatment showed the highest concentration of TSP with AOX. A good EY and EE (%) and storage stability showed the interactions between the carrier agents and the PC present in the calyces. These new interactions were confirmed by the results of UV-Vis spectroscopy and X-ray diffraction. The use of an experimental Taguchi design is a useful methodology that uses a low number of experiments to evaluate the interactions between parameters; this indicates that the maximum TSP content and AOX in the Hibiscus sabdariffa extracts encapsulates were mainly related to the solvent of extraction, extract: carrier agent ratio, and the carrier agents ratio (MD + GA).The results the Taguchi L8 design allowed us to obtain the best values in the studied variables to ensure the quality of the


Climacteric fruits are characterized by a dramatic increase in autocatalytic ethylene production that is accompanied by a spike in respiration at the onset of ripening. The change in the mode of ethylene production from autoinhibitory to autostimulatory is known as the System 1 (S1) to System 2 (S2) transition. Existing physiological models explain the basic and overarching genetic, hormonal, and transcriptional regulatory mechanisms governing the S1 to S2 transition of climacteric fruit. However, the links between ethylene and respiration, the two main factors that characterize the respiratory climacteric, have not been examined in detail at the molecular level. Results of recent studies indicate that the alternative oxidase (AOX) respiratory pathway may play an essential role in mediating cross-talk between ethylene response, carbon metabolism, ATP production, and ROS signaling during climacteric ripening. New genomic, metabolic, and epigenetic information sheds light on the interconnectedness of ripening metabolic pathways, necessitating an expansion of the current, ethylene-centric physiological models. Understanding points at which ripening responses can be manipulated may reveal key, species- and cultivar-specific targets for regulation of ripening, enabling superior strategies for reducing postharvest wastage. Ripening of fruit involves a symphony of transcriptionally and hormonally controlled processes that result in the accumulation of sugars, reduction in acidity, development of aroma, and nutritional profiles . The ripPrunus salicina Lindl.), where a nonclimacteric cultivar and an ethylene-responsive, suppressed-climacteric cultivar were both found to be derived from independent bud sport mutations in a single climacteric plum variety  to inhibit ripening result in developmental patterns that do not follow the classical model of ethylene response and signaling . MechaniThe alternative oxidase (AOX) respiratory pathway has recently garnered interest as a potential target for ripening manipulation ,b. This Solanum lycopersicum) (Early knowledge of the role of ethylene in the ripening process made components of ethylene biosynthesis and transduction some of the first targets for ripening control in model systems such as tomato (ersicum) . Resultiersicum) , ethylenersicum) . Followiersicum) .Pyrus communis), for example, must undergo between 15 and 90 days (depending on the cultivar) of conditioning at 0–10°C to activate the S2 autocatalytic ethylene biosynthesis , and pathways that may be triggered by changes in epigenetic signatures . New advThe hallmark aspect of climacteric ripening is the respiratory rise that occurs prior to the S1–S2 ethylene transition, in which an initially gradual increase in carbon dioxide evolution is followed by a heightened burst in respiratory activity during the ripening climacteric . The resClimacteric respiration represents the combined activity of several mitochondrial pathways that differentially direct electron transport, leading to several possible energetic fates. The first is the cytochrome c (CYTc) pathway. CYTc operates as a result of a proton gradient generated in the mitochondrial intermembrane space and concludes in the production of cellular energy currency via ATP synthase. In plants, CYTc activity also facilitates cellular detoxification via the synthesis of antioxidant compounds, namely, ascorbic acid . The finAdditionally, expression of AOX can also be modulated via retrograde mitochondrial signaling in response to reactive oxygen species (ROS) or metabolic disruption . BecauseThe transition from S1 to S2 ethylene biosynthesis involves numerous metabolic changes, which may occur simultaneously or in series, that require a great deal of regulation and feedback mechanisms to ensure that ripening occurs properly. There is increasing evidence, including respiratory partitioning studies, supporting a role of AOX in the modulation of respiration at various stages around the time of climacteric via induction of S2 ethylene, which thereby influences the development of ripening-associated phenotypes downstream .In several fruits, AOX expression and/or activity has been characterized at the preclimacteric and climacteric stages of fruit development and ripening . Banana,Furthermore, in some fruits, both climacteric and nonclimacteric, AOX activation can be achieved via cold temperature or chemical stimulation . In zuccin vivo via interaction with the two cysteine residues that gate the protein  as a chemical activator of both AOX and ripening . GLA has protein . Further protein . This, i protein ,b. In ad protein . The res2S), though a known phytotoxin, in minuscule doses can enhance alternative pathway respiration and inhibit ROS production  and methyl jasmonate (MeJA) results in increased expression of AOX, correlating with a reduction in chilling injury in sweet pepper. JA is a known activator of MYC, a transcription factor that enhances the function of ethylene signaling factors, particularly within the EIN/EIL family, thus leading to enhanced ethylene responses . It has pression  via a sily genes . In addily genes .Together, these findings reveal interesting insights into chemical and hormonal events that operate in an ethylene-independent space during climacteric ripening, as well as how ripening can be better regulated as a result of this information. Novel discoveries presented in recent studies, which complement and expand upon the foundational knowledge of the role of AOX in ripening, have laid the framework for several exciting hypotheses. First, knowledge of how AOX expression can be induced may provide an avenue for development and testing of ripening strategies in fruits whose respiratory profiles are affected by temperature. Furthermore, knowledge of how temperature preconditioning may mitigate chilling injury via AOX stimulation could allow for improved management practices of papaya, avocado, banana, mango, zucchini, and other temperature-sensitive fruits during storage .It is clear, based on the simultaneity and interdependency of responses, that respiration and ethylene are physiologically correlated during climacteric ripening—climacteric rise in respiration is accompanied by a spike in autocatalytic ethylene production, and blocking ethylene perception prevents respiration from increasing further . While eAOX1a  plants  plants . Reducedpiration . Specifipiration . During piration .Arabidopsis, ethylene-induced signaling by hydrogen peroxide (H2O2), a form of ROS, was shown to activate AOX in response to cold temperatures  facilitate a cascade of downstream signaling activity . In fruirin mutation has become one of the most iconic ripening-associated mutations in studies of climacteric fruit. Rin mutant tomatoes fail to mature beyond the green-ripe stage and do not exhibit the characteristic ripening climacteric of wild-type fruit  and α-galacturonase , an enzyme-associated with cell wall breakdown and fruit softening  transcription factor is a squamosal-promoter binding-like protein, which appears to be necessary for RIN to bind to promoters . With a TAG1, TAGL1, and MADS-box transcription factors FUL1 and FUL2 can form complexes with RIN . MutatioAOX is silenced. When AOX was inhibited via RNA interference, the expression of these transcriptional regulators decreased  and respiration (CYTc and AOX) have been extensively characterized at the physiological level. Studies conducted within the last several years provide new insights with respect to the connection between these two critical pathways at the molecular and metabolic levels. AOX activity begins to increase during the preclimacteric phase, prior to the S1–S2 ethylene transition, particularly in the context of cold temperature or other external stimuli. This activity may be accompanied by the accumulation of ROS as the CYTc pathway capacity reaches a maximum. At the onset of S2 ethylene stimulation, both ethylene response and alternative pathway respiration appear to be interdependent. Ethylene biosynthesis requires ATP, generated via respiration, and activity of respiratory pathways modulated by ERFs in the nucleus; this is evidenced by the inhibition of ethylene production and respiration by the ethylene receptor antagonist 1-MCP. The commencement of both ethylene and respiration-associated processes is likely due to upstream transcriptional and epigenetic regulators, as well as the signaling activity of ROS generated during increased respiration. Mutants of master transcriptional regulators in tomato have provided a means for the study of their effects on both ethylene production and respiration during ripening. Furthermore, studies have investigated the effects of temperature and chemical manipulation of AOX with the aim to understand ways in which the timing of ripening can be controlled. AOX and related processes represent a new frontier in regulating postharvest ripening and, therefore, better management to reduce fruit wastage and development of postharvest management strategies in different types of fruits.The ever-growing fields of genomics, transcriptomics, metabolomics, and epigenomics offer high-throughput strategies for extrapolation of important biological function and expression information from large datasets generated in recent ripening experiments . The relSH and AD conceptualized the review. Both the 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 construed as a potential conflict of interest.


Drosophila dopamine/ecdysteroid receptor (DopEcR) binds the molting hormone 20-hydroxyecdysone (20E) in insects. This review summarizes the research progress on GPCRs as animal steroid hormone cell membrane receptors, including the nuclear and cell membrane receptors of steroid hormones in mammals and insects, the 20E signaling cascade via GPCRs, termination of 20E signaling, and the relationship between genomic action and the nongenomic action of 20E. Studies indicate that 20E induces a signal via GPCRs to regulate rapid cellular responses, including rapid Ca2+ release from the endoplasmic reticulum and influx from the extracellular medium, as well as rapid protein phosphorylation and subcellular translocation. 20E via the GPCR/Ca2+/PKC/signaling axis and the GPCR/cAMP/PKA-signaling axis regulates gene transcription by adjusting transcription complex formation and DNA binding activity. GPCRs can bind 20E in the cell membrane and after being isolated, suggesting GPCRs as cell membrane receptors of 20E. This review deepens our understanding of GPCRs as steroid hormone cell membrane receptors and the GPCR-mediated signaling pathway of 20E (20E-GPCR pathway), which will promote further study of steroid hormone signaling via GPCRs, and presents GPCRs as targets to explore new pharmaceutical materials to treat steroid hormone-related diseases or control pest insects.G protein-coupled receptors (GPCRs) are cell membrane receptors for various ligands. Recent studies have suggested that GPCRs transmit animal steroid hormone signals. Certain GPCRs have been shown to bind steroid hormones, for example, G protein-coupled estrogen receptor 1 (GPER1) binds estrogen in humans, and Video abstract Caenorhabditis elegans genome  ponasterone A ([3H] Pon A), suggesting the existence of an unknown membrane receptor  Pon A. In addition, 20E triggers intracellular rapid increases in Ca2+ and cyclic adenosine monophosphate (cAMP), and increases ERK phosphorylation in the DmDopEcR overexpressing Sf9 cells  Pon A entry into cells relies on ErGPCR-2 localization in the cell membrane -labeled Pon A [H. armigera can bind 20E in the cell membrane or as an isolated protein [GPR30 is suggested as the cell membrane receptor for the steroid hormone estrogen by detection of the binding of fluorescence-labeled estrogen in Cos7 cells . Subtype protein . To prov protein . GPCRs u protein . For exa protein . The cho protein . The cho protein . Therefo protein . A suita protein .GPCRs are considered to function not as a single molecule, but in the form of homo- or hetero-oligomers . Some GPH. armigera [20E regulates gene transcription via its nuclear receptor EcR in armigera . Via theCyanotis vaga, can be used to disrupt the development of insect pests [Drosophila [H. armigera, high concentrations of 20E induce Ca2+ influx to switch autophagy to apoptosis in the midgut [20E, which is produced in both insects and plants, such as ct pests . Insect ct pests . 20E canct pests . 20E chaosophila . In H. ae midgut , 60. TheApproximately 30–35% of marketed drug targets are GPCRs –92. The Studies of the insect steroid hormone 20E show that GPCRs function as cell membrane receptors of steroid hormones. 20E binds to GPCRs to trigger the PKA and PKC pathways via Gαs and Gαq, respectively. 20E induces calcium influx by SOCE via Gαq, PLC, STIM, and Orai-signaling. The increased level of calcium acts as a secondary messenger to induce protein phosphorylation and acetylation to regulate the nuclear receptor transcriptional complex formation for gene transcription and apoptosis. 20E, via the PKA pathway, enhances the PKC pathway. 20E signaling is terminated by desensitization of GPCRs via a negative feedback mechanism. A GPCR-mediated signaling pathway of 20E (20E-GPCR pathway) has been elucidated, which opens a door to further study of steroid hormone signaling via GPCRs Fig. . There a


Objective: Mesenchymal subtype of glioblastoma (mesGBM) is a refractory disease condition characterized by therapeutic failure and tumor recurrence. Hyperactive transforming growth factor-β (TGF-β) signaling could be a signature event in mesGBM, which leads to dysregulation of downstream targets and contribute to malignant transformation. In this study we aimed to investigate the hyperactive TGFβ signaling-mediated pathogenesis and possible downstream targets for the development of novel therapeutic interventions for mesGBM.Methods: GBM-BioDP is an online resource for accessing and displaying interactive views of the TCGA GBM data set. Transcriptomic sequencing followed by bioinformatic analysis was performed to identify dysregulated microRNAs. Target prediction by MR-microT and dual luciferase reporter assay were utilized to confirm the predicted target of novel_miR56. CCK-8 assays was used to assesse cell viability. The miRNA manipulation was proceeded by cell transfection and lentivirus delivery. A plasmid expressing GFP-LC3 was introduced to visualize the formation of autophagosomes. Orthotopic GBM model was constructed for in vivo study.Results: TGFβ1 and TGFβ receptor type II (TβRII) were exclusively upregulated in mesGBM (P < 0.01). Dysregulated miRNAs were identified after LY2109761 (a TβRI/II inhibitor) treatment in a mesGBM-derived cell line, and novel_miR56 was selected as a promising candidate for further functional verification. Novel_miR56 was found to potentially bind to PRAS40 via seed region complementarity in the 3′ untranslated region, and we also confirmed that PRAS40 is a direct target of novel_miR56 in glioma cells. In vitro, over expression of novel_miR56 in tumor cells significantly promoted proliferation and inhibited autophagy (P < 0.05). The expression levels of P62/SQSTM was significantly increased accompanied by the decrease of BECN1 and LC3B-II/I, which indicated that autophagic activity was reduced after novel_miR56 treatment. In addition, over expression of novel_miR56 also promoted tumor growth and inhibited autophagy in vivo, which is associated with worse prognosis (P < 0.05).Conclusions: In summary, we provide novel insight into TGFβ signaling-mediated pathogenesis in mesGBM and TGFβ signaling-induced novel_miR56 may be a novel target for mesGBM management. This characteristic provides numerous potential therapeutic targets and insights into the molecular pathogenesis of GBM phenotypes. The mesenchymal transcriptomic signature can drive GBM cell aggressiveness and chemoradiotherapy resistance, leading to treatment failure and tumor recurrence, which consequently help to develop a refractory disease condition3. The overall survival of GBM patients has remained dismal for decades, and no additional successful treatment options are available to those with the refractory mesenchymal phenotype to improve the clinical outcomes4. Therefore, further interpretation of the mesenchymal profile of GBM and identification of the underlying mechanism responsible for malignant transition are urgently needed.Glioblastoma (GBM) has been classified by The Cancer Genome Atlas (TCGA) research network into four major transcriptomic subtypes; of these, the mesenchymal subtype expresses signature genes functionally correlated with the process of mesenchymal transition6. The TGFβ cytokine superfamily and receptor-activated mothers against decapentaplegic homologue (SMAD) cascades are the core components of TGFβ signaling, which is universally dysregulated in various types of malignancies, including GBM. For example, TGFβ1, an upregulated mesenchymal signature gene, mediates mesenchymal transition and poor prognosis in GBM3. However, phase II clinical trials of galunisertib7, a TGFβ receptor type I (TβRI) inhibitor, and trabedersen8, a TGFβ2 inhibitor, failed to improve the overall survival of GBM patients, which suggests the need for further understanding of the pathological implications of TGFβ signaling in GBM. The SMAD transcription factor-mediated gene profile has been unveiled after years of intense exploration, but most of the efforts focused on protein-coding effectors. With the increasing emphasis on the biological implication of non-coding RNAs (ncRNAs), it is essential to profile novel non-protein-coding effectors to supplement our knowledge of TGFβ signaling-mediated pathogenesis. The discovery of TGFβ signaling-regulated microRNAs (miRNAs), the best-understood class of ncRNAs, has supported this concept9. MiRNAs are 15-27 nt-long single-stranded ncRNAs that mediate mRNA degradation or inhibit translation initiation by seed (guide positions 2-8)-mediated complementation of the 3′ untranslated region (3′-UTR)10. The expression of miR-182 is induced by TGFβ-SMAD binding to the MIR182 promoter, and miR-182 upregulation thus sustains NFκB activation in glioma subsets11. In addition, SMAD proteins control DROSHA-mediated miR-21 maturation at the post-transcriptional level12. It is thus suggested that dysregulated TGFβ signaling may cause the deregulation of a subset of miRNAs as well, and some of those previously unnoticed functional miRNAs within the subset could be responsible for the genesis of certain malignant phenotypes.Transforming growth factor β (TGFβ) signaling is one of the most important and well-studied signaling pathways in metazoan biology. The function of this signaling pathway is highly dependent on different cell contexts. In healthy cells and precancerous cells, TGFβ signaling plays critical roles in maintaining cellular hemostasis and reversing malignant transition, while in cancer cells, this pathway sustains oncogenic transformation, such as epithelial-to-mesenchymal transition (EMT), migration, immune surveillance evasion and chemoresistance13. Mechanistic target of rapamycin complex 1 (mTORC1) and the BECN1-phosphatidylinositol 3 kinase catalytic subunit type 3 (PIK3C3) complex are two main signaling checkpoints that turn autophagy off and on, respectively, and dysregulated pathways influencing these two checkpoints have been linked with various disease conditions, including GBM14. Emerging studies agree that deregulated autophagic activities may result in dichotomous consequences of self-consumption or self-adaption16. However, autophagy-related determination of cell fate is still a factor. Autophagic death is the major GBM cell death pathway because of its ability to resist apoptotic stimuli, but the underlying mechanism remains unclear17. Knowledge of the role of autophagy in GBM pathogenesis has just begun to solidify, and a better understanding of protective and lethal autophagic activities will contribute to mapping the context-dependent functional network, which underlies the molecular basis for selectively targeting autophagy in GBM.Autophagy  is an evolutionarily conserved process by which eukaryotic cells maintain intracellular homeostasis and adapt themselves to cellular stress to survive. Autophagy activities, such as sequestration of endogenous or exogenous cytoplasmic waste and subsequent lysosomal degradation, are tightly controlled by a set of signaling events1, were exclusively upregulated in mesenchymal subtype of GBM (mesGBM). In a cell line derived from a patient-derived tumor xenograft (PDX) of mesenchymal GBM (PDXM), a set of TGFβ signaling-associated miRNAs was identified after LY2109761 (a TβRI/II inhibitor)18 treatment by transcriptomic sequencing. Interestingly, some of these miRNAs were previously unreported miRNAs, which cannot be found in the miRbase database and were temporarily named “novel_miR”. Among those differentially expressed miRNAs, novel_miR56 was chosen as a candidate for functional and mechanistic validation for TGFβ signaling-mediated pathogenesis. Proline-rich akt substrate of 40 kDa (PRAS40), a suppressor of mTORC1 activity20, was experimentally validated as a direct target of novel_miR56, which incorporated TGFβ signaling-induced miRNA into autophagic regulation signaling. Novel_miR56 upregulation impaired autophagic activity and promoted the survival capability of GBM cells. Collectively, we demonstrate a previously unreported mechanism of TGFβ signaling-mediated autophagic regulation, which possibly contributes to the formation of GBM mesenchymal subtypes.In the present study, we noted that in TCGA GBM data, TGFβ1 and TβRII, two of the 840 signature genes for GBM subtyping2 humidified chamber.Human GBM , breast cancer (M231) and renal cancer (ACHN) cell lines were purchased from the China Academia Sinica Cell Repository. Two newly established GBM cell lines, U87-EGFRvIII, a U87 cell line stably expressing EGFRvIII, and N9/N33, were kind gifts from Prof. Huan Ren  and Prof. Tao Jiang , respectively. All cell lines were maintained in appropriate culture media (DMEM or RPMI-1640) supplemented with 10% fetal bovine serum (Gibco), as well as with 0.4 g/mL G418 (EMD Bioscience) for U87-EGFRvIII cells, at 37 °C in a 5% CO21. The following gene-specific primers were used: novel_miR26 ; novel_miR56 ; novel_miR93 ; novel_miR97 ; novel_miR119 ; PRAS40 ; GAPDH ; and RNU6 .Total RNA was extracted using TRIzol (Invitrogen). qRT-PCR assays were performed to measure the expression levels of novel_miR26/56/93/97/119 and PRAS40 according to the manufacturer’s instructions for SYBR Green PCR Master Mix (Promega). Real-time quantification to measure novel miRNAs was performed with a Hairpin-it™ miRNAs qPCR Quantitation Kit (GenePharma). GAPDH and RNU6 were employed for the normalization and quantification of PRAS40 and miRNA, respectively. Real-time PCR was performed using the DNA Engine Opticon 2 Two-Color Real-Time PCR detection system (Bio-Rad Laboratories) in the presence of SYBR Green. Real-time PCR data were analyzed by the comparative Ct methodTransfection was performed using Opti-MEM medium and Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. Novel_miR56 mimics (5′-GGUAGUGUGGCCGAGCGGUCU-3′) and inhibitors (5′-AGACCGCUCGGCCACACUACC-3′) were synthesized and purified by Invitrogen . A PRAS40 3′-UTR reporter plasmid was constructed by inserting the two predicted novel_miR56 binding sites (5′-GGGCCGCGUCCGCC CCGUCCCACACUACG-3′ and 5′-GGCCUUCAAUUU ACGUUCUUUACACUACG-3′) into the 3′-UTR of a promoter-driven luciferase gene. The GFP-LC3 plasmid was used to measure the number of autophagosomes, which is indicated by the formation of GFP-positive granules.Lentiviral vectors expressing nonsense control, novel_miR56 (5′-GGUAGUGUGGCCGAGCGGUCU-3′) or luciferase were all generated by GenePharma. Cell infections were carried out according to GenePharma’s recommendations.22. Antibodies for PRAS40, BECN1, LC3B, Ki-67, SQSTM1, mTOR, LAMP1, GAPDH and β-tubulin were all purchased from Cell Signaling Technology (CST), and the expression of GAPDH and β-tubulin were used as internal controls.Western blot, immunohistochemistry (IHC) and immunofluorescence (IF) assays were performed as previously describedCell viability was assessed using the Cell-Counting Kit 8  assay according to the manufacturer’s instructions. In brief, cells were seeded at 5,000 cells per well in 96-well plates and incubated for the appropriate length of time in an incubator after treatment. CCK8 solution was added to each well of the plate and incubated for 4 h, and a microplate reader was used to measure the absorbance at 450 nm.1. The mesGBM specimens were then implanted subcutaneously into the flanks of female SCID mice, while a portion of each implanted specimen was cryopreserved for long-term storage. The xenografts were passaged or collected when the tumor volume exceeded 1 cm23. For the establishment of the orthotopic mesGBM model, the harvested xenografts were digested into single-cell suspensions. The orthotopic tumor models were constructed as previously described24. Paraffin-embedded sections (8 mm) were stained with hematoxylin and eosin (H&E). The animal studies were performed according to internationally recognized guidelines for animal welfare and experimental regulations. Survival curves were plotted according to the Kaplan-Meier method.Creation of the mesGBM PDX mouse model started with the harvest of pathologically confirmed human glioblastoma specimens from the operating room. The pathological tissue specimens and animals involved in the study were subject to approval by the Affiliated Hospital of Hebei University and the Institute of Animal Care and Use Committee at Tianjin Medical University . Subsequently, RNA extraction was performed, and transcriptomic sequencing was used to classify the clinical GBM samples into four transcriptomic subtypes  based on the 840-gene transcriptomic signaturet-test. A P-value < 0.05 was considered statistically significant.The data are presented as the means ± S.E.M. of three independent experiments or the means ± S.D. of samples assayed in triplicate. Statistical comparisons were made by Student’s P < 0.01), while TGFβ3 was downregulated in proneural and neural subtype . These results suggested that hyperactive TGFβ signaling could be a signature event in mesGBM.TGFβ signaling plays an important role in mesenchymal transition in cancer, and TGFβ1/3 and TβRII are the three TGFβ signaling genes within the list of 840 transcriptomic signature genes that are used to classify GBM subtypes. Using the Glioblastoma Bio Discovery Portal (GBM-BioDP), an online resource for accessing and displaying interactive views of the TCGA GBM data set, we visualized the expression levels of TGFβ1/3 and TβRII in the four GBM subtypes. TGFβ1 and TβRII were exclusively upregulated in the mesenchymal subtype . Deactivation of TGFβ signaling resulted in the downregulation of its downstream miRNA effectors, and interestingly, the top 20 most significantly downregulated miRNAs contained 17 unreported miRNAs, indicating previously ignored functional implications of these potential miRNAs and the significant value of their functional evaluation (data not shown). In addition, the expression of five potential novel miRNAs, novel_miR26/56/93/97/119, was detected in all tested cell lines , indicating that these novel miRNAs were not a cell type-specific phenomenon . In particular, a relatively higher abundance of novel_miR56/119 was found in these cell lines. Recombinant TGFβ1 protein and LY2109761 treatment, which modulated TGFβ signaling activity, could alter the expression of novel_miR26/56/93/97/119 in the N9, PDXM, LN229 and U87 cell lines . However, the changes in novel miRNA expression in response to these modulators were not consistent among different cell lines and miRNAs, and only novel_miR56 was consistently and positively correlated with TGFβ signaling activity. Therefore, novel_miR56 may be the most promising candidate for further functional validation of TGFβ signaling-induced novel miRNAs.TGFβ signaling dysregulation may lead to dysregulated downstream miRNAs. To identify TGFβ signaling-regulated miRNAs in mesGBM, PDXM cells were treated with DMSO or LY2109761 before transcriptomic sequencing. The miRDeep*Figure 2D). Further comparison of the RNA sequence of mature novel_miR56 with miRBase-documented miRNAs revealed that novel_miR56 shared the same seed region (guide position 2-8) with hsa-miR-142-3p and aga-miR-10362-3p, indicating that novel_miR56 might belong to the hsa-miR-142 family. An identical sequence at guide positions 2-13 and 15-16 in novel_miR56 and aga-miR-10362-3p supported this structural feasibility . Therefore, novel_miR56 could be a previously unreported miRNA that serves as a functional effector of TGFβ signaling in mesGBM.Prediction of the secondary structure of the novel_miR56 precursor (pre-novel_miR56) by RNAfold demonstrated that pre-novel_miR56 harbored the typical stem-loop structure of a miRNA precursor . To exogenously modulate the expression of novel_miR56, we employed artificial novel_miR56 mimics and inhibitors, which were experimentally validated to increase or decrease novel_miR56 in the N9 and PDXM cell lines . The novel_miR56 mimics reduced the luciferase activity of the wild-type PRAS40 3′-UTR-expressing reporter in a concentration-dependent manner in the N9 and PDXM cell lines, while no significant change in the luciferase activity was observed in cells transfected with the reporter containing the mutated novel-miR56 binding sites; these results demonstrated the binding between the PRAS40 3′UTR and novel_miR56 . The alterations in PRAS40 mRNA and protein levels in response to novel_miR56 intervention further supported that PRAS40 is a direct target of novel_miR56 . S6K (S6 kinase) and mTOR expression was inversely correlated with PRAS40 after novel_miR56 intervention . Additionally, the change in the immunofluorescent signal intensity of PRAS40 in PDXM cells after novel_miR56 intervention shared a similar trend with its counterpart according to western blot results . Collectively, we proved that novel_miR56 can bind to the PRAS40 3′-UTR as predicted and reduce the protein level, which suggests that PRAS40 may act as a functional target for TGFβ signaling-mediated autophagic regulation.Direct targets of novel_miR56 were predicted based on sequence by an online prediction method, MR-microT. In searching for potential novel_miR56 targets involved in autophagic regulation, we focused on the gene subset correlated with autophagic activity, and PRAS40, an inhibitor of mTORC1 activity, was chosen as a potential target due to its two predicted binding sites at 41-49 bp and 532-540 bp of the 3′-UTR . Rapamycin, a functional partner of FK506-binding protein 12 (FKBP12) that binds to and inhibits the mTOR protein of mTORC1, restored autophagic activity, supporting the assumption that activated mTORC1 mediates the novel_miR56-induced inhibition of autophagy 26. LC3B, a member of the mammalian homologs of yeast Atg8, mostly involved in autophagosome formation and the conversion of LC3-I (cytosolic form) into LC3-II (membrane-bound lipid form) is the characteristic signature of autophagic membranes13. The increase in expression of P62/SQSTM1 and decrease in BECN1 and LC3B-II/I were in accordance with reduced autophagic activity after novel_miR56 treatment . Although novel_miR56-mediated inhibition of autophagy was observed in GBM cells in vitro, the biological influence on GBM cells was unclear due to the dichotomous effect of autophagy. Autophagy plays an important role in GBM cell death, and cell survival ability was measured by CCK8 assay. Surprisingly, novel_miR56 overexpression promoted GBM cell growth, and this pro-survival effect could be partially abolished by rapamycin. These data suggested that novel_miR56-mediated autophagic inhibition could sustain the proliferative phenotype of GBM and that mTORC1 is an important mediator responsible for this oncogenic process . These results demonstrated that novel_miR56 could reactivate mTORC1-mediated autophagy inhibition by targeting PRAS40, and the reduced autophagic activity led to a proliferative phenotype in GBM.PRAS40 has been identified as a partner and inhibitor of mTORC1 as it is an important checkpoint in stopping autophagic initiation. In PDXM cells stably expressing GFP-LC3, an intracellular green fluorescent label recombinant protein for autophagosomes, exogenous upregulation of novel_miR56, a miRNA regulator of PRAS40, resulted in a significant decrease in the number of GFP-LC3-enriched subcellular granules around the nucleus, which indicated decreased autophagic activity in PDXM cells in response to increased novel_miR56 expression. In contrast, reduced expression of novel_miR56 increased the number of GFP-LC3-labeled granules . The survival times were shorter in mice bearing novel_miR56-expressing orthotopic tumors than in those bearing control sequence-expressing orthotopic tumors . Histopathological staining of mesGBM orthotopic tumors revealed larger tumor volumes and a higher ki67 index in novel_miR56-treated mice. Besides, a hyper-staining of SQSTM1, mTOR, and LAMP1, and a hypo-staining of LC3B were observed in orthotopic GBM specimens overexpressing novel_miR56 . The accumulation of intracellular SQSTM1, a selective autophagy substrate and cargo receptor for degradation of ubiquitinated substrates, and LAMP1, a major lysosomal membrane protein, could be the result of impaired autophagosome formation and lysosome consumption. Altogether, these findings revealed that novel_miR56 enhances mesGBM cell growth in vivo and that the underlying mechanism may be attributed to PRAS40 suppression and mTORC1 reactivation, which agrees with in vitro findings on the pro-survival role of novel_miR56 in GBM.Our data showed that novel_miR56 overexpression promotes GBM cell survival 4. MesGBM is subclassified by a subset of transcriptomic signature genes associated with mesenchymal transition and characterized by aggressiveness and resistance to chemoradiotherapies. The mesenchymal signature was considered to contain promising targets for mesGBM; however, after nearly a decade, few successful drugs have been developed for this disease, which thus far remains fatal. To improve the overall survival of GBM patients, or at least patients with the refractory mesenchymal subtype, new insights could be provided by further deepening our knowledge of malignant transformation.GBM is the most common and lethal primary malignant brain tumor. Despite advances in molecular pathogenesis in recent years that have advanced our understanding of the biology of this disease, little progress has been made to change the poor prognosis, even in the era of molecular medicine3. However, trials for drugs targeting TGFβ signaling fail to achieve clinical benefit in patients with GBM, which therefore urges us to reevaluate the pathological relevance of TGFβ signaling in mesGBM. Although the importance of understanding TGFβ signaling has been attached to cancers such as GBM for years, most research efforts were dedicated to protein-coding genes. There is an emerging consensus that ncRNAs are key players in epigenetic regulation, and miRNAs are the most well-studied class of ncRNAs that play functional roles in post-transcriptional regulation. Mature miRNAs are encoded by the genome and undergo step-by-step maturation, which is strictly coordinated by multiple pathways, including TGFβ signaling pathways. TGFβ signaling crosstalks with miRNA machinery at the level of miRNA transcription and DROSHA processing, together increasing expression levels. miRNAs are promising emerging targets for drug development in brain tumors27. The exclusively hyperactive TGFβ signaling in mesGBM may lead to downstream miRNA dysregulation, which will provide numerous potential therapeutic targets for treating mesGBM. We used LY2109761, a TβRI/II inhibitor, to inhibit TGFβ signaling activity in a PDXM cell line derived from a mesGBM PDX model and performed transcriptome sequencing on LY2109761-treated and control cells. There were 113 miRNAs with differential expression in cells after LY2109761 treatment, including 28 unreported miRNAs. These unreported miRNAs, temporarily named novel_miRs, were considered unregistered miRNAs in miRBase. The miRNAome expands gradually over time due to advancements in technology and algorithms of miRNA mining, and the latest version of miRBase (version 22) contains 1917 miRNA precursors and 2654 mature miRNAs for homo sapiens, with an additional 36 miRNA precursors and 66 mature miRNAs compared to the previous version (version 21). Therefore, it is inevitable that novel miRNAs will be discovered, and these miRNAs may play previously unknown roles in particular disease processes, such as the TGFβ signaling-mediated malignant transition of mesGBM, as shown in this study. Here, we paid special attention to the downregulated novel miRNAs that may be direct downstream effectors of TGFβ signaling, and we sought novel insight into mesGBM pathogenesis. Five novel miRNAs (novel_miR26/56/93/97/119) were selected for validation in GBM, mesGBM, breast cancer and renal cancer cell lines. Among these novel miRNAs, novel_miR56 was chosen for functional evaluation due to its relatively higher abundance and consistently positive correlation with TGFβ signaling activity in GBM cell lines.In our study, to search for novel insight into mesGBM pathogenesis, we reviewed the transcriptomic signature genes of mesGBM and found the exclusive upregulation of TGFβ1 and TβRII in the mesenchymal subtype and downregulation of TGFβ3 in the proneural subtype in the TCGA GBM dataset. TGFβ1/3 and TβRII are three important constituents that activate TGFβ signaling. Thus, hyperactive TGFβ signaling could be a signature event in mesGBM. TGFβ signaling is a multifunctional pathway that regulates a variety of oncogenic processes in cancer cells, and it has been reported to correlate with mesenchymal transition and poor prognosis in GBM20. Autophagy in cancer is a rapidly growing research field, and recent studies suggest the possibility of targeting autophagy to fight cancers28. In GBM, due to insensitivity to apoptotic stimuli, autophagy is the major pathway that controls cell death17, and it is possible that dysregulated autophagy can sustain tumor cell survival. TGFβ signaling has been implicated in autophagic regulation in glioma30, but the downstream effectors of TGFβ signaling that crosstalk with the autophagic machinery remain unclear. mTORC1 is an established inhibitor of autophagy induction31, and PRAS40 inhibition by TGFβ signaling-induced novel_miR56 reactivates mTORC1, resulting in autophagy suppression. Our results first link TGFβ signaling-induced miRNA with autophagic regulation in mesGBM.To better understand the functional implications of novel_miR56 in TGFβ signaling-mediated pathogenesis, the functional targets of novel_miR56 should be verified. However, most of the online miRNA target prediction tools are only compatible with miRbase-registered miRNAs, and there are limited options for sequence-based predictions. Here, we used MR-microT, an open access online tool provided by the DIANA lab, to predict novel_miR56 targets. In this study, due to our special interest in autophagy in GBM, PRAS40 was chosen and experimentally validated as a functional target that participates in autophagic regulation by suppressing mTORC1 activity28; however, emerging evidence in recent years suggests that autophagy can mediate tumor suppression via inducing cellular senescence and reducing chromosomal instability32. Rampant genomic instability is one of the hallmark features of GBM, and autophagy suppression mediated by TGFβ signaling-induced novel_miR56 expression may abolish the self-mediating capability to restore chromosomal stability. This process may provide novel insight into mechanism of proliferation maintenance in mesGBM by linking autophagic activity with chromosomal stability regulation.Activation or deactivation of autophagy has opposing, context-dependent roles in cancers, including GBM. In this study, novel_miR56-mediated autophagic inhibition sustained cell growth in GBM cells as well as mesGBM cells, indicating that TGFβ signaling can induce the expression of certain miRNAs to suppress autophagic activity. One of the major reasons for targeting autophagy in cancer is the cytoprotective role in response to various conditions of cellular stressFigure 6). The oncogenic role of TGFβ signaling-induced novel_miR56 provides novel insight into mesGBM formation and a molecular basis for drug development.In summary, our study reveals a novel mechanism for TGFβ signaling-mediated autophagic regulation in GBM. TGFβ signaling-induced novel_miR56 can directly target PRAS40 and reactivate mTORC1 activity, resulting in autophagy suppression but survival maintenance (


Pseudomonas syringae. We also elaborate on how this pathogen releases virulence factors and effectors to gain control over plant jasmonic acid signaling to effectively cause disease. The findings discussed in this review may lead to ideas for the development of crop cultivars with enhanced disease resistance by genetic manipulation.Sensing of pathogen infection by plants elicits early signals that are transduced to affect defense mechanisms, such as effective blockage of pathogen entry by regulation of stomatal closure, cuticle, or callose deposition, change in water potential, and resource acquisition among many others. Pathogens, on the other hand, interfere with plant physiology and protein functioning to counteract plant defense responses. In plants, hormonal homeostasis and signaling are tightly regulated; thus, the phytohormones are qualified as a major group of signaling molecules controlling the most widely tinkered regulatory networks of defense and counter-defense strategies. Notably, the phytohormone jasmonic acid mediates plant defense responses to a wide array of pathogens. In this review, we present the synopsis on the jasmonic acid metabolism and signaling, and the regulatory roles of this hormone in plant defense against the hemibiotrophic bacterial pathogen  Arabidopsis thaliana where the SA-mediated signaling is noted to suppress the JA signaling, thereby protecting plants against biotrophic or hemibiotrophic pathogen infections    , we tentatively placed this branch in the ER.The JA catabolism has been well-demonstrated in higher plants for its role in maintaining the JA homeostasis during a switch from inducible to non-inducible conditions. Three distinct JA-catabolic pathways are well demonstrated in bidopsis b. In theCOI1 complex for the 26S proteasome-mediated degradation [CORONATINE INSENSITIVE 1 (COI1) receptor and JASMONATE ZINC-FINGER EXPRESSED IN INFLORESCENCE MERISTEM (ZIM)-DOMAIN (JAZ) proteins are the integral components of the JA signaling ,30. COI1radation ,30. NINJA-TPL-TPR repressor complex tightly holds the bound TFs and inhibits them from activating the transcription of downstream JA-responsive genes [In the absence or scarcity of endogenous JAs, JAZ proteins form a complex with the co-repressor TOPLESS (TPL), TPL-RELATED (TPR) proteins and HISTONE DEACETYLASE (HDA) a. This cve genes ,31,32,335) molecule functions as a co-receptor for JA-Ile and stabilizes the association of COI1–JAZ complex [However, upon elicitation, once the JA-Ile accumulates in the cytoplasm, it is imported into the nucleus by JASMONATE TRANSPORTER 1  located in the nuclear envelope . The imp complex ,37  [The MYC branch specifically regulates the wounding- and insect-induced JA responses and is governed by the MYC2 TF. In addition, other members of the MYC family, specifically MYC3 and MYC4, act in conjunction with MYC2 to activate the transcription of JA-responsive marker genes such as 2 (VSP2) ,44  ,45,46. T2 (VSP2) ,45,46. M2 (VSP2)   , therebyERF1, OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2 (APETALA2)/ERF-DOMAIN PROTEIN 59 (ORA59) and PDF1.2) and suppressing JA signaling [ORA59 and ERF1 [The ERF branch is activated specifically upon necrotrophic pathogen attack and is characterized by the transcriptional activation of the gene encoding a PLANT DEFENSIN 1.2 (PDF1.2) ,48. Thisignaling   and represses their expression, inhibiting JA biosynthesis [PDF1.2 gene [BPM E3 ligases) and are stabilized by JAs. These BPM proteins interact with MYC2, MYC3, and MYC4 proteins and target the MYCs, particularly MYC2 and MYC3, for CUL3BPM E3 ligases-mediated proteasome breakdown [JA signaling is tightly regulated in plant cells and is negatively regulated at multiple stages in the absence of the inducer or during the recovery phase . In addiynthesis  -mediated stomatal reopening, which makes plants more vulnerable to this bacterial strain [Arabidopsis mutants display a drastic reduction in the expression of plant defense-related genes (such as PDF1.2) and consequently exhibit enhanced resistance against P. syringae [Arabidopsis coi1 mutant displays enhanced SA levels and transcript level of the SA-inducible PATHOGENESIS-RELATED PROTEIN 1 (PR1) gene, as well as increased resistance to Pto DC3000 infection [The synergism between ET and JA signaling pathways is a requisite for the activation of plant defense against necrotrophic pathogens ,48,58. Hed genes ,60,61,62l strain ,64. Notasyringae . The mossyringae . For exanfection .Arabidopsis jaz5/10 double mutant plants display aggravated chlorotic symptoms with increased growth of P. syringae, suggesting that both JAZ5 and JAZ10 mutually function to restrict P. syringae proliferation [P. syringae infection [Arabidopsis jaz2 mutant plants are partially impaired in a microbe-associated molecular pattern (MAMP), from the crude extract of Pto DC3000-induced stomatal closure, and are susceptible to Pto DC3000 [Pto DC3000 produced a phytotoxic virulence factor coronatine (COR), which led to the degradation of JAZ2 and the release of JAZ-repressed MYC2 functioning [The JAZ repressors are important mediators of early basal and subsequent secondary plant defense responses ,68. For nfection . Arabidoo DC3000 . Furtherctioning .P. syringae by inhibiting SA-dependent responses. The suppressive effects of JAs on SA biosynthesis or signaling are mostly mediated by the MYC2. Reportedly, MYC2 represses SA signaling in Arabidopsis as evidenced by the fact that myc2 mutants exhibit increased SA levels, upregulated PR1 expression, and enhanced resistance to Pto DC3000, compared with wild-type plants [P. syringae [Arabidopsis thaliana ACTIVATING FACTOR 1/2 (ATAF1/2), and CUP-SHAPED COTYLEDON 2 (CUC2) domain NAC TFs, such as ANAC019, ANAC055, and ANAC072 in Arabidopsis, and the two homologous NAC TFs JASMONIC ACID 2 (JA2) and JA2-LIKE (JA2L) in tomato, and activate their transcription [SALICYLIC ACID INDUCTION DEFICIENT 2  but induce the transcription of BENZOIC ACID/SALICYLIC ACID CARBOXYL METHYLTRANSFERASE 1 (BSMT1) that encodes an SA-methylating enzyme. This reprogramming of transcriptional regulation leads to reduced SA accumulation, activation of stomatal reopening, and increased in planta proliferation of P. syringae [SID2 gene and repress its transcription to negatively regulate Arabidopsis resistance against Pto DC3000 [Pto DC3000. In addition to MYC2 and EIN3–EIL1, MITOGEN-ACTIVATED PROTEIN (MAP) KINASE 4 (MPK4) is another node that governs the antagonism between JAs and SA, while positively regulating JA signaling but suppressing the SA signaling [Arabidopsis mpk4 mutant plants exhibit impaired expression of JA-responsive PDF1.2 and are insensitive to JAs; however, these mutant plants exhibit increased accumulation of endogenous SA, constitutive expression of SA-dependent PR1 gene, and consequently are more resistant to P. syringae [In addition, the JA signaling regulates plant response to invading e plants . Similarsyringae . MYC2 bicription ,63,69. Tsyringae ,69. In ao DC3000 . Moreoveo DC3000 . Thus, iignaling ,72. The syringae ,72.defenseless-1 (def-1) mutant plants displayed enhanced susceptibility to the Pto DCT6D1 and Xanthomonas campestris pv. versicatoria DC93-1 [Pto DC3000 carrying the avrRpt2 (a type III effector gene encoding a cysteine protease) [Pto DC3000 avrRpt2-mediated ETI induction, although the SA accumulates to a very high level, such accumulation does not lead to the inhibition of JA signaling, however [P. syringae 2 (RPS2)-mediated ETI [Increasing evidence is also available for the positive role of JA-mediated signaling in regulating plant immunity. For example, the JA-deficient tomato a DC93-1 . The plaa DC93-1 . More sta DC93-1 . Moreoverotease) , which c however . The SA ated ETI . In the ated ETI .P. syringae infection also relies on the intracellular ratio of these two hormones, and both JA- and SA-dependent signaling pathways can potentially coexist during a hemibiotrophic infection [PDF1.2) and SA-dependent  signaling [P. fluorescens N21.4 in Arabidopsis activated the expression of genes associated with both JA  and SA  pathways as well as quenched the reactive oxygen species (ROS)-mediated oxidative stress and provided protection against Pto DC3000 [P. syringae as well as the JA-dependent resistance responses against the necrotrophic fungal pathogens Alternaria brassicicola and Botrytis cinerea [Not only this, but the relationship between JAs and SA during avirulent nfection . For exaignaling . Similaro DC3000 . The MED cinerea .ENHANCED DISEASE SUSCEPTIBILITY 5 , encoding a member of the MULTIDRUG AND TOXIN EXTRUSION (MATE) transporter family, which transports SA synthesized in the chloroplasts to the cytoplasm, and reduced expression of PHYTOALEXIN DEFICIENT 4 (PAD4) in a MYC2-dependent manner [PAD4. However, in the case of loss-of-function of PAD4 , JAs positively regulate EDS5 and stimulate SA accumulation [P. syringae in the presence of PAD4 but positively regulate plant defense responses in the absence of PAD4 [JAs not only regulate SA biosynthesis and signaling but also influence the intracellular homeostasis of SA. For example, the MeJA application led to the activation of MYC2-dependent signaling and enhanced the expression of t manner ,80,81. Nvsp1Arabidopsis constitutive expression of  (cev1) mutant, which displays a constitutive expression of the JA-dependent ERF branch-regulated PDF1.2 gene, is also more resistant to P. syringae pv. maculicola ES4326 [B. cinerea or A. brassicicola infection in Arabidopsis plants led to the activation of both JA- and ET-signaling pathways [Arabidopsis, the functional NPR1 is required for SA-mediated suppression of JA-dependent defenses [PDF1.2 and VSP2 expression [In another example, the a ES4326 . In addipathways . In thispathways . In Arabdefenses ,83. HowePto DC3000 secretes COR and hijacks JA signaling. COR is a mimic of the bioactive JA-Ile and perceived through the COI1-JAZ co-receptor complex, it directs JAZ proteins, such as JAZ2, JAZ5, and JAZ10, for degradation . These NAC TFs, in turn, inhibit SA accumulation and SA-dependent plant resistance responses to facilitate the bacterial infection in the host plants [Pto DC3000 thus hijacks the COI1–JAZ2–MYC2/3/4–ANAC module that is involved in the regulation of stomatal aperture during the infection process [P. syringae pv. tabaci 11528, interacts with the ZIM domain of all the members of the JAZ protein family and triggers their breakdown in a COI1-independent manner to activate the JA signaling, favoring P. syringae infection [P. syringae type III effector HopBB1 interacts with both TEOSINTE BRANCHED, CYCLOIDEA, AND PROLIFERATING CELL FACTORS 14 (TCP14), a negative regulator of JA signaling, and JAZ3, a repressor component of JA signaling, and glues them together to facilitate their degradation in a COI1-dependent manner. The breakdown of JAZ3 and TCP14 thus activates the JA-signaling pathway that aids bacterial pathogenesis (P. syringae pv. syringae A2 (Psy) and is a member of a widely distributed YopJ effector family of acetyltransferase. Upon Pto DC3000 infection in Arabidopsis, HopZ1a interacts with JAZ proteins  through the conserved C-terminal Jas domain to acetylate JAZ proteins and direct their degradation in a COI1-dependent manner [P. syringae virulence. Both HopBB1 or HopZ1a could partially complement the virulence defects of a COR-deficient P. syringae mutant [Pto DC3000 mutant.While plants are well equipped with defense mechanisms to thwart away pathogens, it has also been demonstrated that pathogens employ a cocktail of virulence factors and effectors to manipulate plant defense responses for their own benefits ,64,84,85radation a. As a rt plants ,69,86. P process . As opponfection  -INTERACTING 4 (RIN4) to activate Arabidopsis H(+)-ATPase (AHA1), a plasma membrane-localized proton ATPase , which results in changes in membrane potential. Alteration in plasma membrane potential increases guard cell turgor pressure to induce stomatal opening [P. syringae [Pseudomonas by disrupting JA signaling in an rpm1 mutant background [ opening . The Avr opening   or those that are generally vulnerable to JA-dependent defense . The literature on JA signaling suggests extensive redundancy among different JAZ protein-mediated signaling modules. JAZ proteins not only regulate distinct TFs and an array of downstream responses based on the nature of the pathogens, the host plant developmental stages, tissues, or cell types, but they are also a major hub for molecular crosstalk and the integration of JAs with other signaling pathways. In addition, given the small number of examples in which most pathogens evolved multiple effectors to target JAZ repressors, these JAZ proteins are the best candidate targets for manipulation to develop better defensive plants. Hence, detailed knowledge of JA signaling opens up exciting opportunities for translational research to improve crop resistance under a multi-attacker situation.