Giardia duodenalis of Slab51 probiotic supernatants were evaluated in vitro and ex vivo. In vitro, Slab51 (101 UFC) was cultured and the obtained supernatant was filtered, adjusted at pH 7, and added (100μl/ml) as such (Slab51 FS) or after heat-treatment, to G. duodenalis cultures to evaluate its effects on G. duodenalis trophozoites growth and adherence. For comparison, negative and metronidazole (20μg/ml) treated controls were used. The morphological and ultrastructural alterations of G. duodenals trophozoites following treatment with Slab51 FS supernatant were investigated by transmission electron microscopy. Ex vivo, mice duodenal portions were cultivated in standard conditions with 5x105G. duodenalis trophozoites/ml, while to further five duodenal portions similarly cultured and infected, Slab51 FS 200μl was added. After 12 and 18h, samples were fixed in 10% buffered formalin and histologically processed to score Giardia infection and cell damage. Cell proliferation/apoptosis was scored by Ki67, TUNEL and Caspase–3 tests. All experiments were conducted in triplicate throughout the study. All data were statistically evaluated (P< 0.05). Results showed that Slab51 FS significantly reduced Giardia growth and adherence respect to negative controls, but its efficacy was overall lower than that of metronidazole. Moreover, the effects of Slab51 FS were significantly lowered by heat-treatment and this reduction was statistically higher at 90°C than at 56°C, indicating a heat-sensitive nature of active Slab51 FS compounds. At the ultrastructural level, Slab51 FS treated Giardia trophozoites were swelling, increased in size and showed alterations of their cellular membrane and vacuole patterns, loss of the nuclear envelope and nuclear architecture. In ex vivo trials, viable G. duodenalis trophozoites and enterocyte TUNEL+ and Caspase-3 expression were significantly reduced in intestinal sections added with Slab51 FS, while enterocyte Ki67 expression was significantly increased, confirming the anti-G. duodenalis activity of Slab51 FS observed in vitro. In conclusion, results from this study showed that the fresh culture supernatant of the commercial probiotic Slab51 has anti-G. duodenalis properties both in vitro and ex vivo in a mouse model.The effects on  Giardia are found in the digestive tract of vertebrate hosts worldwide in which they are the cause of giardiasis  enlargement or epithelial-associated lymphocytes increase).G. duodenalis trophozoites, and Ki67 positive cells at different times in treated and untreated samples, ten random fields of the sample were examined under a dry-X40 objective. The total number of protozoans and Ki67 stained epithelial cells was recorded. The mean value obtained per histological section per time was considered. To enumerate the TUNEL positive nuclei and cleaved Caspase-3 expression, tissues were graded in five categories by two independent blinded observers according to the number of detected apoptotic cells as follows: 0: without any apoptotic signal; 1: low level of apoptotic signal (<5%); 2: moderate level of apoptotic signal (5–10%); 3: high level of apoptotic signal (10–20%); 4: very high level of apoptotic signal (>20%). For the evaluation of these parameters indicating the apoptotic rate, 10 random fields of the sample were examined under a dry-x40 objective. The number of positive enterocytes was normalized to the number of enterocytes per field and expressed as a percentage of these values. Similarly, Ki-67 slides were visually scanned and scored: 0, negative (<5% positive cells); 1, sporadic (5%-20% positive cells); 2, moderate (20–50%); 3, diffuse (50–75%), and 4, strongly diffuse (>75% cells). For all parameters, cells on the margins of the tissue sections were not considered for evaluation to avoid possible artifactual staining.Paraffin sections were used for immuno-histochemical tests. Rehydrated sections were treated for endogenous peroxidases neutralization with 3% hydrogen peroxide for 1h followed by rinsing for 5min in deionized water. Antigen retrieval was achieved by incubating slides in antigen retrieval solution in a steamer  for 20min. Nonspecific immunoglobulin binding was blocked by incubation of slides for 10min with a protein-blocking agent  before application of the primary antibody. Slides were incubated overnight in a moist-chamber with polyclonal rabbit anti-human Ki67 antibody , used as primary antibody at dilutions of 1:50. A goat anti-rabbit byotinilated antibody (Dako), was used as secondary antibody at standard dilution of 1:250 in buffer. A streptavidin–immunoperoxidase staining procedure  was used for immunolabeling. The immunoreaction was observed with 3,3'-diaminobenzidine (DAB) or VIP substrate . Sections were counterstained with Mayer's hematoxylin. Positive immunohistochemical controls included mouse mammary carcinoma sections. Negative immunohistochemical controls were known mouse mammary carcinoma or intestinal sections, treated identically as routine sections with 20min antigen retrieval and 10min protein blocking, except that the overnight incubation with primary antibodies was replaced by an overnight incubation with buffer. Expression of cleaved Caspase-3 in paraffin-embedded tissue sections was investigated using the Anti-active Caspase-3 antibody  directed against a peptide from the p17 fragment of the active (cleaved) human Caspase-3, and after an O/N incubation with this antibody, sections were treated routinely as described above. Sections from mouse mesenteric lymph nodes were subsequently selected as the positive control for further tests. The primary antibody was replaced by phosphate buffered saline solution (PBS) as a negative control. In small intestinal sections, pro-apoptotic effect induced by Giardia in crypts, and mucosal lining epithelial cells were highlighted through a TUNEL colorimetric staining  according to the manufacturer’s instructions. To score the intensity of in vitro experiments were repeated in triplicate in two independent assays. Values were expressed as mean ± sd and compared by repeated measures analysis of variance, followed by Bonferroni’s multiple comparison [All mparison .ex vivo experiments were repeated in triplicate. Descriptive and comparative statistical analyses of ex vivo tests were performed and results  were described and tested for normal distribution with the Kolmogorov–Smirnov test and normal probability plots. As they were not normally distributed, the non-parametric Wilcoxon Signed-Rank test was used to compare median values for these variables between the treated and untreated control biopsies. Correlations between degrees of expressions of these different variants in the two groups of samples were analysed with Spearman rank tests [All nk tests .The level of statistical significance was set at P <0.05 throughout the study.G. duodenalis trophozoites respect to untreated controls, but its effects were generally lower than that of metronidazole, the reference drug. In fact, in the growth inhibition assay the number of trophozoites (35.48±8.16x104) found after 48h in cultures treated with fresh Slab51 supernatant was significantly (P <0.05) reduced respect to that counted in untreated cultures (54.15±6.58x104). However, this reduction was significantly lower (P <0.05) respect to that observed in cultures treated with metronidazole where the number of trophozoites was extremely low (5.05±4.47x104)  observed in Slab51 FS-treated cultures was significantly reduced (P <0.05) respect to that observed in untreated cultures (48.58± 7.31%) but similar to that counted in culture treated with metronidazole (5.64±4.75). However, after 48h the number of adherent G. duodenalis trophozoites (12.85±2.26%) counted in Slab51 FS-treated cultures was still significantly lower (P<0.05) respect to that observed in untreated cultures (52.91±8.64%), but significantly higher (P<0.05) than in culture treated with metronidazole (3.43±3.44%)  ; Fig 1. 3±3.44%) ; Fig 1. ozoites 1.85±2.26%G. duodenalis growth and adhesion ability of fresh Slab51 supernatant. In fact, while after 48 h the inhibiting activity of 56°C heat-treated Slab51 supernatant (42.54± 5.02%) was significantly different (P<0.05) both from treated (5.05±4.47x104) and untreated controls (54.15±6.58 x104), as well as from Slab51 FS-treated cultures (35.48±8.16x104). After the same time-period, results observed for 90°C heat-treated Slab51 supernatant cultures (55.42±10.96 x104) were comparable to that of untreated controls . Indeed, at this time treated with Slab51 ultrafilterd fresh supernatant showed a significant reduction of viable G. duodenalis trophozoites at the end of the observation period, as evidenced in ex vivo intestinal tissue cultures after 18h of incubation with Slab51 ultrafilterd fresh supernatant. In untreated controls, G. duodenalis trophozoites showed an intact morphology also after 18h (ex vivo. These observations were also supported by the general morphology of bioptic samples. In fact, even if well preserved, untreated samples showed a clear loss of epithelial cells with a different pattern of inflammatory cell distribution throughout the mucosal corion, as evidenced in haematoxylin-eosin stained tissues (Figs In fter 18h  while thfter 18h . As showues Figs  and 5A.G. duodenalis is a common fecal-oral parasite of the small intestine and one of the most important causes of human and animal diarrheal disease worldwide. Indeed, G. duodenalis infection can be asymptomatic, or cause acute or chronic diarrhea, dehydration, intestinal malabsorption, malnutrition and steatorrhea [G. duodenalis infections, [G. duodenalis infection through different mechanisms, including competition for limited adhesion sites, competition for nutrients that would otherwise be utilized by G. duodenalis, stimulation of the host immune response and by producing substances that may inhibit G. duodenalis [G. duodenalis activity of probiotic compounds, mainly derived from Lactobacilli, has been demonstrated [Lactobacillus acidophilus were found able to inhibit in vitro the adhesion and the growth of G. duodenalis trophozoites [G. duodenalis trophozoites, a decline of the intestinal parasite density and amelioration of intestinal pathology in infected mice treated with L. acidophilus bacteriocins [L. johnsonii strain LA1 and Lactobacillus gasseri CNCM I-4884, may represent a further mechanism contributing to the inhibition of Giardia trophozoite growth in vitro [atorrhea , 4–7. Chections, , 29, whiections, . Probiotodenalis , 7, 11. nstrated , 24. In in vitro , 32.G. duodenalis showed by the fresh supernatant of the commercial probiotic evaluated in vitro and ex vivo in the present study, are similar or higher to those reported in most of these previous studies. In fact, the fresh Slab51 supernatant was able to inhibit in vitro the adhesion and the growth of G. duodenalis trophozoites, although this inhibition was significantly lower than that of metronidazole. In the study of Perez et al. [Lactobacillus johnsonii was able to control G. duodenalis growth in vitro but it was unable to inhibit the adhesion of the parasite, while six Lactobacillus acidophilus strains tested did not show any noticeable effects. These data could be indicative that Slab51 constituent probiotic strains, mainly L. plantarum DSM 32244, possibly produce more effective active anti-G. duodenalis compounds with respect to those produced by L. johnsonii LA1. Negative effects on G. duodenalis adherence here observed are consistent with the ability reported for some lactobacilli to modulate G. duodenalis infection in vivo by minimizing or preventing the adherence of trophozoites to the intestinal mucosal surface [Negative effects on z et al. , the cul surface , 34.G. duodenalis trophozoites [G. duodenalis trophozoites from in vitro cultures treated with Slab51 fresh supernatant, including profound alterations of cellular and nuclear membranes, nuclear disorganization and formation of intra-cytoplasmic cavities. These in vitro cytopathic effects are very similar to those caused by L. acidophilus bacteriocins in vivo, possibly indicating a similar mode of action [in vitro.In agreement with previous studies in which some probiotic compounds were found able to induce morphological changes of hozoites , 24, impf action  and may in vitro inhibiting effects on G. duodenalis showed by Slab51 fresh supernatant were greatly reduced by heat treatment at 56°C and completely annulled at 90°C, indicating that active metabolites contained in Slab51 supernatant are likely term labile compounds. These results agree with those of a previous report [Giardia effects of fresh Slab51 supernatant observed in the present study.Trials performed in this study showed that the s report  and encoex vivo trials confirmed the inhibiting effects of the fresh supernatant of Slab51 on G. duodenalis trophozoites observed in vitro. The best observations were obtained after an 18h incubation period. In fact, after this period a significant reduction of G. duodenalis trophozoites and a significantly greater vitality of intestinal epithelial cells was evidenced in treated intestinal cultures respect to the untreated controls. Moreover, G. duodenalis was found capable of slowing or damaging the intestinal epithelial cell turnover in untreated ex vivo cultures, since in these cultures the apoptotic rate was increased. On the other side, obtained results showed that the number of G. duodenalis trophozoites was significantly lowered by the fresh Slab51 supernatant. Moreover, in ex vivo cultures the apoptosis and the death of intestinal epithelial cells was higher in G. duodenalis-inoculated cultures respect to those inoculated with G. duodenalis and the fresh supernatant of Slab51, indicating that the damage to the epithelial cells induced by G. duodenalis was reduced by the fresh supernatant of this commercial probiotic. Considering that the increase in the rate of enterocyte apoptosis and enterocyte damage are included among the main pathogenic mechanisms of G. duodenalis [ex vivo trials are promising about possible in vivo protective effects of the fresh culture supernatant of Slab51 against G. duodenalis. This is the first study in which negative effects on G. duodenalis by metabolites from lactobacilli were demonstrated both in vitro and by using a murine ex vivo model. In previous studies, a variety of different systems have been used to evaluate the adherence and growth of G. duodenalis, including synthetic surfaces, human cells and non-human cells, as isolated rat enterocytes and rat enterocyte cell lines [G. duodenalis trophozoites under different conditions [G. duodenalis trophozoites to the intestinal mucosa and of associated inflammatory cells, while the murine ex vivo model performed in this study allowed these evaluations, by preserving the tissue architecture and the cellular complexity over several days. Indeed, alterations, damage and inflammation herein observed in ex vivo negative controls, i.e. intestinal tracts inoculated with G. duodenalis trophozoites only, were not so different from that observed in in vivo rodent models and at histopathological examination of intestinal biopsies taken from symptomatic human patients [ex vivo mouse model used in this study allowed to assess the anti-Giardia activity of the fresh supernatant of Slab51 by the evaluation of several positive effects on intestines inoculated with G. duodenalis trophozoites, although this model cannot be able to mimic the complexity of whole living organisms.Results from odenalis , resultsll lines –37. Amonll lines . Therefonditions . Howeverpatients , 35. MorG. duodenalis both in vitro and ex vivo in a mouse model. These antagonistic effects may suggest that this probiotic may likely represent a further and interesting approach for the prevention of giardiasis and/or the reduction of the pathogenic effects and proliferation of this protozoan parasite in infected hosts. However, further studies aimed to evaluate its efficacy in vivo on experimentally and/or naturally infected animals, are needed.In conclusion, results from this study showed that the fresh culture supernatant of the commercial probiotic Slab51 has negative effects on


This study investigated the moderating role of household registration in the relationship between housework division and second-child fertility anxiety among Chinese couples of childbearing ages. Multilevel cluster sampling was used to select 1834 respondents aged 20–45 years from Jilin Province in China between 2016 and 2017. A sample of 542 adults who were married and had only one child was included in the final analysis. Hierarchical multiple regression was used to examine the proposed hypothesis. The results showed that the association between housework division and second-child fertility anxiety was significant in rural areas. However, the above association was not significant in urban areas. Household registration status was found to have a moderating effect on the relationship between housework division and second-child fertility anxiety. Differences in gender and fertility ideology have led to different housework divisions in urban and rural areas, which in turn have led to different effects on the second-child fertility anxiety of couples of childbearing ages in these areas. The division of housework in families is an important part of the cooperation between couples . At the Different from Western countries, China’s typical hukou system has created a two-tier urban–rural system in China. Hukou refers to household registration status, which is generally based on an individual’s birthplace. Rural populations generally hold agricultural household registration status, and those living in urban areas hold non-agricultural household registration status. Individuals with different hukou status have different levels of access to education, employment, housing and social welfare services and benefits. After the implementation of the policy of reform and opening up (Chinese economic reforms in 1978 led to a significant transition of China’s economy from a planned economy model to a market-oriented economy), China’s urban–rural differences have become increasingly prominent . The proIn urban China, economic and scientific spheres are rapidly developing, and domestic labor market resources are sufficient. These factors have alleviated the burden of domestic work among women who live in urban areas. However, in rural China, living conditions are poorer, social service levels are limited, and domestic market resources are relatively scarce. Compared with urban women, rural women need to take on more housework . ResearcFollowing its reform and opening up policy, China’s public and private sectors have gradually been divided, and the division of housework has always been regarded as a problem in the private sector without attracting sufficient attention. In fact, there is no clear boundary between the private and the public sector, and the two are always infiltrating each other. However, research on the division of housework in China is very limited. At present, related research has mainly focused on issues such as gender equality ideology, labor time allocation, and income distribution. Few studies have paid attention to the impact of housework division on fertility. Especially in the context of the urban–rural dual structure, the urban–rural differences of division of housework have differing effects on fertility intention in urban and rural populations of childbearing age, including varying influences on their mental health. This study explores the moderating role of household registration in the relationship between the division of housework and fertility anxiety. According the results of the model, we propose suggestions to improve the fertility anxiety of couples of childbearing ages.Anxiety has a psychological definition. The original meaning of anxiety refers to a kind of emotional response of an individual facing uncertain danger and a lack of effective self-protection ,18. In cTo release the stress of the transformation of the population structure in China, in 2016, the Chinese government implemented its universal two-child policy. Every couple of childbearing age could have a second child. However, most couples did not respond to the policy by having a second child. In modern society, the cost of parenting a child is much higher than ever, and with the increase in women’s public labor participation, most families lack child care resources . HoweverHousework refers to unpaid labor in the family setting, such as laundry, cleaning, food preparation, and other tasks necessary to maintain a “livable” home . HousewoGiven the dramatic increase in women’s education level and paid labor force participation, there has been escalating sociological attention paid to gender differentials in unpaid housework division ,32,33,34In China, the transformation of the economic structure changed traditional gender ideology and promoted more equal housework division; however, the housework labor time of women is still much longer than that of men. Wives are still the main bearer of daily housework ,40. At tAfter World War II, the education level of women in Western countries increased rapidly. In the context of promoting productivity and the transformation of labor type, women got more opportunities to participate the public labor sector . Thus, tIn China, before the implementation of the policy of reform and opening up, most enterprises were state-owned and had their own nurseries for their employees, which greatly eased the family burden of women who participated in the public labor sector. However, given the market economy reforms since late 1970s, enterprises began to seek maximization of profits. In order to save production costs, enterprises gradually reduced the subsidies and benefits in terms of family reproduction . FurtherIn China, traditional culture significantly influences people’s fertility ideology, and different generations have different fertility ideologies . In receAccordingly, we posit the following hypotheses: Housework division influences second-child fertility anxiety. Household registration has a moderating effect on the relationship between housework division and second-child fertility anxiety. The effects of housework division on second-child fertility anxiety are more significant in rural China.The data for this study were derived from a survey named The Influence of the Universal Two-Child Policy on Female Fertility Behavior conducted in Jilin Province by the Institute of Gender and Culture at Changchun Normal University. A secondary data analysis was used in this study. The survey occurred from October 2016 to April 2017 in eight cities. Jilin Province is in Northeast China, where the main financial resource is agriculture. Thus, in the rural areas of Jilin Province, most women need to do not only housework but also some agricultural work. Compared to their counterparts in urban areas, the labor burden of women in rural areas is much heavier. In addition, because women in rural areas have few opportunities to participate in public labor, they have no independent income, leading to low family status. They often must do extensive housework and take care of their children. They frequently do not want to have a second child to add their work burden; however, they have less power in their marital and family relationships. These factors can produce conflicts in the family and lead to second-child fertility anxiety. Data collection involving human respondents was conducted according to the ethical standards of the institutional research committee and in accordance with the Helsinki declaration. Oral consent was obtained from all the respondents. Ethics approval was obtained from the Institute of Gender and Culture, Changchun Normal University.The survey used multilevel cluster sampling as the sampling method. First, the researchers calculated the relative population of the eight cities according to the population proportions of the local representative samples from the sixth national consensus. According the proportions, sample size was calculated and allocated in the eight cities. The inclusion and exclusion criteria are as follows: Eligible respondents needed to (a) be between 20 and 45 years of age (through October 2016); (b) be married; (c) have at least one child. The respondents needed to have adequate cognitive function and listening capacities to complete the survey. Of 1920 questionnaires distributed, 1834 valid questionnaires were returned. More than 80% of questionnaires were valid. The survey assessed the respondents’ basic situation, fertility intentions, effects of the universal two-child policy on their work and family lives, and factors affecting their decision to have one more child. The survey used four options to measure fertility intentions: “I want to have a second child,” “I am not sure,” “I am thinking about it,” and “I do not want to have a second child.” Respondents who chose the first and last answers indicated that they had confirmed their fertility decision—whether they did or did not want to have a second child, they were unlikely to have second-child anxiety. In contrast, those who chose “I am not sure” or “I am thinking about it” would likely have second-child fertility anxiety, because they have some second-child fertility intention and have not made a final determination. Accordingly, we selected the participants who chose the second and third answers—i.e., 542 individuals. We calculated sample size based on the average effect size of 0.20 reported in the literature . ResearcFirst, we used a question—“After the implementation of the universal two-child policy, do you want to have a second child?”—to screen the participants. As previously described, we selected respondents who chose the answers “I am not sure” and “I am thinking about it.” Then, we used another question to measure these respondents’ second-child fertility anxiety: “Imagining that someone else has two children and you do not have the ability to have a second child, tell me the degree of anxiety you feel.” The variable was measured on a 5-point Likert scale .We used one survey question to measure this variable: “What is the mode of the housework  division in your family? How much housework does the wife in your family need to undertake?” Participants had three answer options: (a) The wife does not to undertake the housework at all; (b) the wife undertakes about half of the housework; and (c) the wife has to undertake all of the housework. We coded the answers as 1–3, respectively; thus, higher scores indicated that the wife undertook more housework in the family.We coded household registration as a dichotomous variable. In China, household registration is mainly divided into two categories: Agricultural  and non-agricultural  household registration. Agricultural household registration was coded as 0, and non-agricultural household registration was coded as 1.Control variables included age, gender, education level, type of occupation, family per capita income, parenting costs, fertility ideology, the husband’s status as the only son, and the gender of the first child. Age was calculated by year of birth. Male was coded as 1, and female was coded as 2. Four categories were used to indicate the education level of the respondents, coded as 1 (primary school and below), 2 (junior high school), 3 (high school), and 4 (college and above). The type of occupation was divided into two categories: “In-system work” was coded as 1, and “out-system work” was coded as 2. The former included civil servants, workers in public institutions, and workers in state-owned enterprise; the latter included farmers, employees of private and foreign companies, and freelancers. We divided family per capita income into six levels, from 1 (less than 1000 yuan) to 6 (greater than 5000 yuan). Parenting costs were measured as direct and indirect parenting costs. Three questions were used to measure direct costs: (a) Do you think having a second child will reduce your family life quality? (b) Do you think having a second child will have negative impact on your family’s economic situation? (c) Do you think having a second child will reduce your personal life quality? We used “yes” (coded as 1) and “no” (coded as 0) as the answer options. A summed score for the three questions was used. Higher scores indicated greater direct parenting costs. To measure indirect parenting costs, we used two questions. First, “How do you think having a second child will affect your career?” Participants had four answer options: Affecting personal career development, affecting economic income, affecting individual career advancement, and reducing opportunities for individual re-education and training. The more answers the respondents chose, the greater impact on their occupation. The other question was, “Do you think that having a second child will reduce your entertainment time and space?” The alternative answers were “yes” (coded as 1) and “no” (coded as 0). We added the scores of the two questions; higher scores reflected greater indirect costs. Another question was used to measure fertility ideology: “The following options are about fertility ideology, which ones do you agree with?” Answers representing traditional fertility ideology were: “The more sons, the more blessing;” “Bring up sons to provide for one in old age;” “Having a child could carry on the family line;” and “Fertility is the value of a women.” Answers representing modern fertility ideology were: “The aim of fertility is participating in the process of a child growing up” and “Fertility is a method to promote the relationship between husband and wife.” Each affirmative response resulted in one point, and we used the score of modern fertility ideology minus the score of traditional fertility ideology to measure respondents’ fertility ideology, because the scores we calculated included negative number and zero (the scope of the score was from –4 to 3); for convenience, we recoded the scope of the score from 1 to 8. We determined whether the husband was the only son in his family, measured as “yes” (coded as 1) or “no” (coded as 0). Finally, the gender of the couple’s first child was coded as 1 for male and 2 for female.We used a hierarchical regression analysis to test the moderating role of household registration in the relationship between housework division and second-child fertility anxiety. Hierarchical regression analysis was conducted in three steps. First, the sociodemographic characteristics and socioeconomic status of the respondents, the gender of the first child, and household registration status were entered in the regression model. Second, second-child fertility anxiety was regressed on housework division. Third, a two-way interaction term of household registration and housework division was entered in the model to test whether the association between housework division and second-child fertility anxiety varied by household registration. Regression coefficients for housework division were calculated by conducting two separate regression models for those with different household registration status. A data analysis was conducted using SPSS 22.0 . First, a descriptive analysis of all variables was performed  using di2 = 0.198, Durbin–Watson statistic = 1.889). In the second model, we added the variable of housework division , and the model did not change significantly. We found that housework division had no statistically significant effects on second-child fertility anxiety . According to gender equality theory [2 = 0.205, Durbin–Watson statistic = 1.906). We found household registration had a moderating effect on the relationship between housework division and second-child fertility anxiety . Therefore, we concluded that the moderating effect of household registration offset the influence of housework division on second-child fertility anxiety.We constructed three models to analyze the data . First, y theory , housewo2 = 0.241 and Durbin–Watson statistic = 1.917; for the rural group, R2 = 0.186 and Durbin–Watson statistic = 1.872. The results of the two models further verified the moderating role of household registration on the relationship between housework division and second-child fertility anxiety. Household division was positively associated with fertility anxiety only among rural couples .After exploring the moderating role of household registration, we tested the relationship between housework division and second-child fertility anxiety in urban and rural groups through a hierarchical regression analysis . The staThis study analyzed the influence of housework division on second-child fertility anxiety in couples of childbearing ages and further analyzed the moderating role of household registration. The results show that in urban areas, housework division had no significant impact on second-child fertility anxiety, whereas in rural areas, housework division significantly influenced second-child fertility anxiety.Fertility intention and fertility behavior are two different concepts; fertility intention influences fertility behavior through some mediators, such as fertility attitude, fertility decision, and fertility anxiety . The majIn urban China, most people have accepted the gender equality ideology; thus, most couples in urban areas tend to embrace a relatively equal mode of housework division . HoweverSome limitations exist in this study. First, the data used in the study were cross-sectional. Thus, we could not determine a clear causal relationship between the dependent variable and independent variable. However, the findings of the study are supported by theories, and the independent variable influenced the dependent variable at a statistically significant level, indicating that we can accept the conclusions of the study. Second, the data were derived from a survey in Jilin Province, China. The economic development, social development, and cultural context of Jilin Province differ from many other regions in China, excluding provinces in Northeast China and some western provinces. Therefore, the conclusions of the study are likely representative of Jilin Province and some other regions with similar economic and social development, cultural contexts, and population structure. These results might not be generalizable to regions that are significantly different than Jilin Province. In future research, longitudinal national data should be used to analyze related issues, such as housework division, gender equality, and fertility-related issues. Finally, conditions related to fertility anxiety might be different for women and men in the Chinese contexts, which need further explorations in future qualitative studies. The findings suggested that in the whole model, housework division had no significant influence on second child fertility anxiety. This is because the moderating effect of household registration offset the influence. For further explore the moderating role of household registration in the relationship between housework division and second-child fertility anxiety according to related theories, we added the interaction variable of housework division by setting into the model and found the moderating role of household registration. Finally, we constructed regression on rural and urban groups separately. In the rural group, housework had a significant effect on second child fertility anxiety. In the urban group, however, housework had no significant effect on second child fertility anxiety. Policy makers and intervention designers should publicize gender equality ideology, especially in rural China, to promote the equalization of housework division and to increase the rate of male participation in housework. In this way, couples could reduce conflicts about fertility. In addition, females should fight for the right to speak and make fertility decisions based on their own intentions, and husbands should respect their wives’ rights to reduce conflicts about fertility and relieve second child fertility anxiety.


To further improve the electrochemical sensing properties, discal Fe2O3 NPs with the highest electrocatalytic activity were coupled with graphene oxide (GO) nanosheets. The surface morphology, microstructures and electrochemical properties of the obtained Fe2O3 NPs and Fe2O3/GO nanohybrids were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. As expected, the electrochemical performances were found to be highly related to morphology. The discal Fe2O3 NPs coupled with GO showed remarkable electrocatalytic activity toward the oxidation of dopamine (DA) and uric acid (UA), due to their excellent synergistic effect. The electrochemical responses of both DA and UA were linear to their concentrations in the ranges of 0.02–10 μM and 10–100 μM, with very low limits of detection (LOD) of 3.2 nM and 2.5 nM for DA and UA, respectively. Moreover, the d-Fe2O3/GO nanohybrids showed good selectivity and reproducibility. The proposed d-Fe2O3/GO/GCE realized the simultaneous detection of DA and UA in human serum and urine samples with satisfactory recoveries.Various morphologies of iron oxide nanoparticles (Fe Dopamine (DA) and uric acid (UA) usually coexist in the serum and extracellular fluids of the central nervous system and play a significant role in regulating human metabolism activity . As an iTo date, several analytical techniques have been proposed for the detection of DA and UA, including, but not limited to, high performance liquid chromatography ,10, fluo2O3) has become one of the most versatile transition metal oxides not only due to low cost, more abundant, good biocompatibility and excellent electrochemical performances, but also for its widespread applications ), potassium ferrocyanide (K4[Fe(CN)6]), ammonia (NH3·H2O), sodium hydrate (NaOH), concentrated hydrochloric acid , concentrated sulfuric acid  and absolute ethanol (CH3CH2OH) were supplied by Sinopharm Chemical Reagent Co. Ltd. . All chemicals were analytically pure and directly used as received. Human serum samples were provided by Zhuzhou People’s Hospital . The stock solutions of DA and UA (1 mM) were prepared by dissolving appropriate amount of DA and UA in 500 mL 0.1 M PBS. Then lower concentration series of the standard solution were obtained by appropriately diluting the stock solution with 0.1 M PBS. Deionized water  was used in all the experiments.Dopamine (DA), uric acid (UA), Iron (III) nitrate nonahydrate  and powder X-ray diffractometry (XRD), respectively. SEM images were recorded on a cold field-emission SEM  at an accelerating voltage of 5.0 kV. The XRD patterns of α-Fe2O3 NPs was recorded using a powder X-ray diffractometer system  with monochromatized Cu Kα radiation (λ = 0.1542 nm) operating at 40 kV and 40 mA. The patterns were collected in a 2θ range from 10° to 80° with a scanning step of 0.02°2θ s−1. A digital precision pH meter  was used to measure solution pH.All the electrochemical measurements were performed on a CHI 760E electrochemical workstation . Surface morphologies and crystalline structure of α-Fe2O3 NPS were synthesized by a meta-ion mediated hydrothermal route (3)3·9H2O was adequately dissolved into 10 mL of DI water under magnetic stirring, and then 1 mM of acetate precursor was added to the above solution. After 15 min of stirring, 10 mL NH3·H2O was added and continuously stirring for another 15 min. Next, the mixture was poured into a 50 mL Teflon-lined stainless-steel autoclave and reacted at 160 °C for 16 h. Subsequently, the autoclave was naturally cooled down to room temperature. Finally, the resultant α-Fe2O3 product was alternately washed three times with absolute alcohol and DI water, and allowed to dry under vacuum at 60 °C for 12 h. Herein, cupric acetate, zinc acetate, and aluminum acetate were separately used as acetate precursor to obtain thorhombic, cubic and discal α-Fe2O3 NPs.Referring to previous reports ,47,48, tal route . In brie2O3 products was added into five aliquots of GO dispersion (1 mg/mL), and then ultrasonicated for 0.5 h to form uniform α-Fe2O3/GO dispersion.GO was synthesized by a slightly modified Hummer’s method according to our earlier reports ,23,24. 12) was carefully polished to mirror-like with 0.3 μm and 0.05 μm fine alumina slurry. Then the polished GCE was alternately washed by absolute ethanol and DI water several times and allowed to dry under an infrared lamp. The Fe2O3/GO/GCE was prepared by a simple drop-casting method. More specifically, 5 μL Fe2O3/GO dispersion was carefully dropped and coated on the GCE surface with a micropipette, then dried with an infrared lamp to form sensing film. For comparison, the discal, thorhombic, and cubic α-Fe2O3 NPs decorated GCEs  and GO decorated GCE (GO/GCE) were also fabricated by similar procedure.At first, bare GCE 6]3−/4− (1:1) in 0.1 M KCl as an electrochemical probe. The electrochemical responses of 10 μM DA and UA (1:1) mixture standard solution at different electrodes were recorded by CV. Differential pulse voltammetry (DPV) was used for the individual and simultaneous detection of DA and UA. A conventional three-electrode assembly was immersed into a 10 mL electrochemical cell for electrochemical measurements and detections, consisting of a bare or modified GCE as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as auxiliary electrode, respectively. To increase response peak current, a suitable accumulation period was applied before CV and DPV measurements. DPV was performed from 0 V to 0.8 V, with step potential of 4 mV, the amplitude of 50 mV and pulse width of 0.06 s and pulse period of 0.5 s, respectively. 0.1 M PBS was used as the supporting electrolytes unless otherwise specified. All the electrochemical measurements were performed at 25 °C.The electrochemical behaviors of various decorated electrodes were measured by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the 0.1 M PBS (pH 7.0) containing 0.5 mM 3−/4− as probe solution. The corresponding CV curves are plotted in Ipa = 12.73 μA, Ipc = 12.16 μA) are observed on the bare GCE with peak potential separation (∆Ep) of 0.156 V, suggesting that the electron transfer rate is very slow. At the GO/GCE, a pair of wide and weak redox peak occurs with the lowest redox peak currents  and maximum ∆Ep (∆Ep = 0.182 V). It was mainly due to the presence of poor electrical conductivity of GO. At the d-Fe2O3/GCE, a pair of well-shaped and sharp redox peak appears. Both anodic peak current (Ipa) and cathodic peak current (Ipc) increase greatly  while the ∆Ep decreases to 0.136 V, indicating d-Fe2O3 can effectively promote the redox process due to its high electrocatalytic activity. As expected, d-Fe2O3/GO/GCE displays remarkable electrochemical properties with the highest redox peak currents  and minimum potential separation (∆Ep = 0.104 V), attributing to the synergistic effect from d-Fe2O3 NPs and GO nanosheets. As well known, electrochemical active area is highly related to the electrochemical sensing properties. The electrochemical active areas of various electrodes were also estimated using the Randles-Sevcik equation 3−/4− happened on the surface of d-Fe2O3/GO nanohybrids. The results verify that the d-Fe2O3/GO nanohybrids can effectively lower the Rct.EIS is a valuable tool to acquire interfacial properties, which has extensively used in the various electrochemical sensors ,57,58,59Ipa of DA and UA is 0.2724 μA and 0.1555 μA, respectively. After the modification of GO nanosheets, the Ipa of DA and UA increased a little (Ipa(DA) = 0.2915 μA, Ipa(UA) = 0.2356 μA). The large specific area of GO could provide abundant adsorption sites for target analytes, which enhances the electrochemical response signals. But the poor electrical conductivity of GO severely retards the redox process, which impedes electrochemical oxidation of DA and UA. When d-Fe2O3 NPs were modified on the GCE, the Ipa of both DA and UA boosts greatly (Ipa(DA) = 0.5519 μA, Ipa(UA) = 0.4770 μA), suggesting d-Fe2O3 NPs have high electrocatalytic activity toward DA and UA. Among all the electrodes, d-Fe2O3/GO nanohybrids show a remarkable electrocatalytic capacity toward the electrochemical oxidation of DA and UA, with the highest Ipa of 1.077 and 1.234 μA for DA and UA, respectively. The synergistic effect from d-Fe2O3 NPs and GO nanosheets contributed to the significant enhancement in the response currents. Specifically, abundant active sites presented in the GO surface greatly facilities the adsorption of target analystes; d-Fe2O3 can efficiently catalyzed the electrooxidation of DA and UA. Besides, the d-Fe2O3 NPs/GO nanohybrids remarkably decrease the electron transfer resistant  at different electrodes were recorded by DPV in 0.1 M PBS (pH = 5.65). As presented in Ipa/Ipc approaches 1 for all the electrodes, meaning the electrode undergoes a quasi-reversible process. Among all electrodes, d-Fe2O3/GO nanohybrids also have largest response peak currents for DA and UA. The CV result is in good agreement with the results obtained by DPVs. It is worth pointing out that DPV is more sensitive than CV, which is very suitable for multiple analystes detection. Therefore, DPV was used for the quantitative analysis of DA and UA.The electrochemical responses of various electrodes were also measured by CV. As shown in EDA = −0.06079 pH + 0.5762 (R2 = 0.9988) and EUA = −0.06130 pH + 0.7184 (R2 = 0.9986). The slopes of these linear plots  are very close to the theoretical value from the Nernst equation (−59 mV/pH), indicating that the equal numbers of protons (H+) and electrons (e−) participate in the electrooxidation process of DA and UA, which is in good agreement with previous works.It is well known that pH is a vital parameter that directly influences the electrochemical responses. The DPVs of 10 μM DA and UA mixture solution (1:1) at different pH are depicted in To enhance the response electrochemical peak currents, accumulation was applied before electrochemical measurements. The influence of accumulation time on the response anodic peak currents of DA and UA was explored A. The re2O3/GO/GCE. CVs at different scanning rates are plotted in v1/2), suggesting the electrooxidation of DA is a diffusion-limited process. The linear regression equations are Ipa(DA) = 13.6968 v1/2 −0.5756 and Ipc(DA) = −8.7663 v1/2 + 0.0129, with the correlation coefficient (R2) of 0.985. The anodic peak currents of UA are in positive proportion to the square root of scanning rate (v1/2), indicating the oxidation of UA limited by the diffusion in bulk solution. The linear regression equation is Ipa(UA) = 11.9383 v1/2 − 0.7370 with the correlation coefficient (R2) of 0.999 (−) coupled with protons (H+) process, a two electron and two proton  mechanism was proposed for the oxidation of DA and UA  mixture solution were recorded at various scanning rates using d-Feof 0.999 C. Since A and UA .2O3/GO/GCE, DPVs were performed in the potential range from 0–0.8 V in 0.1 M PBS (pH 5.65). In this case, only the concentrations of the target species were changed, while the concentrations of the other species remained unaltered. As plotted in CDA) at higher concentration domain (4–100 μM). For individual detection of UA, the response anodic peak currents of UA are in proportion to the all working concentrations from 0.1 to 100 μM (2O3/GO/GCE in the DA and UA mixture (specific concentrations (CDA/CUA) range of 0.01–100).For the individual detection of DA and UA on the d-Feo 100 μM . The lim2O3/GO also showed simultaneous detection of DA and UA using DPV technique in 0.1 M PBS (pH 5.65)  = 0.1062CDA (μM) + 0.0122 (R2 = 0.975) and IDA(μM) = 0.0186CDA (μM) + 0.7835 (R2 = 0.985). The linear regression equations for UA are IUA(μM) = 0.0862CUA (μM) + 0.0036 and IUA(μM) = 0.0320CDA(μM) + 0.4804, with correlation coefficient of 0.995 and 0.990, respectively. The LODs are calculated as 3.2 and 2.5 nM for DA and UA, respectively. All results demonstrate that the proposed d-Fe2O3/GO/GCEs feature wider linear response ranges and lower LOD for the electrochemical oxidation of DA and UA. Hence, the simultaneous detection of DA and UA can be realized on d-Fe2O3/GO/GCE with high sensitivity and good selectivity. The sensing performances are compared to those in previous reports  . Two welectively B–E. The  reports . Clearly R2 = 0.95 and IDA2O3/GO/GCE toward the DA and UA, the DPV responses of the DA and UA in the presence of potential interfering species were recorded. Negligible interference with accepted relative error (less than 5.70%) even in the presence of 100-fold ascorbic acid, citric acid, alanine, glutamic acid, and lysine  for the anodic peak current are 4.56% and 3.03%, respectively, indicating that the electrode fabrication has high reproducibility. To evaluate the repeatability, seven successive measurements for detection of 1 μM DA and UA (1:1) were carried out. The RSD for DA and UA are 5.07% and 4.81%, respectively, suggesting good repeatability.Prior to the simultaneous determination of DA and UA in actual samples, the selectivity, repeatability, stability and reproducibility were also investigated. In order to assess the anti-interference of the proposed d-Fed lysine . To asse2O3/GO/GCE. Since DA and UA always coexist in most biological fluids, the practicability of d-Fe2O3/GO/GCE in actual sample detection was assessed with human urine and serum samples. The determined results listed in To verify applicability, the concentrations of DA and UA in actuate samples were also detected on d-Fe2O3/GO morphologies on electrochemical sensing performances was studied systematically. Cubic, thorhombic and discal Fe2O3 NPs with a uniform size and controllable structure were successfully prepared by a facile meta-ion mediated hydrothermal route. When coupled with GO nanosheets and then worked as sensing films for the simultaneous detection of DA and UA, the α-Fe2O3 NPs with discal morphology displayed the highest electrocatalytic activity. This remarkable electrocatalytic behavior was highly correlated with the discal shapes with more surface defects and a rougher surface. The synergistic effect from d-Fe2O3 NPs and GO nanosheets contributed to a significant enhancement in the response currents. As a consequence, two wide detection ranges (0.02–10 μM and 10–100 μM) were obtained for DA and UA, with very low limit of detection (LOD) of 3.2 and 2.5 nM for DA and UA, respectively. Moreover, the d-Fe2O3/GO nanohybrids showed good selectivity and reproducibility. The proposed d-Fe2O3/GO nanohybrids have become one of the most competitive candidates as sensing materials for the simultaneous determination of DA and UA in various real samples.In summary, the influence of Fe


The transforming growth factor-β (TGFβ) family factors induce pleiotropic effects and are involved in the regulation of most normal and pathological cellular processes. The activity of different branches of the TGFβ family signaling pathways and their interplay with other signaling pathways govern the fine regulation of the self-renewal, differentiation onset and specialization of pluripotent stem cells in various cell derivatives. TGFβ family signaling pathways play a pivotal role in balancing basic cellular processes in pluripotent stem cells and their derivatives, although disturbances in their genome integrity induce the rearrangements of signaling pathways and lead to functional impairments and malignant transformation into cancer stem cells. Therefore, the identification of critical nodes and targets in the regulatory cascades of TGFβ family factors and other signaling pathways, and analysis of the rearrangements of the signal regulatory network during stem cell state transitions and interconversions, are key issues for understanding the fundamental mechanisms of both stem cell biology and cancer initiation and progression, as well as for clinical applications. This review summarizes recent advances in our understanding of TGFβ family functions in naїve and primed pluripotent stem cells and discusses how these pathways are involved in perturbations in the signaling network of malignant teratocarcinoma stem cells with impaired differentiation potential. The transforming growth factor-β (TGFβ) family factors are involved in the regulation of most normal and pathological cellular processes from early development, morphogenesis and histogenesis to various metabolic diseases and cancers. More than 40 factors of the TGFβ family support a dynamic equilibrium of processes during the normal functioning of various cells. TGFβ family factors can activate several signaling pathways that induce pleiotropic and even antagonistic effects when they interact with other signaling pathways ,2.Maintaining a balance between proliferation, differentiation, and cell death in stem cells is the basis of self-renewing tissue homeostasis. The activity of different branches of TGFβ family signaling pathways and their interactions with other signaling pathways and intracellular targets govern the fine regulation of the self-renewal, differentiation onset, and step-by-step specialization processes of stem cell descendants in various cell derivatives ,4. HowevPluripotent and teratocarcinoma stem cells are well characterized and stable cell lines that represent unique models for studying the initiation, progression, and development of cancer. These paired models of normal and cancerous stem cells allow us to uncover the normal and transformed cell lineages and understand the mechanisms that regulate the signaling network during each step of multi-lineage differentiation and tumor progression .Identifying critical nodes in the regulatory cascades initiated by TGFβ family factors and those that are shared by TGFβ family factors and other signaling pathways, as well as analyzing the rearrangements of the regulatory network during stem cell state changes, are key issues for understanding the fundamental mechanisms of both stem cell biology and cancer initiation and progression. This review summarizes recent advances in our understanding of the biological roles of TGFβ family factors in pluripotent stem cells and discusses how TGFβ family signaling pathways are involved in perturbations in the regulatory network of teratocarcinoma stem cells, which are the malignant counterparts of pluripotent stem cells.TGFβ family factors , and growth differentiation factors (GDF)) act through type I and II transmembrane serine-threonine receptors, which are associated with the kinases of the Activin receptor-like kinases (ALKs)  Figure . There aThere are two main canonical TGFβ family signaling pathway branches: TGFβ/ActivinA/Nodal/Smad2/3 and BMP/GDF/Smad1/5/8, which activate different target genes and often have opposite functional roles . Non-canTGFβ family signaling pathways are modulated by various agonists and antagonists at different cellular levels . ExtraceAt the same time, BMPER-2 can act as both an activator and inhibitor of BMP signaling, depending on its interactions with extracellular matrix proteins ,36,37. MSeveral TGFβ family members can modulate the activity of the different branches of TGFb family signaling pathways. Lefty 1 and 2 antagonize Nodal, GDF-1, and GDF-3, but not Activin or TGFβ signals, by preventing receptor interactions, and GDF-3 and BMP-3 inhibit the signaling of BMP and Activin but not Nodal . Importaink4B by reducing MYC-MIZ-1 interaction in its promoter, thereby enhancing anti-proliferative effects. In addition, TGFβ indirectly inhibits the expression of E-cadherin and Inhibitor of differentiation-2 (ID2) by inducing the expression of their repressors: a zinc-finger protein SIP-1 and activating transcriptional factor 3 (ATF3), respectively [TGFβ factors also contribute to indirect regulation of the expression of a number of master transcriptional factors. Thus, TGFβ directly and indirectly regulates C-myc expression and function through transcriptional repression by Smad3 and through derepression of cyclin D-dependent kinases (CDK), and p15TGFβ family signaling pathways are integrated in the cell regulatory network through synergistic and antagonistic interactions with other signaling pathways. The cross-talk between TGFβ family signaling branches and the MEK/ERK, PI3K/AKT, WNT/GSK, JAK/STAT, and NF-κB signaling pathways define the ultimate cell responses in cell-specific context ,19.ink4b, p21Cip1, and Id1-3, are also components and direct and indirect targets of TGFβ/BMP/Smads signaling [The interplay between the TGFβ family and different MAPK pathways  includes the cooperation of both canonical and non-canonical cascades. MAPK pathways are activated through receptor tyrosine kinases (RTK) and a small membrane-bound GTPase, RAS, which transduces the signal to serine-threonine kinase, RAF, and the extracellular signal-regulated kinases ERK1/2 . The ERKignaling ,43,44,45ignaling . ERK1/2,ignaling ,48,49,50Cip1 expression [The PI3K/Akt and TGFβ family signaling pathways can antagonize each other to support the balance of cell growth, death and differentiation, but can also cooperate to promote cell survival. The PI3K/Akt signaling pathway, which is also activated through the RTK/RAS cascade, can promote cell growth and survival or facilitate TGFβ-induced cell cycle arrest and apoptosis, depending on physiological context. Akt can physically interact with Smad3, but not Smad2, and prevent TGFβ/Smad3-induced apoptosis ,52,53. Tpression . In contpression ,57,58. Tpression ,60.Msx1, Msx2, Emx2, and gastrin [The TGFβ/BMP and WNT/β-catenin signaling pathways’ crosstalk in the nucleus or cytoplasm is also essential for the regulation of the balance of cellular proliferation, differentiation, death and migration. The Smad-β-catenin-Lef1/Tcf protein complex, which forms in the nucleus during the interaction of the TGFβ/BMP and WNT cascades, is able to synergistically regulate the expression of target genes, such as  gastrin ,62,63. T gastrin , and WNT gastrin . In addi gastrin , whereas gastrin . The can gastrin . Further gastrin ,70. Dire gastrin . Through gastrin .The crosstalk between the TGFβ/BMP and Notch signaling pathways varies depending on the cell context and the activity of other signaling pathways . The TGFTGFβ can activate NF-kB signaling, which also can mediate the transcription of both TGFβ and NF-kB target genes ,84. ActiTGFβ family factors induce diverse cellular responses that depend on the cell type and physiological status. These context-dependent effects are governed by the complex multi-level regulation of TGFβ family signaling pathway components and interactions with other signaling pathways. Therefore, the outcomes of TGFβ family signaling-based regulation of proliferation, apoptosis, differentiation and migration vary significantly in different cells .ink4b and p21Cip1, as well as repressing the growth-stimulating transcription factors C-myc and Id1-3 [Inhibition of the cell growth in response to TGFβ in various cell types is associated with Smad3-mediated mechanisms that activate the expression of the CDK inhibitors, p15nd Id1-3 . An addind Id1-3 . On the nd Id1-3 . Howevernd Id1-3 ,19,92.Bcl-2 and Bcl-X1 or Bad and Bax [TGFβ family factors affect cell death and survival by modulating the expression of both anti-apoptotic and pro-apoptotic genes, such as  and Bax ,96,97,98 and Bax , DAP kin and Bax , growth  and Bax , connect and Bax  and prog and Bax .Goosecoid (Gsc) and Mix-like homeodomain protein 1 (Mixl1) [TGFβ family factors are widely involved in the regulation of differentiation and morphogenesis during development and histogenesis. TGFβ family factors are able to initiate or inhibit the differentiation of certain cell types by regulating the expression of lineage-specific genes. Different branches of the TGFβ family signaling pathways may also have various effects in the same cell type during differentiation ,103. Mor (Mixl1) ,110,111. (Mixl1) ,112,113.TGFβ signaling plays a key role in the epithelial-mesenchymal transition (EMT) during development and cancer metastasis . TGFβ caDeregulation of TGFβ/BMP signaling pathways during the progression of various types of cancer, resulting from genetic and epigenetic changes in the genes of the components of TGFβ family signaling pathways or their repressors, leads to changes in the intensity and duration of signals and to consistent reorganization of the cell regulatory signaling network. These changes are associated with decreased anti-proliferative and pro-differentiation responses to TGFβ/BMP signals in cancer cells ,127,128.Pluripotent stem cells—embryonic stem (ESCs), embryonic germ (EGCs), epiblast stem (mEpiSCs) and induced pluripotent stem cells (iPSCs)—can be maintained indefinitely in a metastable and dynamic undifferentiated state in vitro by modulating the interplay of different signaling pathways. Undifferentiated pluripotent stem cells can exist in naїve and primed states, which represent the pre- and post-implantation epiblast developmental stages, respectively . HoweverThe self-renewal of naїve pluripotent stem cells, represented with mouse ESCs (mESCs) as the in vitro prototype, requires the activity of the Leukemia inhibiting factor (LIF)/Jak/Stat3 signaling pathway in cooperation with the TGFβ/BMP and PI3K/Akt signaling pathways ,151,152,Nanog expression [For both naїve and primed pluripotent stem cells, the MEK/ERK signaling pathways are involve in supporting the proliferation of differentiating cell descendants, but not in the self-renewal of pluripotent stem cells. Inhibition of MEK1/2 and ERK1/2 by chemical inhibitors enhances the proliferation and prevents differentiation of ESCs ,162,163.pression . In addipression , and it pression ,166.In both phases of pluripotency, the threshold levels of activity of the serine/threonine protein kinase Gsk3β, implicated in the PI3K/Akt, MEK/ERK and Wnt/β-catenin signaling pathways, result in pleiotropic biological effects. In naїve mESCs, PI3K/Akt suppresses Gsk3β, which antagonizes self-renewal by targeting Myc and Nanog; in primed hESCs, Gsk3β is also involved in suppression of canonical Wnt signaling ,168. WntTGFβ family signaling is critical for supporting the identity and self-renewal of both naїve and primed pluripotent stem cells, while the contribution of the underlying mechanisms of the ActivinA/Nodal and BMP signaling branches might differ. Noteworthy is that, in both naїve and primed pluripotent stem cells, ActivinA/Nodal/Smad2/3 signaling activates the expression of the key pluripotency transcriptional factor Nanog ,171. AddThe activity of the BMP/Smad1/5/8 signaling branch contributes to the self-renewal of naїve mESCs by inducing the expression of Id1 and Id3, which inhibit the expression of the transcription factors MyoD and NeuroD and differentiation into the mesodermal and neuronal lineages, respectively . Smad1 wLefty1 in the mESCs and for TGFβ1 and BMP4 in hESCs; additionally, NODAL, TGFβ1, BMP4, and GDF3 expression was higher, while ACTIVINA and LEFTY1 expression was lower, in hESCs than in mESCs. However, with the onset of spontaneous differentiation, the expression profiles of the TGFβ family factors in hESCs and mESCs became similar: high levels of BMP4 and TGFβ1 and significantly decreased expression levels of ACTIVINA/ActivinA, NODAL/Nodal, LEFTY1/Lefty1, and GDF3/Gdf3. Therefore, the pro-differentiation effects of BMP signaling in hESCs can be governed by significantly higher endogenous BMP4 expression compared to mESCs. Given that plenty of potential targets are activated by the signaling pathways of TGFβ family members, the time-dependent concentrations of each factor and interactions between their targets contribute to the final cellular status/fate.Furthermore, differences in the regulation of TGFβ family signaling pathways in naїve and primed pluripotent stem cells may be associated with the endogenous expression levels of these factors. A comparative quantitative analysis of the endogenous gene expression of TGFβ family factors identified significant differences between the naїve and primed pluripotent stem cells grown in serum-free medium . Thus, tDuring early development pluripotent cells transit from the naїve to primed phase and then move toward the exit from the pluripotent state and toward the onset of differentiation. The natural fluctuations in the expression levels of key transcription factors at the single-cell level can induce rearrangement of the entire signal-regulatory network and trigger a transition to the next state ,185,186.Klf4, Gbx2, c-Myc, and Tcfp2l1 [Fgf4 and Erk2, in mESCs leads to significant disturbances in differentiation and retention of pluripotency factor expression [Suppression of MEK and GSK3β with chemical inhibitors , together with LIF supplementation (2iL), stabilize naїve pluripotency and block differentiation stimuli in mESCs ,163. LIF Tcfp2l1 ,187,188. Tcfp2l1 . Therefopression . The effpression . The cripression ,192. In pression ,184,193.Klf4, Klf2, and Nanog [OCT4, SOX2, NANOG, KLF4, and KLF2 [When modeling interconversions of pluripotency phases in vitro, naїve mESCs transit to primed mEpiSCs after changing conventional culture conditions by supplementing the media with Activin A and bFGF . At the nd Nanog ,195,196 and KLF2 ,198,199 and KLF2 ,139,140.and KLF2 ,204,205.and KLF2 ,202,203.Noteworthy is that, when reprogramming the somatic cells of different mammals into iPSCs, the resultant cultures are stabilized in those phases of pluripotency that are characteristic of their ESCs . Thus, mThus, the stabilization of the naїve state requires the weakening of TGFβ family signaling pathways to balance pro-differentiation stimuli, although their activity is necessary to maintain the expression of core pluripotency transcriptional factors. The reverse transition from the primed to the naїve phase of pluripotency occurs under conditions where the MEK/ERK signaling pathway and GSK3β activity are weakened, LIF/STAT3/KLF2/4 signaling is enhanced, and the cells remain partially dependent on bFGF and Activin A/TGFβ.The gradually increasing heterogeneity of the expression of internal and external regulators during the transition from the naїve to the primed state of pluripotency and to the subsequent onset of differentiation is a necessary condition for the irreversibility of differentiation and development. Pluripotent stem cells are capable of initiating multilineage differentiation into somatic and germ cells, as well as extraembryonic tissues, in vivo and in vitro due to modulation of the spatiotemporal expression patterns of the TGFβ family factors. TGFβ family factors contribute to the maintenance of identity and self-renewal of undifferentiated pluripotent stem cells, but they become lineage-promoting drivers when the cells exit from the pluripotent state and enter differentiation .Balancing self-renewal and differentiation in pluripotent stem cells, TGFβ family signaling pathways regulate cell cycle progression and initiate cell proliferation arrest. Cell cycle regulators, including cyclin D and CDK4 and CDK6, can directly affect Nodal/Activin/Smad2/3 signaling through the regulation of site-specific phosphorylation and nuclear translocation of the Smads2/3 ,217. MorThe Nodal/Activin/Smad2/3 signaling branch is critical for the activation of Nanog expression, and therefore, for the self-renewal and supporting of both naїve and primed pluripotency ,171,218.Gsc and Mixl1. The Smad2/3-TRIM33-H3K9me3 complex displaces the chromatin-compacting factor HP1γ and thereby makes Activin/Nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment [During early differentiation, Smad 2/3 cooperate with new dominant transcriptional partners and activate the expression of quiescent lineage-specific master regulators that previously were in a so-called “poised” state ,172,223.ruitment ,223.The multilevel interactions between the TGFβ signaling branches and PI3K/Akt, MEK/ERK and WNT/GSK3β/β-catenin signaling cascades play an important role in the formation of heterogeneity in pluripotent stem cell populations entering differentiation. Reduced PI3K/Akt activity weakens the suppression of the MEK/ERK and Wnt signaling pathways and redirects ActivinA/Smad2/3 signaling from a pro-self-renewal to a pro-differentiation function ,168. In c-Ki-ras-2, N-ras, and N-myc oncogenes [The malignant counterparts of the pluripotent stem cells—the embryonal carcinoma (teratocarcinoma) cell lines (ECCs)—have a germ cell /embryonic stem cell origin because they were isolated from mouse and human gonadal or extragonadal teratocarcinomas ,237,238.ncogenes ,243,244.ncogenes ,251,252.ncogenes .POU5F1, NANOG, LDB2, GABRB3, FGF4, FGF13, DNMT3B, LDB2, and CD9 genes and decreased levels of lineage marker genes in hECCs, compared to the hESCs [NANOG, OCT4/POU5F1, GAL, DPPA4, NALP7, etc.). At the same time, the analysis of normal hESCs and genetically altered (“adapted”) hESCs with genetic disorders did not reveal significant differences in their transcriptional profiles [The ECC lines share many characteristics with pluripotent stem cells  maintained in vitro: expression of core transcriptional factors  and cell surface antigens , and cell cycle similarity ,254. Howhe hESCs . The genhe hESCs . Quantithe hESCs . In addihe hESCs ,259. Comhe hESCs ,261,262.profiles .Comparative studies of mouse and human ECC lines have revealed variable characteristics and differences from normal and “adapted” pluripotent stem cells; however, how ECC states correspond to the naїve and primed states of normal pluripotent stem cells remains unclear. Given their germ cell/embryonic stem cell origin, ECC lines can be considered malignant counterparts of pluripotent stem cells, which partially or completely lose their ability to differentiate into embryonic somatic and germ cell lineages, although they retain proliferative potential comparable to that of normal cells. The imbalance of the proliferative and differentiation potentials in ECC lines is a result of the selection of genetically altered cells with high viability and growth rate and restricted differentiation capacities during cancer progression.The deregulation of pro-mitogenic and pro-differentiation signal balance and intrinsic transcriptional networks impairs ECC differentiation . The selInterestingly, unlike mESCs, F9 and P19 mECCs are not able to restore the naїve state when monolayer cultures are maintained in the naїve state-promoting culture conditions with 2iL. The ECC clusters in both lines became multilayer, like the colonies of mESC and mEGC, and the alkaline phosphatase activity significantly increased in the treated ECCs compared to the control cultures. However, no significant differences in the growth rates, cell distributions among cell cycle stages or gene expression were identified .ActivinA in F9 and P19 mECCs than in mESCs and mEGCs, whereas the expression levels of Nodal, Lefty1, TGFβ1, BMP4, and GDF3 were similar. In nullipotent PA-1 hECCs, ACTIVINA, NODAL, LEFTY1, BMP4, and GDF3, but not TGFβ1, were expressed at significantly lower levels than in ESM01 hESCs [The activity of TGFβ family signaling pathways varies in different ECC lines, but these pathways are critical for supporting cellular identity and initiating differentiation . The gen01 hESCs . Moreove01 hESCs ,280. Add01 hESCs . In cont01 hESCs ,282,283.The BMP/Smad1/5/8 signaling branch contributes to the self-renewal and viability of PA-1 hECCs via stimulation of Id1/Id3 expression, whose inhibition by a peptide aptamer, Id1/3-PA7, induces apoptosis and cell-cycle arrest through increased expression level of cyclin-dependent kinase inhibitor the CDKN2A . SimilarActivinA and Nanog expression in ECCs and ESCs, a positive correlation was found between the expression levels of ActivinA and Nanog, but not Oct4, in both types of cell lines, indicating that similar mechanisms regulate Nanog expression via the Activin/Smad2/3 signaling branch [Gata-6, Gata-4, Hnf1, and LamininB1 [Nanog expression, enhanced expression of Gata-4, Pax6, and Bry was detected [BRY expression, and its expression decreased slightly after RA-stimulated neuronal differentiation but without differentiation into the mesodermal derivatives [Despite significant differences between the levels of g branch ,278. NanamininB1 ,288. In detected . Similarivatives . TherefoKi-ras, N-ras, and N-myc oncogenes and tumor suppressors that lead to changes in their expression levels play a pivotal role in the formation of an imbalance between the self-renewal and differentiation of ECCs [c-Ki-ras2 gene amplification [N-ras mutation can also lead to a significant reorganization of the signaling network by reducing the endogenous expression of ACTIVINA, NODAL, LEFTY1, BMP4, and GDF3 and the activity of the corresponding signaling pathways [Mutations in the  of ECCs ,243,244.fication , sustainpathways ,278,280.Most ECC lines are not capable of spontaneous differentiation in monolayer cultures, but can be induced to differentiate with RA, hexamethylene bisacetamide (HMBA) and dimethyl sulfoxide (DMSO) or culturing in spheroids—embryoid bodies ,290,291.Thus, the stabilized signal-regulatory network configuration for each ECC line is based on the altered functions of mutant components and has a characteristic weak response to pro-differentiation signals, particularly TGFβ family factors .When compared with pluripotent stem cells, undifferentiated ECCs exist in the intermediate states that are not consistent with both pluripotency states: their self-renewal does not depend on either the LIF/STAT3 or ActivinA /Nodal pathways, but relies instead on both the PI3K/Akt and MAPK signaling pathways. Notably, the culture systems for the maintenance of both mouse and human ECCs are similar, unlike mouse and human ESCs. In addition, BMP/Smad1/5/8/Id1/3 can regulate cell cycle progression, apoptosis and early differentiation events similar to both naїve and primed pluripotent cells. Therefore, these fundamental differences between the gene regulatory networks of pluripotent and teratocarcinoma stem cells determine the different regulatory pathways for the onset of differentiation.Pluripotent stem cells and ECCs transplanted ectopically into adult animal tissue initiate rapidly growing tumors—teratomas and teratocarcinomas. ECCs’ limited differentiation and tumorigenicity are caused by their imbalanced signaling pathways. Although most ECC lines are only capable of partial differentiation, some ECC lines and sublines can completely differentiate in the course of long-term stimulation in vitro, and their descendants lose tumorigenicity after transplantation into recipients. Paradoxically, genetically normal pluripotent stem cells can give rise to teratomas that consist of the cell derivatives of different differentiation degrees. Teratoma growth initiation is also associated with the residual undifferentiated cells among in vitro differentiated pluripotent stem cell descendants. Why the residual undifferentiated ESCs retain among differentiated cells remains unclear but is a great challenge for the development of safe stem cell-based therapies.The tumorigenicity of pluripotent stem cells is a consequence of the deregulation of the differentiation in ectopic tissue sites and autonomous regulation of cellular processes in the grafts. Moreover, the degree of cell differentiation in grafts that occurs after the transplantation of pluripotent stem cells is associated with chromosomal and gene abnormalities, as occurs in genetically abnormal teratocarcinoma cells ,308,309.The differentiation patterns of ESCs and ECCs in the grafts are very similar to those of their embryoid bodies during spontaneous in vitro differentiation . NullipoA detailed comparative study of RA-induced differentiation of mESCs and F9 mECCs revealed differences in the expression dynamics of TGFβ family factors and the loss of tumorigenicity in F9 mECC descendants after 10 days of RA exposure ,315. In The TGFβ family members are highly implicated in all the ontogeny steps of pluripotent stem cells, maintaining pluripotent stem cell identity in naїve and primed states, balancing between self-renewal and cell fate signals, initiating differentiation, and specializing lineage derivatives. A significant advance in understanding the TGFβ family signaling-based regulation of these basic cellular processes was achieved using in vitro cell models of pluripotent stem cells, which were obtained from early embryos and by reprogramming somatic cells, as well as ECC lines that were previously isolated from germ cell tumors. Moreover, normal and adapted pluripotent stem and ECC lines are the only well-standardized models for studying the role of TGFβ family signaling in various aspects of cancer stem cell biology: the mechanisms of cancer transformation, cancer evolution and progression, restructuring of signaling pathways and gene regulatory networks, and interactions with various signaling pathways that stabilize normal and abnormal cellular statuses.Recent studies have improved our understanding of the contribution of the ActivinA/Nodal/Lefty/Smad2/3 and BMP/Smad1/5/8 signaling branches to maintaining pluripotent stem cells in naїve and primed states and to interstate transitions, as well as in switching from self-renewal to lineage commitment. The ActivinA/Nodal/Lefty/Smad2/3 branch was shown to be involved in the regulation of self-renewal and pluripotent stem cell identity through Nanog expression stimulation, whereas the BMP/smad1/5/8 signaling branch demonstrates different contributions to the regulation of proliferation and differentiation of naїve and primed pluripotent stem cells. The final outcome of the activity of each branch is highly dependent on its interaction with the PI3K/Akt, MAPK, and Wnt/Gsk3 signaling pathways and feedback within both branches of the TGFβ family signaling pathway that provide a dynamic equilibrium in the signal network of pluripotent stem cells.In addition, TGFβ family factors can trigger differentiation in different embryonic and extraembryonic lineages due to the wide repertoire of signaling ligands and their antagonists, dose and time of exposure and interplay with other signaling pathways through common intermediate regulators. High and low level activity of Activin signaling stimulates differentiation of different lineages, into definitive endoderm and mesoderm, respectively . BMP/SmaComparative analysis of signaling pathways in ESCs/iPSCs and ECCs revealed different expression patterns of the TGFβ family factors and the activity of the corresponding signaling pathways. The most pronounced difference was found in the activity of the ActivinA/Nodal/Lefty/Smad2/3 branch, whose reduction in ECCs promotes proliferation and inhibits differentiation. Such signaling rearrangements, together with increased activity of the mutant oncogenes Ras and Myc, can lead to the capture of their proliferative status and to complete or partial blockade of differentiation. Multipotent and nullipotent ECCs exist in intermediate states relative to naїve and primed pluripotent states and their differentiation potential is defined by peculiarities in their signal-regulatory networks. Therefore, further studies of TGFβ family signaling pathways and their targets in pluripotent stem cells and ECCs are needed to dissect the functional impairments that lead to the deregulation of proliferation and differentiation in normal and cancerous pluripotent stem cells. Despite significant progress in understanding the biology of pluripotent stem and teratocarcinoma cells, the full picture of the functioning of the signaling pathways and gene regulatory networks in them is still unclear and numerous unresolved issues still warrant further investigation. This knowledge is critical for the development of safe and effective pluripotent stem cell-based technologies for regenerative medicine and new cancer therapies.