The gap between fertility outcomes and fertility ideals is notably higher in sub-Saharan Africa (SSA) than elsewhere, relating to both under- and overachievement of fertility ideals. We consider the extent to which the relationship between fertility ideals and fertility outcomes is related to educational achievement. Further, we consider if these educational differentials are the same or different in SSA, and thereby consider the extent to which increasing levels of education in SSA may decrease fertility.We use 227 Demographic and Health Surveys (DHSs) from 58 countries worldwide to look at population- level measures of the mismatch between fertility ideals and fertility outcomes. Population level measures are used to assess whether the correspondence between fertility intentions and achievements differ by level of education. We then look at the individual-level determinants of both under- and overachieving fertility intentions. Data from the most recent DHS in 54 of the original countries is used for the individual level analysis, with five countries excluded due to the most recent available survey being out of date.An average of 40% of women in SSA underachieve their stated fertility intentions compared to 26% in non-SSA countries. Furthermore, compared to other LMICs, higher levels of education are not related to better correspondence between fertility intentions and outcomes in SSA. In Middle/Western Africa countries, on average, 48% of women with secondary or higher education have fewer children than their ideal, compared to just 24% who have more children than their ideal.We argue that the phenomenon of underachieving fertility ideals  may be of particular importance for the ongoing fertility transition throughout SSA, especially as more highly educated groups do not appear to be following the patterns observed elsewhere. The misalignment of childbearing goals and childbearing outcomes has long been seen as an important area of research, but in low and middle income countries (LMICs) this research has predominantly focussed on unintended fertility i.e. women having more children than they would like . The othPrevious research has found that the mismatch between fertility outcomes and fertility ideals at an aggregate level is notably higher in sub-Saharan African (SSA) countries as compared to other LMICs . In partIn sub-Saharan Africa fertility ideals (the number of children people say they want) are particularly high, as has been noted in previous research , 9. FurtThis paper considers educational differentials in the extent to which fertility ideals are successfully translated into fertility outcomes. Specifically we compare responses to the question “If you could go back to the time you did not have any children and could choose exactly the number of children to have in your whole life, how many would that be?” and responses to questions about actual number of children ever born.One common argument is that increasing education will reduce desired fertility, thus reducing fertility. Educational differentials in fertility have long been a feature of demographic research, with Caldwell arguing that mass education was the primary driving force behind fertility transitions . A numbeIn sub-Saharan Africa while both fertility outcomes and desired family size fall with increasing education, achieved fertility appears to fall much more substantially than desired fertility. Even relatively highly educated women have high desired fertility in much of sub-Saharan Africa  . Despite their high desired family size more highly educated women do have fewer children than their less educated counterparts . In factWhat does ideal family size mean if more educated women consistently have fewer (often far fewer) children than they say is ideal? Previous research in four African countries found that neither education nor standard women’s empowerment indicators show a strong or consistent relationship with achievement of fertility ideals . HoweverThe concept of underachieving fertility ideals has been seriously neglected in the context of low and middle income countries, while the literature on this phenomenon within high income countries is relatively well developed –25. To dOur paper argues that the phenomenon of underachieving fertility ideals is a potentially important, but neglected, aspect of future fertility decline in sub-Saharan Africa. The gap between ideal and actual fertility is important in that it might signify the potential for future fertility decline or the potential for a future stall—or even rebound—in fertility. The particular tendency for highly educated women in sub-Saharan Africa to underachieve fertility intentions, given that we would expect such women to be empowered to achieve their reproductive ideals, is puzzling. It has implications for future research concerning the nature of the data that we are collecting on fertility ideals as well as well as how we choose to analyse and interpret that data.1.1Does any educational variation differ across regional LMIC groupings?Does the relationship between fertility ideals and fertility outcomes vary by educational level?2.1Does this differ across regional LMIC groupings?Are changes in fertility ideals driven more by increasing levels of education, or changes in fertility ideals within educational groups?3.1Does this relationship differ across regional LMIC groupings?What is the relationship between education and underachieving, equalling, and overachieving fertility ideals?Demographic and Health Surveys (DHSs) were used from every country with available data, with a small number of exclusions. This amounted to 227 surveys across 58 countries. For descriptive analyses we split the countries into four regions: western/middle Africa, eastern/southern Africa, Asia and North Africa, and Latin America and the Caribbean. For individual level analyses, we only used the most recently available DHS for each country in order to provide an overview of the current state of achieved fertility compared to fertility ideals . This measure uses the answers provided to the following question: “If you could go back to the time you did not have any children and could choose exactly the number of children to have in your whole life, how many would that be?” This has some important limitations. Firstly, non-numeric responses (such as “up to God”) are problematic, though the make up less than 10% of the responses in the majority of surveys. Non-numeric responses have been found to be much more common amongst those with less education and a lower level of contraceptive knowledge, though such responses are increasingly rare . We exclPrevious research has tended to use constructs such as called the wanted TFR (WTFR), the desired TFR (DTFR) and the wanted fertility rate (wTFR) to consider the effect of fertility ideals on actual fertility , 28–31. At an individual level the ideal number of children can be compared to the number of children ever born to establish if an individual has overachieved, underachieved or equalled their fertility ideals. Of course, it is only possible to do this after childbearing is completed. In line with previous research we restrict our sample to women aged over 40 for individual level analyses. It would also be possible to use the question about intending to have another child to consider whether a woman had underachieved her fertility ideals. The relevant question within the DHS is: “Now I have some questions about the future. Would you like to have (a/another) child, or would you prefer not to have any (more) children?” A possible response to this is for the woman to say that she can’t get pregnant, which would be the likely response for a woman aged 40 or over. This is not to say that her ideal number of children has been achieved. Thus, this question, which has frequently been analysed, is not as useful for analysing achievement of fertility ideals amongst those who have completed childbearing.In terms of education, for the majority of analyses we split education into four levels: none, primary, secondary and higher. However, for some surveys the sample within these educational groupings is very small. This is especially the case for older women in some sub-Saharan African countries, where it has been necessary to collapse the secondary and higher education categories. For the country level analysis the variables (TFR and AINC) were calculated separately for each educational level. For the individual level analysis we took the decision to combine secondary and higher level education to reduce the number of countries removed from our analysis due to small sample sizes. In addition we exclude European countries that were surveyed  because the levels of education are so high that a comparison is not possible for less educated individuals.We conducted a decomposition analysis to study the effects of education on overall changes in desired family size. Following the method used by Weinberger et al. , the oveWe use four geographical regions within our analysis: Western/Middle Africa, Eastern/Southern Africa, Asia/Northern Africa and the Middle East, and Latin America and the Caribbean. Initially we conducted analyses with sub-Saharan Africa as one geographical region, but a sensitivity analysis indicated that there were substantial differences between Western/Middle and Eastern/Southern Africa, which is in line with previous research .We conducted a series of two-way 4 (region) x 3 (level of education) mixed ANOVAs with repeated measures within country for the different levels of education; a separate ANOVA was conducted to look at the percentage of women who overachieved fertility ideals, the percentage who equalled fertility ideals, and the percentage who underachieved fertility ideals. We used these to test whether there were significant differences between geographic regions, and education levels, and to test whether there was a significant interaction between these two factors.There are several limitations to our analysis. Firstly, we were limited in the countries that we could include in our analysis as DHSs were not conducted in every country at every time point. Furthermore, the countries for which we have data may not be representative of the region as a whole. Coverage is particularly low in Latin America (e.g. Argentina and Mexico are missing). Some of the surveys included are not recent (e.g. the latest survey in Kazakhstan was conducted in 1999). In some surveys only women who are currently in a union are asked about their fertility preferences. Perhaps most importantly, though, aggregate analyses mask country level trends that may be substantially different to overall and regional trends. Information on the countries and survey years included in the analyses are provided in the supplementary material .Desired family size (as measured by the AINC) is higher in sub-Saharan Africa for women of every educational level, but this is particularly the case in Western/Middle Africa compared to Eastern/Southern Africa see . For womOne explanation for the high desired family sizes observed amongst highly educated women in sub-Saharan Africa is that these countries have much higher levels of overall fertility than most other LMICs. At earlier stages of the fertility transition perhaps other countries exhibited a similar pattern. Looking at earlier DHSs in non-African countries we find that the AINC for women with higher education was still very low . For couResults of the decomposition analysis are shown in The data show that African countries are not homogenous and that Middle/Western Africa is particularly pronatalist at all levels of education, even when compared to other countries with similar fertility levels. However, we have already noted that the fertility levels of more highly educated women in sub-Saharan Africa are not as high as the AINC would suggest.So far we have studied only macro patterns, which is in line with previous work looking at the mismatch between fertility ideals and achievements. However, this misses the important issue of individuals’ ability to translate their fertility preferences into reality. We would expect, a priori, that more highly educated individuals would be better equipped to translate their preferences correctly. The country level mean of overachieving, underachieving and equalling fertility preferences is summarised in From The educational gradient seen in most countries  is weak or inconsistent, which is at odds with what we would expect to see. That said, the ANOVA analysis indicated that the main effect of education was significant (p<0.001), while the interaction between education and region was not. However, the individual contrasts indicated that there was no significant difference between no education and primary education, but there was a significant difference between no education and secondary/higher education (p = 0.001). Overall, it is clear that a mismatch between fertility ideals and outcomes (including both under- and overachievers) was common across all regions and educational levels, with the highest observed mean regional proportion of women saying that their fertility outcomes equalled their ideals at only 40% for those with secondary or higher education in Asia/North Africa .It is notable that the proportion of underachievers is particularly high amongst all educational levels in Western/Middle Africa, with 48% of those with secondary or higher education underachieving compared to 24% overachieving . In EastWe conducted further two way mixed ANOVAs to compare the main effects of education and region and the interaction between education and region on the percentage of women who overachieved and underachieved their fertility ideals. In both cases the main effect of education was found to be highly significant (p<0.001). The individual contrasts in both cases indicated that there was no significant difference between no education and primary education, but there was a significant difference between no education and secondary/higher education (p<0.001). The main effect of region was also highly significant for both overachieving (p = 0.004) and underachieving (p<0.001), making it clear that a woman’s ability to overachieve or underachieve her fertility ideals is related to which region she lives in. For overachieving the interaction between education and region was also significant (p = 0.039). This provides some evidence that education does have a differential effect depending on region, which is over and above the additive effects of education and region.The lack of research in underachieving of fertility ideals  in low and middle income countries within the demographic literature is surprising, especially when this theme can clearly be seen within the literature of other subjects such as anthropology and sociology .There is large variation visible in sub-Saharan Africa both between regions and between individual countries, with particularly high desired fertility seen in countries in the Western/Middle region. Western/Middle Africa is particularly distinctive in terms of the large proportion of women who say that their ideal fertility is higher than their achieved fertility, that is underachievers; it is also distinctive for the extremely high desired family sizes (above four children) visible even amongst those with higher education, and the fact that education-specific desired family sizes have not shown substantial decline. One possible explanation is that the quality of schooling in many places within sub-Saharan Africa is relatively low and highly variable, and thus the effect it has on fertility desires may be lower than elsewhere , 33, 34.Women underachieving their fertility ideals is common amongst women of all educational groups in sub-Saharan Africa. When highly educated women are not achieving their reported fertility desires due to underachieving, then that is an interesting scenario. These results indicate, first, that fertility has been declining faster than fertility preferences, in particular for those with higher education. This result is in line with Lam’s  finding Second, in sub-Saharan Africa, while there is considerable scope for family planning programs (or other policies) to decrease unwanted births and to improve the transformation of fertility preferences into fertility outcomes for women with low levels of education, this is not the case for more highly educated women. Indeed, one might ask the question as to why more educated women in sub-Saharan Africa have relatively few children when they still report a desire for relatively large family sizes; it seems that there may even be an unmet need for children amongst more educated women in many sub-Saharan African countries. This is something which is yet to be well explored in the demographic literature. While high fertility desires are most certainly an obstacle to fertility decline, education appears to be associated with a reduction in fertility without the concomitant reduction in desires. All this lends further weight to the argument of African exceptionalism in the fertility transition. That said, within the demographic literature there are two broad explanations of why women might reach the end of their reproductive lifecourse without achieving their fertility ideals: the first of these is that women have competing life goals and it is not possible to pursue all those goals simultaneously (e.g. having many children and being in full time employment). The other broad explanation is that women “leave it too late”, which could be the result of many things including pursuing other life goals such as education. The delay in starting childbearing resulting from more education has been extensively discussed in the Western context, along with the implications for rising numbers of women who find that they have “left it too late” (see e.g. ), but thAn important methodological point to make is that the idea that women employing ex post rationalisation to report wanting the same number of births that they actually have is assumed to upwardly bias the AINC. However, given the large proportion of women who report wanting more children than they had, this bias may not have such a substantial effect as previously thought. Relatively small numbers of women report a desired family size that equals their actual family size. It may also be the case that the extent of such ex post rationalisations vary substantially depending on the cultural context as is the case with non-numeric responses .Another possibility is that the high levels of underachieving fertility ideals in sub-Saharan Africa have a more traditionally demographic explanation; it could be that high levels of infant and child mortality are behind this trend. We do not have a thorough qualitative understanding of precisely what women mean when they answer questions about their retrospective fertility ideals. It is often assumed that women are reporting their ideal number of surviving children, but it could be that they are reporting their ideal number of children ever born and thereby taking into account the mortality levels that they have experienced. What we need is a thorough qualitative understanding of how women in different contexts understand and answer these questions. A deeper qualitative understanding of the consequences of underachieving fertility ideals in low and middle income countries is also needed. As Casterline and Han point out, the goal of reducing unintended fertility  is seen as advancing individual and national agendas, whereas to reduce underachieving of fertility ideals will merely advance individual goals . DespiteS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


The first description of Hippo signaling in mammals a little more than 10 years ago showed a striking phenotype in the liver, linking the role of this signaling pathway to organ size control and carcinogenesis. Even though Hippo signaling has been extensively studied in the liver and other organs over the recent years, many open questions remain in our understanding of its role in hepatic physiology and disease. The functions of Hippo signaling extend well beyond cancer and organ size determination: components of upstream Hippo signaling and the downstream effectors YAP and TAZ are involved in a multitude of cell and non-cell autonomous functions including cell proliferation, survival, development, differentiation, metabolism, and cross-talk with the immune system. Moreover, regulation and biological functions of Hippo signaling are often organ or even cell type specific – making its role even more complex. Here, we give a concise overview of the role of Hippo signaling in the liver with a focus on cell-type specific functions. We outline open questions and future research directions that will help to improve our understanding of this important pathway in liver disease. Drosophila and mammals, the importance of Hippo signaling in the liver became evident with a striking phenotype: overexpression of YAP or expression of activated YAP resulted in dramatic overgrowth of the liver, identifying Hippo signaling as an important determinant in organ size control , that have been shown to influence regeneration and fibrosis through angiocrine signaling to stellate cells in acute and chronic liver damage . Co-cultWith the emerging importance of Hippo signaling in HSC, LSECs, and possibly endothelial cells in fibrosis and injury, any considerations to therapeutically target this pathway should take into account that liver regeneration will likely be impaired – possibly exacerbating liver injury in the long run.In any tissue, cell proliferation and growth are dependent on nutrient availability. In this context, regulation of pro-proliferative YAP and TAZ by cellular metabolism is likely. Indeed, low energy levels trigger inhibition of YAP and TAZ, while high levels of glucose and fatty acids activate downstream YAP/TAZ transcription. On the other hand, YAP/TAZ themselves have been shown to promote key metabolic processes such as glycolysis to ensure an adequate energy supply in proliferating cells . In the Timp1, and Col1a1 (Sav1 in NAFLD-associated tumors, but not those that developed in viral hepatitis (In murine models of NAFLD and in samples of human steatohepatitis an increase of YAP/TAZ levels can be observed – mainly in regenerative ductular reactions. In mice, viral expression of activated TAZ promotes inflammation in the liver, NAFLD, and tumor formation, linking TAZ – but not YAP – to an inflammatory signature in tumor development . Mechanid Col1a1 . Interesepatitis . One mecepatitis . To dateCirrhosis and chronic inflammation in the liver are leading risk factors for liver cancer . While YGiven the increase in liver stiffness that occurs in cirrhosis, mechanical inputs are key candidates for oncogenic YAP activation that contributes to fibrosis progression and liver cancer. Mechanistically, several membrane-associated proteins such as Nf2/Merlin, scaffolding proteins of the angiomotin family, and WWC proteins have been shown to positively and – for certain members of the angiomotin family – negatively regulate Hippo signaling  – howeveMcp1 triggers accumulation of tumor-infiltrating macrophages that impair immune clearance of transformed hepatocytes and promote HCC development (Downstream of activated YAP/TAZ, unchecked proliferation and deregulated cell cycle control are one of the key mechanisms driving tumor development. Several studies showed that YAP/TEAD co-operate with E2F transcription factors downstream of retinoblastoma signaling to promote proliferation in cancer, including in liver cancer . While lelopment . If the elopment .in vivo – either to promote liver regeneration or to inhibit fibrosis and cancer progression – presents an unmet need in liver disease. Hepatic inactivation of MST kinases or YAP by microencapsulated siRNA as well as YAP silencing by AAV-delivered shRNA has yielded promising efficiency in murine models, but a transition into clinics is not foreseeable yet (Effective targeting of Hippo signaling able yet . Recentlable yet .Nf2-deficient livers (in vivo YAP inhibition (in vitro, their efficiency in the liver cells is not known and the toxicity associated with interference of the cytoskeleton will likely limit their in vivo use (In contrast to regeneration, inhibition of YAP/TAZ is a promising therapeutic approach in cirrhosis, steatohepatitis, or hepatocellular carcinoma. Outcomes of YAP activation in the liver are mostly dependent on its interaction with TEAD transcription factors, making YAP/TEAD complexes a promising target. Verteporfin, a substance identified in a compound screen for YAP/TEAD inhibitors, impeded HCC development in t livers . Howevert livers . Recentlhibition . In esophibition . Howeverhibition . In HCC hibition . While cvivo use . In Wnt-vivo use , while ivivo use . Again, Our knowledge about Hippo signaling in different hepatic cell types has expanded over the recent years (summarized in SM and UE drafted and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Emerging studies highlight the importance of metabolic reprogramming in T cell development and function, although how these processes are regulated remains to be fully understood. Recent advances in dissecting the roles of Sin1–mTORC2 signaling in early thymocyte development provide new insight into the dynamic interplay between immune signaling and cell metabolism, as well as the crucial role in directing thymocyte proliferation and development.T cell metabolism is shaped by immune signals (antigens and co-stimulatory factors) and diverse environmental cues, including cytokines, growth factors, hormones, and nutrients . SpecifiThe mammalian target of rapamycin (mTOR) integrates environmental cues by functioning via two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2 . Both mTin vitro and in vivo treatments with a PKM2 activator partially rescue the developmental defects of Sin1-deficient thymocytes, indicating a crucial role of PKM2 in mediating Sin1–mTORC2 signaling in thymocyte development (Figure How does Sin1–mTORC2 regulate PKM2 expression? Previous studies in non-immune cells show that insulin or IGF treatment promotes PKM2 expression by signaling through AKT, although the specific signaling mechanisms remain poorly understood . Ouyang In summary,


The human primed pluripotent state is maintained by a complex balance of several signaling pathways governing pluripotency maintenance and commitment. Here, we explore a multiparameter approach using a full factorial design and a simple well-defined culture system to assess individual and synergistic contributions of Wnt, FGF and TGFβ signaling to pluripotency and lineage specification of human induced pluripotent stem cells (hiPSC). Hierarchical clustering and quadratic models highlighted a dominant effect of Wnt signaling over FGF and TGFβ signaling, drawing hiPSCs towards mesendoderm lineages. In addition, a synergistic effect between Wnt signaling and FGF was observed to have a negative contribution to pluripotency maintenance and a positive contribution to ectoderm and mesoderm commitment. Furthermore, FGF and TGFβ signaling only contributed significantly for negative ectoderm scores, suggesting that the effect of both factors for pluripotency maintenance resides in a balance of inhibitory signals instead of proactive stimulation of hiPSC pluripotency. Overall, our dry-signaling multiparameter modeling approach can contribute to elucidate individual and synergistic inputs, providing an additional degree of comprehension of the complex regulatory mechanisms of human pluripotency and commitment. Human induced pluripotent stem cells (hiPSCs) have an incredible potential for regenerative medicine therapies, drug-screening and disease modeling ,2,3. UndHuman pluripotency is usually associated with a primed state, controlled by a complex balance between multiple signaling pathways that govern pluripotency maintenance and exit from pluripotency towards differentiation ,5,6. ThiFGF, TGFβ and Wnt signaling pathways are among the most important pathways controlling hiPSC fate ,5,6. TheWe previously used a multifactorial analysis approach that revealed a significant contribution of Wnt signaling to mESC pluripotency under physiological oxygen tensions . Here, w+ cells of healthy donors were used to validate results as described in the different sections and figure legends.In this work, the hiPSC cell line iPS-DF6-9-9T.B, purchased from WiCell Bank, was mainly used. This cell line is vector free and was derived from foreskin fibroblasts with a karyotype 46, XY. Both the hiPSC cell line F002.1A.13 provided by TCLab  that was generated using a retroviral system and the hiPSC line Gibco™  derived from CD34Maintenance of hiPSC culture was performed using an mTeSR1 medium  in 6-well tissue culture plates coated with Matrigel  and diluted 1:30 in DMEM/F12. The medium was changed daily. Human iPSC passaging was performed using an EDTA  solution diluted in PBS at a concentration of 0.5 mM. Cells were incubated for 5 min with EDTA at room temperature and flushed with culture medium. For maintenance cultures, splits from 1:3 to 1:8 were usually performed. For cell counting, a sample of 100 µL was incubated in 400 µL of Accutase for 7 min at room temperature and samples were diluted 1:2 in Trypan Blue  for counting using a hemocytometer. Culture photos were obtained using a Leica DMI 3000B microscope  and a digital camera Nikon DXM 1200 .3 full factorial design consisting of 27 culture conditions, corresponding to different concentrations of three different soluble factor activators of FGF, TGFβ and Wnt signaling , as well as three concentration levels , was performed using E6 medium  as the basal medium. FGF2  concentration levels ranged from 0, 35 ng/mL to 100 ng/mL; TGFβ1  concentration levels ranged from 0, 0.7 ng/mL to 2 ng/mL; and CHIR99021  concentration levels ranged from 0, 2 µM to 6 µM  using trypan blue. Cells were washed with PBS, centrifuged, and the cell pellets were stored at −80 °C to perform real-time PCR afterwards.A 3 to 6 µM . Three b5 cells/cm2 and maintained in pluripotency conditions with daily medium changes. When confluence reached percentages around 95%, hiPSC cardiac differentiation was induced following the Wnt signaling modulation protocol previously described by Lian et al. [Human iPSCs were seeded at a density of 1 × 10n et al. . Experim5 cells/cm2 using E8. For E6 differentiation, after overnight growth, the medium was changed to E6 as previously described by Lippmann et al. [Human iPSCs were seeded at a density of 2 × 10n et al. . For duan et al. ,38. For v/v) PFA for 20 min at room temperature. Cells were centrifuged and resuspended in 90% (v/v) cold methanol, incubated for 15 min at 4 °C. Samples were then washed 3 times using a solution of 0.5% (v/v) BSA in PBS (FB1). Primary antibody Cardiac Troponin T (CTNT) monoclonal mouse IgG antibody  or Primary antibody T/Brachyury polyclonal goat IgG antibody  were diluted in FB1 plus 0.1% (v/v) Triton (FB2) and incubated for 1 h at room temperature. Cells were then washed and the cell pellet resuspended with the secondary antibody goat anti-mouse Alexa-488  for CTNT or secondary antibody donkey anti-goat Alexa-488 for T/Brachyury , both diluted 1:1000 in FB2 and incubated for 30 min in the dark. Finally, cells were washed twice and cell pellets were resuspended in 500 µL of PBS and analyzed in a FACSCalibur flow cytometer . Data were analyzed using the software “Flowing Software” at http://www.flowingsoftware.com (version 2.5).Cells were washed with PBS, singularized and fixed using 2% (v/v) PFA for 15 min, washed with PBS and incubated with blocking solution  for 1 h. After incubation, for hiPSC-CM differentiation, Cardiac Troponin T (CTNT) monoclonal mouse IgG antibody  was diluted 1:250 in staining solution  and incubated for 2 h at room temperature. For hiPSC-Neural commitment, NESTIN monoclonal mouse IgG antibody  and PAX6 polyclonal rabbit IgG antibody  were used both diluted 1:1000 in staining solution and incubated for 2 h at room temperature. After washing with PBS, secondary antibodies goat anti-mouse IgG Alexa-546 and goat anti-rabbit IgG Alexa-488  were diluted 1:500 in staining solution and incubated for 1 h at room temperature. Samples were then washed 2 times with PBS, incubated for 2 min with 3 µg/mL of DAPI diluted in PBS, washed again 3 times, and stored at 4 °C. Samples were analyzed using a fluorescence optical microscope  and a digital camera . Images were processed using ImageJ/Fiji (http://fiji.sc) [+ cells were quantified using CellProfiler .Cells were fixed with 4% (fiji.sc)  and PAX6−ΔΔCT method. GAPDH gene expression was used as endogenous control and relative expression was calibrated for each gene using mTeSR1 gene expression values. For hiPSC-CM differentiation, relative expression was calibrated using day 0 of differentiation.RNA from each condition and controls was extracted using the High Pure RNA Isolation Kit  following the instructions provided with the Kit. RNA was quantified using a nanodrop, and 1 µg of RNA was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit  following the instructions provided with the kit. Relative gene expression was evaluated using 10 ng of cDNA, 250 µM of each primer  and usinOCT4/POU5F1 (Hs00999634_gH), NANOG (Hs02387400_g1), PAX6 (Hs00240871_m1), SOX1 (Hs01057642_s1) and GAPDH (Hs02758991_g1). GAPDH gene expression was used as endogenous control and relative expression was calibrated using day 0 of differentiation.For hiPSC-Neural commitment, real-time PCR was performed using the TaqMan Gene Expression Assay  for the genes OCT4 and NANOG), ectoderm , mesendoderm (MIXL1 and T), mesoderm (NKX2.5 and MESP1) and endoderm (SOX17 and PDX1) were created by averaging the expression value of each gene. Then, scores for pluripotency and for each lineage were empirically calculated as follows:Relative expression values were normalized using the minimum and maximum value obtained for each gene. Then, panels for pluripotency  were performed using Clustvis, a web tool based on R . ClusteriY corresponds to the specific score; β0 is the intersect coefficient; β1, β2 and β3 are the coefficients correspondent to the linear main effects; β11, β22 and β33 are the quadratic coefficients and β12, β13 and β23 are the coefficients for factor interactions. The full factorial design with three replicates of Sample 1 (E6) resulted in a total of 28 degrees of freedom. Statistical significance for each model was assessed by ANOVA using Fisher’s statistical test, in which factors with p-values lower than 0.05 were considered to have a statistically significant contribution to the model [2-adjusted (R2-Adj), a modified version of R2, is also showed for every model. R2-Adj compares the explanatory power of the regression models calculated with many prediction factors by discarding factors that do not significantly improve model prediction, and therefore helps to assess the true quality of the model.A model for each score was created using Statistica Software. Models were obtained by fitting the data to a full quadratic model  with centered and scaled polynomials, as follows:he model ,41. ModeTo expose the impact of FGF signaling, TGF/Nodal signaling and Wnt signaling in human pluripotency and exit towards differentiation, a full factorial design was conceived to detect dual signaling roles by combining three concentration levels of each signaling input: Zero, lower activation (1/3 of higher activation) and higher activation, using E6/VTN , a dry-sIn our multiparameter approach, FGF pathway was modulated using FGF2 at concentrations of 0, 35 and 100 ng/mL. Both TeSR and E8 medium use 100 ng/mL of FGF2 to maintain hiPSC pluripotency ,42. At tEach condition of the full factorial design was assessed by analyzing the overall fold increase in total cell numbers, colony morphology and by real-time PCR, allowing the attribution of scores to each condition and the assessment of the data by clustering and modeling tools B. To assTo assess the effect on cells exposed to each signaling input, real-time PCR was performed to analyze the expression of a set of genes , correspTo emphasize the main path that hiPSCs were following after exposure to the signaling cocktails, scores to each lineage commitment and pluripotency were attributed to each sample. This data was hierarchically clustered using Pearson correlation and average linkage, resulting in two main clusters mainly explained by the presence or absence of Wnt signaling activation A. Cocktap-values < 0.05. As expected, CHIR supplementation contributed negatively for pluripotency scores , which can be observed in Similarly, CHIR linear and quadratic terms also contributed significantly for the mesoderm score model A–C. In aMESP1 expression, with a peak at day 3. Furthermore, NKX2.5 expression was not significantly affected by the level of Wnt signaling stimulation, but CTNT expression was one order of magnitude higher at 6 µM when compared with 1 µM of CHIR  and 0.7 ng/mL of TGFβ (1/3 of input). These values are in concordance with the level of input provided by both commercial media E8 and TeSR used to maintain hiPSCs pluripotency ,42. In aIn the presence of Wnt signaling stimulation, TGF and FGF signaling effects were secondary, with all models showing CHIR terms as the most significant contributors. This dominance contributed negatively for pluripotency and ectoderm scores , and posPAX6 [Synergies observed between Wnt signaling and FGF signaling in our models are also coherent with previously reported data. The positive contribution for the ectoderm model is in line with the role of FGF in repressing PAX6  and, togPAX6 ,30. The PAX6 ,59,66,68PAX6 ,63,64,65In conclusion, using a multifactorial, multiparameter modeling approach we predicted a dominant role of Wnt signaling over FGF and TGF signaling in our dry-signaling culture system. This modeling methodology also allowed the construction of models providing a rational understanding of hiPSCs pluripotency and commitment, allowing to discriminate the different synergies between FGF and Wnt signaling, in agreement with previously reported studies. Following this proposed framework, carefully designed 5-level fractional factorial designs coupled with multiple signaling activation dynamics should contribute to the construction of models with increased sensitivity and reduced variance and, consequently, providing an extra degree of comprehension of the complex regulatory system of human pluripotency and commitment.


The results of the flotation and contact angle experiments showed that the addition of Ca2+, Mg2+ and Fe3+ significantly decreased both the recovery and contact angle of cassiterite with pH ranged from 6.0 to 12.0 in the presence of OHA collector. ζ-Potential measurements, solution chemistry analysis and FTIR measurements indicated that the flotation recovery of the cassiterite declined due to the CaOH+, MgOH+ and Fe(OH)3 sites on the cassiterite surface. XPS results indicated that the chemisorption of OHA and calcium ions on the cassiterite surface finally changed its chemical properties. The AFM images also revealed that new species Fe(OH)3 of Fe3+ formed and adsorbed on the cassiterite surface at pH 9.0. The adsorption of Fe(OH)3 reduced the adsorption of OHA on the cassiterite surface, thus the hydrophobicity of cassiterite was deteriorated.The existence of metal ions should not be ignored in both hydrometallurgy and flotation. In this study, the effects of Ca Cassiterite is an economically important mineral of tin in the Earth's crust . Gravity2+, Ca2+, Mg2+, Cu2+ and Al3+, commonly exist in flotation pulp due to the use of groundwater, recycled water or seawater [Froth flotation is an effective method for separating fine target minerals from gangue minerals depending on their surface hydrophobicity differences ,7. Thus,seawater . Therefoseawater –14. Calcseawater ,15.et al. [2+ effectively improved the flotation of hemimorphite. The adsorption/precipitation of the hydrolysed species of lead cations promoted sodium oleate adsorption and formed lead oleate on the surface of hemimorphite. Potapova et al. [Previous studies indicate that metal ions adsorbed on mineral surfaces can affect flotation. Liu & Zhang  found thet al.  found tha et al.  investig2+, Mg2+ and Fe3+ on cassiterite flotation when octanohydroxamic acid (OHA) is used as the collector.Alkyl or alkaryl hydroxamic acids and their salts are well-known collectors for the froth flotation of oxide minerals such as scheelite , rhodoch2+, Mg2+ and Fe3+ on the flotation of pure cassiterite with OHA as the collector was intensively investigated. The experiments were carried out in a mechanical agitation flotation machine and the effects of metal ions on the adsorption of OHA on cassiterite surfaces were fundamentally investigated via ζ-potential determination, Fourier transform infrared (FTIR) analysis, solution chemistry analysis, X-ray photoelectron spectroscopy (XPS) analysis and atomic force microscopy (AFM) imaging.In the present work, the effect of Ca2.2.1.2 was of high purity. Chemical analysis showed that the resultant powder contained 95.58% SnO2. For the micro-flotation tests, the used cassiterite samples with a particle size distribution of 20–38 µm were obtained by screening. The remaining ones were ground continuously for ζ-potential, XPS analysis and FTIR tests. In addition, 1 × 1 cm block samples used for the AFM experiments were finely polished and further cleaned by rinsing thoroughly with ethanol.The cassiterite samples were purchased from Yunnan Bao Yun Jewelry Company, China. The X-ray diffraction (XRD) result is shown in 2 was used as the source of Ca2+. H2SO4 and NaOH were used for pH adjustment of the suspension. All the chemicals were of analytical grade except frother pine oil , and distilled water was used for all experimental work.The OHA of 99% purity was purchased from Aladdin Bio-Chem Technology, China. CaCl2.2.2.2.1.Micro-flotation tests were conducted in a mechanical agitation flotation machine with a 40 ml cell. The mixture was stirred at 1650 r.p.m. by a four-bladed impeller. In each test, 2.0 g of pure cassiterite particles were dispersed in the cell with 30 ml distilled water for 1 min. The pH was adjusted by adding a pH regulator for 3 min. The metal ions were added and the pulp was conditioned for 3 min, then the collector was introduced and conditioned for 3 min, the frother was added, followed by flotation for 4 min. Finally the flotation products were filtered, dried and weighed for recovery calculation.2.2.2.ζ-Potential measurements of cassiterite were carried out using a Zetasizer Nano ZS90 . All measurements were done in a 1 mM KCl background electrolyte solution. A solid concentration of 0.05% by weight was used and conditioning procedures were similar to that used for the micro-flotation tests. The ζ-potential results were obtained using the average of three independent measurements with a typical variation of ±2 mV.2.2.3.XPS analysis was conducted using an ESCALAB250Xi  with Al Kα X-ray source. The mineral samples were taken from flotation experiments and then washed with distilled water to a similar pH to remove any suspended colloidal particles; finally, they were introduced immediately into the fore vacuum of the spectrometers as a slurry.2.2.4.The contact angles of cassiterite were measured using an instrument . The cassiterite was treated with OHA and/or metal ions; the conditioning procedures were similar to that used for the micro-flotation tests, and modified cassiterite powder was taken from flotation experiments. The samples were vacuum-dried in a desiccator, model Sotelem, at approximately 25°C and then transferred into a tablet machine to be pressed into a wafer with a diameter of 12 mm.2.2.5.−1 using 32 scans with a resolution of 4 cm−1.FTIR measurements were taken on a Nicolet 6700 . Pure cassiterite was ground to −5 µm for FTIR tests. Conditioning procedures were similar to that used for the flotation tests. After conditioning, the solid samples were washed three times using deionized water with the same pH and dried prior to FTIR analysis. Approximately 1% (mass fraction) of the solid sample was mixed with spectroscopic grade KBr. The analysis was conducted from 4000 to 500 cm2.2.6.−1 were used for surface image measurements. The AFM measurement was commenced after the cassiterite plate had made a good contact with the chemical solution for a specific time. The images of the cassiterite surface were obtained at a scan rate of 1 Hz and a scan area of 5 × 5 µm. The images were processed offline using Nanoscope v. 5.31R1 software. Flattening and low-pass filtering were applied to remove noise.Multi-mode 8  AFM was used in the contact mode for imaging of the cassiterite surface. Triangular cantilevers with a nominal spring constant of 0.12–0.58 N m3.3.1.−1 and metal ion concentration of 20 mg l−1. The maximum recovery, 77.63%, was obtained at a pH of 9.0 in the absence of metal ions. The floatability of cassiterite was significantly depressed by Ca2+, Mg2+ and Fe3+, while the pH ranged from 6 to 12.Mineral floatability is highly related to solution pH values, which influences the component of collectors and ions in the solution. −1 and metal ion concentration of 20 mg l−1. The recovery of cassiterite approached 90% at high collector dosage levels without metal ions. As can be seen, the floatability of cassiterite decreased significantly in the presence of the metal ions.With the solution pH maintained at a value of 9.0, −1 OHA dosage at a pH of 9.0. Cassiterite recovery decreased exponentially with an increase in metal ions. The recovery of cassiterite dropped from 84.42% to 27.14%, 24.98% and 17.88% with an elevation in the Ca2+, Mg2+ and Fe3+ concentrations, respectively. Therefore, the Ca2+, Mg2+ and Fe3+ cations present in the pulp significantly reduced the floatability of cassiterite, even at a pH range of 6–12.3.2.The contact angle is useful for the determination of the wetting characteristic and floatability of a solid mineral . The conThe results in 3.3.ζ-potential of cassiterite as a function of pH in the presence of metal ions, and the IEP of cassiterite alone was located at a solution pH of 3.2, which is lower compared with that of 4.2, 5.0 reported in the literature [42− on the surface of cassiterite and hence the IEP of cassiterite is influenced [2+, Mg2+ and Fe3+, the ζ-potential of the cassiterite was increased significantly and the IEP of cassiterite increased from a pH of 3.2 to 5.3, 9.1 and 7.2, respectively. The influence of Ca2+ and Mg2+ on the electrokinetic properties of cassiterite was more evident in basic solutions, while that of Fe3+ was more evident in acidic solutions. The significant increase in the IEP of cassiterite provided evidence of metal ions adsorbing/precipitating onto the negatively charged cassiterite surfaces.Electrokinetic phenomena play an important role in understanding the flotation phenomena based on the isoelectric point (IEP) of mineral particles. The surface charge and surface potential determine the particle–chemical interactions (e.g. collector adsorption) and particle–particle interactions (e.g. hetero-aggregation) . Figure terature ,24,27. Ifluenced . In the ζ-potentials of cassiterite as a function of pH in the presence or absence of both metal ions and OHA. The IEP of cassiterite decreased from a pH of 3.2 to 2.7 after OHA was added into the pulp suspension. We can attribute the decrease in the IEP values to a specific adsorption of anionic collector OHA on the cassiterite surface. Moreover, in the presence of OHA and Ca2+, Mg2+ and Fe3+, the IEP of the cassiterite was still increased significantly, which varied from a pH of 2.7 to 4.1, 3.8 and 4.2, respectively, even though the adsorption of OHA had a negative effect on the IEP. This indicated that the presence of metal ions in the solution played a more important role in determining the IEP of the cassiterite than the OHA.2+, CaOH+ and Ca(OH)2(aq) in a solution containing 20 mg l−1 (1.8 × 10−4 mol l−1) of total calcium ions were calculated and are shown in 2+ was the dominant species at the pH range of 3–12, while concentrations of CaOH+ increased with a rise of the solution pH value. At the pH range of 8–10, the ζ-potential of cassiterite in the presence of Ca2+ increased significantly compared to the cassiterite alone, which can be attributed to the adsorption of positively charged CaOH+ on the cassiterite surface. In addition, micro-flotation tests in −1 Ca2+. It is reasonable to suggest that the cassiterite surface was covered with a layer of CaOH+ attached via hydrogen bonding and electrostatic attraction [The equilibrium concentrations of Catraction .Figure 2+, MgOH+ and Mg(OH)2(aq) in a solution containing 20 mg l−1 (2.1 × 10−4 mol l−1) of total magnesium ions were calculated and are shown in 2+ existed as free ions throughout the pH range of 2.0–10.0; concentrations of MgOH+ increased significantly with the increase in pH; magnesium ions were predominantly in the form of Mg(OH)2(s) when pH > 10.27. The ζ-potential results showed that the influence of Mg2+ on the electrokinetic properties of cassiterite was more evident in basic solutions. Combined with flotation results, it was determined that hydroxy complexes (MgOH+) and precipitates (Mg(OH)2) could adsorb onto the mineral surface.The equilibrium concentrations of Mg−1 (1.2 × 10−4 mol l−1) Fe3+ was calculated, and the result is shown in 3+, Fe(OH)2+ and Fe( OH)2+ species all existed across the acidic pH range. The ζ-potential tests in 3+ changed the surface charges of cassiterite from negative to positive at an acidic pH range, which indicated that the species of Fe3+, Fe(OH)2+ and Fe(OH)2+ were expected to adsorb on the cassiterite mineral surfaces at this pH range. Iron ions were predominantly in the form of Fe(OH)2(s) and Fe(OH)4− when pH > 8. According to the theory of electrostatic attraction, a positive surface can attract a negatively charged species. The flotation of cassiterite should also be enhanced in the presence of Fe3+ [3+. It was determined that Fe(OH)3 colloids adsorbed on the cassiterite surfaces, while Fe(OH)3(s) had similar zeta potentials to that of cassiterite measured with Fe3+ alone [Similarly, the species distribution of 20 mg l of Fe3+ . However3+ alone .Figure 3.4.a shows the spectrum of OHA. The peak at 3259 cm−1 was the N–N stretch, and the peak at 3060 cm−1 was the Fermi resonance of the C–N stretch and the N–H bend. The peaks at 2954 and 2915 cm−1 were the C–H antisymmetric stretch for CH2 and CH3, and the peak at 2846 cm−1 was due to the C–H symmetric stretch for CH2/CH3. The peaks at 1666 and 1619 cm−1 were the C=O stretches. The bands at 1564 and 1468 cm−1 were related to the C–N stretch. The bands at 1421 and 1115 cm−1 were related to the N–O–H bend and C–H symmetric deformation, respectively. The peak at 1080 cm−1 resulted from the C–C–C stretch. The bands at 1031 and 972 cm−1 were attributed to the C–O stretch and the N–O stretch, respectively. Figure 11b shows the spectrum of cassiterite. The bands of cassiterite at 640 and 538 cm−1 are assigned to the antisymmetric vibration peak of Sn–O.FTIR analysis was used to evaluate the chemical changes of cassiterite surfaces before and after the adsorption of OHA. Figure 112+, Mg2+ and Fe3+ are plotted in figures figure 12a, after the adsorption of OHA, new bands at 1510 and 1618 cm−1 appeared, indicating the occurrence of chemical adsorption. OHA spectra showed that the peaks at 1666, 1619 and 1564 cm−1 represented the C=O stretch and the C–N stretch. It is reasonable to suggest that the chemical reaction which occurred between OHA and cassiterite involved the C–N and C=O groups. As can be seen from figure 12b, when Ca2+ was added to the cassiterite system, the peak at 1618 cm−1 shifted to 1626 cm−1 and the peak at 1510 cm−1 disappeared. Figure 13b shows that the peak at 1618 cm−1 shifted to 1610 cm−1 and the peak at 1510 cm−1 shifted to 1430 cm−1 in the presence of Mg2+. Figure 14b shows that both the peaks at 1618 and 1510 cm−1 disappeared in the presence of Fe3+. It could be inferred that the presence of additional species on the surface of cassiterite can be detected upon the addition of Ca2+, Mg2+ and Fe3+.FTIR spectra of cassiterite with OHA at a pH of 9.0 in the presence and absence of Ca3.5.a, no Ca signal is displayed in the XPS survey spectra of untreated cassiterite, indicating the high purity of the cassiterite samples. For the cassiterite treated with calcium ions, the Ca spectral peak is displayed in figure 15b,c, which indicates that the calcium ions interacted with the mineral surface.XPS is one of the most useful techniques for surface analysis in order to obtain chemical information . XPS anafigure 15a, there are two pairs of Sn3d doublets, with a binding energy of 486.58 eV for Sn3d5/2 and 494.98 eV for Sn3d3/2. These peaks are ascribed to the Sn species from cassiterite surfaces [figure 16b, the binding energies of Sn3d5/2 and Sn3d3/2 on a calcium ion-treated cassiterite surface were decreased by 0.1 eV and 0.2 eV, respectively. Moreover, the binding energies of Sn3d5/2 and Sn3d3/2 on the cassiterite surfaces treated by calcium ions and OHA (figure 16c) were decreased by 0.2 eV and 0.1 eV, respectively, compared to the one treated with calcium ions. These results indicate that a weak interaction occurred between the calcium ions and Sn sites on the cassiterite surface, and the Sn sites on the cassiterite surfaces exhibited low reactivity [figure 16surfaces . As showactivity ,33.Figuc, the O1s spectrum also displayed two peaks in the presence of OHA and calcium ions. The peak at 531.27 eV was assigned to the O=C oxygen atom of the OHA molecule [figure 17molecule ,36; it s3.6.3+ on cassiterite surfaces. The AFM images of OHA and Fe3+ adsorbed on cassiterite are shown in AFM is a powerful tool for surface chemistry research. It can describe the surface topography without damaging the delicate surface. In this study, AFM was used to investigate the adsorption of OHA and Fea, the fresh cassiterite surface was flat and smooth except one small bright area and another dark area. In figure 18b, many small patches appeared on the cassiterite surface after the conditioning with 45 mg l−1 OHA. It was certified that OHA directly adsorbed on the mineral surface. This agreed well with the FTIR analysis (a). When Fe3+ was added during the conditioning, it was expected that Fe3+ would adsorb on the cassiterite surface. As shown in figure 18c, the surface became rougher than that of pure cassiterite and the cassiterite surface after OHA conditioning. However, the height of figure 18c is lower than that of figure 18b. The reason for this may be the adsorption of Fe3+ on the cassiterite surface reducing the adsorption of OHA on the cassiterite surface. The results of the chemical calculation of solution in 3(s) was the dominant species at a pH of 9.0. It was concluded that the Fe(OH)3 colloids impeded the adsorption of the collector and made the cassiterite surface hydrophilic. It also indicated that the surface of cassiterite was covered with Fe(OH)3(s), and then a hydrophilic film formed which prevented the adsorption of OHA. Thus, the flotation was inhibited.As revealed in figure 18analysis a. When F4.2+, Mg2+ and Fe3+ on flotation of cassiterite was investigated in this study. Flotation results showed that the recovery of cassiterite dropped from 84.42% to 27.14%, 24.98% and 17.88% in the presence of 30 mg l−1 Ca2+, Mg2+ and Fe3+, respectively. The presence of Ca2+, Mg2+ and Fe3+ cations in the pulp significantly reduced the floatability of cassiterite at pH ranging from 6 to 12. Contact angle results indicated that the contact angle of cassiterite decreased in the presence of both OHA and metal ions compared to OHA alone at the pH range of 6–12. The decrease of the contact angle was the basis of the decrease in cassiterite recovery in flotation experiments. ζ-potential, FTIR measurements and solution chemistry analysis revealed that the addition of Ca2+, Mg2+ and Fe3+ in the flotation pulp produced some additional species on the surface of cassiterite. The new sites including CaOH+, MgOH+ and Fe(OH)3 on the cassiterite surface decreased the recovery of cassiterite. In addition, XPS results indicated that tin and oxygen atoms on the cassiterite mineral surface were changed under the combined action of calcium ions and OHA. AFM images offered direct evidence that the Fe(OH)3 colloids formed on the surface of cassiterite at a pH of 9.0 impeded the adsorption of OHA and made the cassiterite surface hydrophilic.The effect of Ca


Cationic polymers are often employed in conjugation with nanomaterials, and the resultant hybrids are useful for various bioapplications. Here, a single-step metal-free method for the synthesis of fluorescent nanodiamonds (FNDs) conjugated with cationic polymer brushes is reported. Distinct from the common methods such as atom transfer radical polymerization and reversible addition fragmentation chain transfer, our ring-opening-polymerization-based method is simple and less time consuming and hazardous. Infrared spectroscopy, thermogravimetric analysis, zeta potential, and dynamic light scattering confirmed the synthesis. The produced FND-polymer brushes showed markedly higher cell labeling and internalization efficiency without noticeable cytotoxicity. Our method is general and applicable to other nanoparticles as well for uses in diverse research areas. Since these transition metal ions are toxic to living organisms, it is better to avoid them in the synthesis. To our knowledge, the development of a facile, ideally single-step, and metal-free method for producing nanoparticles with cationic polymer brushes has not been reported so far, but is critically important for applications in the life sciences.Surface modification with cationic polymer brushes is one of the key technologies to control the properties of nanoparticles. To date, a variety of polymer brushes have been developed and used for diverse bioapplications ,2. AmongTo address this challenge, ring-opening polymerization (ROP) offers a solution . It has The nanoparticle platform used to demonstrate this method is the fluorescent nanodiamond (FND). FNDs are carbon-based nanoparticles, having stable fluorescence, low toxicity, and physicochemical inertness ,14,15. T+ ion (40-keV), annealed at 800 °C for 2 h under reduced pressure, and air-oxidized at 450 °C for 2 h, yielding FNDs. Further details are described elsewhere [2SO4:HNO3 (3:1 v/v) at 70 °C for 3 days and then washed with deionized distilled water (DDW) three times. Next, the solution of FND slurry was replaced with 0.1 M NaOH and stirred for 1 h at 90 °C. After centrifugation and wash process, the solution of FND slurry was replaced with 0.1 M HCl and stirred for 1 h at 90 °C. Finally, the FNDs were centrifuged and washed three times with DDW, yielding bare FND (FND-bare).Nanodiamond powders  containing ~100 ppm of atomically isolated nitrogen were irradiated by Helsewhere . To elimFNDs (5 mg) were dispersed in glycidyltrimethylammonium chloride (GTMA) or the mixture solution of GTMA and glycidol  at a different volume ratio of 1.5/0.5, 1.0/1.0, and 0.5/1.5 (mL/mL). Note that moisture in GTMA and glycidol was removed by rotary evaporation before use. The mixtures were then sonicated for 5 min, and stirred for 24 h at 120 °C under nitrogen atmosphere. After the reaction, the resulting gel was diluted with DDW, and centrifuged at 15,000 rpm for 5 min to collect FND-PEGTMA or FND-HPGTMA. Then they were washed four times to remove by-products and unreacted materials.FNDs (1 mg) and Polyethylenimine  were dissolved in DDW (1 mL), sonicated, and gently mixed for 1 h. Unbound polymers were removed from the FND-PEI conjugates by repeated centrifugation/wash processes with DDW.−1 with a resolution of 4 cm−1. Thermogravimetric analysis (TGA) measurements were carried out in air at a heating rate of 10 °C min−1 using a Thermal Analyzer . The average sizes and zeta potentials of FNDs were measured using a Delsa Nano C . Size distributions are represented by the particle number.Fourier transform infrared spectroscopy  spectra were obtained on a Bruker VERTEX 70 using standard KBr-pellets in the range of 2200–400 cm2 incubator. For magnetically modulated fluorescence analysis, HeLa cells were plated at a density of 5 × 105 cells per 35 mm dish in DMEM and cultured for 22–26 h in the incubator. After incubation, the culture medium was subsequently replaced with an FND-containing medium . Note that the concentrations of FNDs were determined in terms of the fluorescence intensity, namely, polymer coating was not taken into account. After incubation for 1 h, cells were washed with PBS extensively and further incubated for three hours. The cells were then trypsinized, collected, counted, and suspended in DDW. After sonication, the fluorescence intensity of FNDs were measured and analyzed by home-built MMF spectrometer [5 cells per 35 mm dish, treated with FND samples, and stained with Hoechst 33342. Fluorescence images were observed with a Leica SP8  equipped with an oil-immersion objective , a white light laser , a photomultiplier  and hybrid detector . Logical sizes and pixel sizes of X and Y dimensions were 512 × 512 and 0.361 μm × 0.361 μm, respectively, and voxel size of Z dimension is 0.5 μm.HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and 1% penicillin at 37 °C in 5% COtrometer . BrieflyDehydrogenase activity detection in viable cells was measured with the colorimetric assay cell counting kit-8 . HeLa or MCF7 cells (5000 cells in 100 μL culture medium) were seeded in 96-well plates for 22–26 h. FND samples were then added to cells and incubated for 22–26 h at 37 °C. The absorbance was measured at 450 nm by using a plate reader  one hour after the addition of CCK-8.−1 in the FTIR spectra were found to increase for both FND-PEGTMA and FND-HPGTMA, compared to that of bare FND (FND-bare) as shown in Results of the synthesis were examined by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), zeta potential, and dynamic light scattering (DLS). After the reaction, the absorption intensity of the –C–O–C– stretching modes of the polymer around 1100 cmWe next investigated the zeta potentials of the samples c. Acid-tCollectively, we have successfully developed a single-step, metal-free method for the synthesis of FNDs with cationic polymer brushes. Under the experimental conditions described herein, the GTMA/glycidol ratio of 75% gave the highest positive zeta potential value and these conjugates, indicated as FND-HPGTMA, are used in subsequent experiments. Stability of FND-HPGTMA under various ion concentration and pH condition was studied .We performed the CCK-8 assay to test the cytotoxicity of FND-HPGTMA  with HeL4 FND particles, assuming that the particles are spherical and uniform in size. In comparison with the cell labeling with protein-coated FNDs [Next, we demonstrated a markedly high cell labeling efficiency of FND-HPGTMA. For obtaining a quantitative measure, we performed magnetic modulation to realize background-free fluorescence detection of FNDs ,22. Detated FNDs , FND-HPGFinally, using confocal fluorescence microscopy, we investigated whether FND-HPGTMA is taken up by cells or anchored on the cell membrane for the treatment at two different concentrations . For theWe have successfully developed a single-step metal-free method for facile production of FNDs with cationic polymer brushes. Compared to ATRP or RAFT, our method is simple and less time-consuming and hazardous. Since the method involves only ring-opening polymerization, it is widely applicable to other nanoparticles as well ,25,26,27


Mitogen-activated protein kinases (MAPKs) include ERK, p38, and JNK MAPK subfamilies, which are crucial regulators of cellular physiology, cell pathology, and many diseases including cancers. For the MAPK signaling system in pituitary adenomas (PAs), the activation of ERK signaling is generally thought to promote cell proliferation and growth; whereas the activations of p38 and JNK signaling are generally thought to promote cell apoptosis. The role of MAPK in treatment of PAs is demonstrated through the effects of currently used medications such as somatostatin analogs such as SOM230 and OCT, dopamine agonists such as cabergoline and bromocriptine, and retinoic acid which inhibit the MAPK pathway. Further, there are potential novel therapies based on putative molecular targets of the MAPK pathway, including 18beta-glycyrrhetinic acid (GA), dopamine-somatostatin chimeric compound (BIM-23A760), ursolic acid (UA), fulvestrant, Raf kinase inhibitory protein (RKIP), epidermal growth factor pathway substrate number 8 (Eps8), transmembrane protein with EGF-like and two follistatin-like domains (TMEFF2), cold inducible RNA-binding protein (CIRP), miR-16, and mammaliansterile-20-like kinase (MST4). The combined use of ERK inhibitor  plus p38 activator  and/or JNK activator , or the development of single drug  to target both ERK and p38 or JNK pathways, might produce better anti-tumor effects on PAs. This article reviews the advances in understanding the role of MAPK signaling in pituitary tumorigenesis, and the MAPK pathway-based potential therapeutic drugs for PAs. Pituitary adenomas (PAs) are commonly benign tumors, accounting for about ten percent of intracranial tumors , 2, and The MAPK signaling pathways play important roles in cell dissemination, survival, and drug resistance of human cancers including PAs , 10–12. The ERK/MAPK pathway delivers signals from cellular surface receptors via ERK pathway. Briefly, different cellular surface receptors such as EGFR, GPCR, and RKT are activated by the corresponding extracellular factors  to activate Ras and small GTPase. The activated Ras-small GTPase complex recruits Raf kinase to the cell membrane and activates it. Then, Raf activates MEK (MAPK and ERK kinase) through phosphorylation. The phosphorylated MEK subsequently activates ERK through phosphorylation  Figure . Rafs inin vitro  In lactotroph cells, the ERK signaling exerts different effect on cell proliferation based on the exposure time. Short-time activation of the ERK (24–96 h) leads to increased proliferation in rat pituitary lactotroph or somatolactotroph cell lines in vitro , 43. Howin vitro . Thus, pin vitro . ERK patin vitro . In addiin vitro . Somatosin vitro . (iii) Iin vitro . Most ofin vitro , 49. (ivin vitro . And (v)in vitro .in vitro  can inhibit the release of GH, prolactin (PRL), and their α-subunit in GH-secreting PAs, PRL-secreting PAs, ACTH-secreting PAs, and NFPAs, respectively –18. Theiin vitro –59. The in vitro –62. Morein vitro , 64. Somin vitro .Hypothalamic dopamine suppresses the production of pituitary PRL . DopaminTGFβ is widely considered to be a tumor suppressor . HoweverComponents and regulators of the ERK MAPK pathway are all potential targets for treating pituitary tumors. (i) Raf kinase inhibitory protein (RKIP). RKIP is a modulator of MAPK signaling, which inhibits Raf-1 phosphorylation to inhibit Ras/Raf-1/MEK/ERK signaling pathway –74. RKIP(ii) Epidermal growth factor pathway substrate number 8 (Eps8). Over-expression of Eps8 and over-activation of Raf, MEK, and ERK in the ERK signaling pathway promote cell proliferation and survival in PAs . Also, E(iii) Retinoic acid (RA). RA has antiproliferative effect in corticotroph cell, and long-term treatment with RA has some clinical efficacy in patients with Cushing's disease . The mec(iv) Cold inducible RNA binding protein (CIRP). The underlying mechanism of cold-shock protein (CIRP) in its role in tumorigenesis is through its induction of cyclinD1 which decreases p27 expression via ERK1/2 signaling , 78. CIRThe p38 MAPK includes isoforms p38α, p38β, p38γ, and p38δ, with ~60% of sequence similarity among four isoforms . Of thesPrevious studies demonstrate that p38 MAPK is associated with apoptosis, and drugs that activate this pathway can thereby induce apoptosis in pituitary tumor cells. A study found that dopamine agonists such as BRC and CAB activate p38 pathway to induce cell apoptosis; for example, when BRC is used to treat rat lactosomatotroph GH3 cells, BRC activates p38 MAPK and promotes cell apoptosis. Moreover, p38 MAPK inhibitors  completely inhibit BRC-induced p38 MAPK activation and cell apoptosis . In addiIn recent years, with the development of next-generation sequencing (NGS), a large number of non-coding RNAs have been identified , 93. NonThe JNK MAPK pathway is mainly activated by various stress stimuli, including oxidative stress, UV irradiation, osmotic shock, heat shock, and proinflammatory cytokines , and plaNESΔ) are used to study the effects of JNK1 signaling on glucose metabolism. Unexpectedly, the decreased somatic growth and increased thyroid axis activities are observed in JNK1NESΔ mice with decreased levels of circulating GH and IGF1 , a triterpenoid compound found in food, medical herbs, and other plants , which hERK, p38, and JNK signaling pathways both independently and concordantly contribute to pituitary tumorigenesis. Thus, some chemotherapeutic drugs for PAs may target several subfamilies of the MAPK signaling pathway at the same time. For example, fulvestrant is an estrogen receptor antagonist without agonist effects , which iThe comprehensive pathway-network analysis of multiple sets of proteomic data in PAs –124 reveIn the MAPK network system in PAs, the activation of ERK is generally thought to promote cell proliferation and growth; whereas the activations of p38 and JNK are generally thought to promote cell apoptosis. The MAPK signaling pathway can be targeted by several mechanisms. Two types of combination strategies can be used: (i) A single drug to target ERK pathway and p38 or JNK pathway. For example, BIM-23A76 inhibits cell proliferation through targeting ERK1/2 pathway, and promotes cytotoxic effects through targeting p38 pathway . And (iiThis review summarized the studies of MAPK signaling in pituitary tumorigenesis. We discussed some important molecules involving in MAPK signaling pathway and potential drugs targeting the MAPK signaling . The ERKML and YW collected and analyzed literature, prepared figures and tables, and wrote and revised manuscript. XZ conceived the concept, designed, coordinated, wrote and critically revised manuscript, and was responsible for its financial supports and the corresponding works. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human T-cell lymphotropic virus (HTLV) belongs to a larger group of primate T-cell lymphotropic viruses (PTLVs) within the family Retroviridae. It is estimated that 10 to 20 million people worldwide may be infected with HTLV-1. Although most of them are asymptomatic, around 5% of infected individuals may develop either HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) or Adult T-cell Leukaemia/Lymphoma (ATLL). Public Health authorities in many countries have implemented routine blood-donor tests for HTLV-specific antibodies; but this is not the case for Germany since the reported prevalence is very low . With the aim to evaluate retrospectively the presence of HTLV-1 among oncology patients in this country, samples stored at the Universitätsklinikum Freiburg, were analyzed. For this purpose, two different nested-PCR (n-PCR) protocols have been modified and set up for HTLV-1 detection. One positive case was detected by n-PCR among 406 samples  in a period of 5 years (2008–2012) corresponding to a T-Cell Lymphoma. Despite the low prevalence, this virus is circulating in Germany, probably due to the increasing numbers of immigrants in these last years. Physicians should consider HTLV-1 infection and suspect it taking in account the ethnic and relation to endemic regions regardless the patient's residence. Human T-cell lymphotropic viruses (HTLVs) belong to a larger group of primate T-cell lymphotropic viruses (PTLVs) within the family Retroviridae. To date, four different types have been identified: HTLV types 1, 2, 3 and 4 [HTLV-1 was the first human retrovirus to be isolated from a patient with a T-cell malignancy in 1979 . It is eHTLV-1 is the causative agent of serious illnesses with unsatisfactory treatment options, including inflammatory syndromes such as HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), uveitis, Adult T-cell Leukaemia/Lymphoma (ATLL), opportunistic infections such as Strongyloides stercoralis hyperinfection and a range of lung diseases, including bronchiectasis, that is causing high mortality in Central Northern Australia  among otSeveral South American countries have naturally occurring endemic regions of HTLV-1, while most European countries present low prevalence (0.001 ± 0.03%), with Romania being the only endemic country for HTLV-1 infection in this continent. It is believed that over 80% of infected people in Europe are originally from endemic regions such as West Indies (Caribbean), Africa and South America , 10. HTLVarious Public Health authorities in many countries have implemented routine blood-donor tests for HTLV-specific antibodies; the screening for HTLV-1/2 is mandatory in Japan, Canada, the Caribbean and some South America countries. In Europe, it is mandatory in several countries such as UK, France, Demark, Greece, Portugal, Romania and Sweden; and recommended in others such as Ireland, Luxembourg and Norway. In contrast, for example, in Italy and Germany, the HTLV-1/2 blood screening is not even recommended . Further6 non-infected cells [Considering that the routine screening for HTLV-1/2 cannot distinguish between these two viruses, repeatedly reactive samples by screening tests are further tested by Western blot (WB), which may allow to ascertain the specific HTLV-type-seropositivity or -seronegativity. Nevertheless, the indeterminate serological results by WB are a frequent problem in HTLV-1 testing and this often leads to difficulties in the interpretation and counseling , 17. Oneed cells , 19.The focus of this study was to evaluate retrospectively the presence of HTLV-1 in oncology samples stored at the Universitätsklinikum Freiburg, Germany. For this purpose, two different n-PCR protocols have been modified and set up for HTLV-1 detection.A blind study was conducted. A total of 410 paraffined biopsies and/or blood samples stored in the Pathology Department at Freiburg Uniklinik, Germany, during the period 2008–2012, with a presumptive diagnosis of malignancy were selected and analyzed for HTLV-1. To set up the ideal n-PCR conditions, the HTLV-1-producing cell line MT2 and 6 samples from the INBIRS Institute in Argentina were used: 2 samples from 2 healthy individuals and 4 samples from 4 HTLV-1-positive individuals .6 MT2 cells or b) 2 x 106 PBMCs from HTLV-1-infected persons or c) 200 μl of blood obtained from HTLV-1-infected persons. For the negative controls, DNA was extracted from the 2 healthy individuals’ cells (either 2 x 106 PBMCs or 200 μl of blood).DNA was extracted from tissue samples in paraffin using the DNA FFPE Tissue Kit . DNA from cell lines, peripheral blood mononuclear cells (PBMCs) or blood was obtained by column extraction . For the HTLV-1-positive controls, DNA was extracted from a) 2 x 102 2,5 mM, dNTPs 0,2 mM, Primers 0,3 mM and Taq polymerase 0,5 units from Qiagen, Hilden, Germany), and 5 μl of DNA (20 ng/μl). The second round of PCR used 2 μl of the first round and the same Master Mix 2 , with 10 mM of pol and actin primers, in a final volume of 25 μl (dx.doi.org/10.17504/protocols.io.zjyf4pw).The first PCR amplification was performed in a 25 μl reaction volume containing Master Mix 1 .The first PCR amplification was performed in a 50 μl reaction volume containing Master Mix 1 with one unit of Taq DNA polymerase enzyme, and 2 μl of DNA (20 ng/μl) from either the MT2 cell line or from pooled DNA isolated from 10 different samples. The second round of the PCR was performed taking 5 μl of the first round and using the same amount of Master Mix 2 in a final volume of 25 μl (pol primers PolEF (5´ TTTAGGTGCCCAAACTGGAG 3´) and PolER (5´GCAGGATATTGGAAGCCTCAG3´) only anneal with HTLV-1 virus sequence. As inner primers PolIF (5´GCCCTCATGCCAGTGTTTAC3´) and PolIR (5´CCTGGAGATGGGATCAGGTAG3´) were selected. The forward (5´ATCGAGCACGGCATCGTCACCAAC3´) and reverse (5´GTTGAAGGTCTCAAACATGATCTG3´) actin primers were designed based on their location in the sequence GenBank NG_006672.1.Primers were chosen based on their location within the prototype HTLV-1 genomic sequence available in the GenBank J02029.1, considering conserved sequences, and designed to avoid complementary sequences and strong internal secondary structures. Outer PCR cycles. Cycling conditions on a TProfessional TRIO Thermocycler  were 10 sec 94°C, 20 sec 54°C, 45 sec 70°C for the first round and 10 sec 94°C, 20 sec 52°C, 45 sec 70°C for the second round. Both PCRs were amplified for 40 cycles. Before the cycling started, the PCR tubes were heated to 94°C for 4 min. After the last cycle, they were heated to 70°C for 5 min and then kept at 4°C. Amplified products were loaded onto a 2% agarose gel stained with Gel Red Nucleic Acid Gel Stain . Samples were run at 110 mV for 50 min. Bands were observed under ultraviolet (UV) luminometer . The 50 and 100 bp DNA Ladders  were used.pol gene with the co-amplification of actin gene were combined to confirm the presence of DNA in the sample. Amplification of pol and actin genes were observed in the control sample (MT2) and the HTLV-1 positive samples (lines 1–4). Only actin gene amplified in the healthy blood donors, lines 5 and 6  of the DNA obtained from the MT2 cell line. The  MT2 DNA .actin was also performed separately, to verify the correct extraction of DNA.As another modification, we set up the conditions to analyze a pool of 10 samples in a single n-PCR detecting the presence of HTLV-1. As a control, the amplification of pol fragment amplification in MT2 (positive control) and in lines 1–4 (HTLV-1-positive samples).Each PCR tube contained 18 μl of pooled DNA from 9 negative HTLV-1 samples and one HTLV-1 positive sample. pol amplification was measurable at 0.01 ng/μl, but not at 0.001 ng/μl, of MT2 DNA (data available at https://figshare.com/s/b24ff1bedb99814d4136).In subsequent experiments, the sensitivity of the pooled-PCR, was evaluated using serial dilutions (1/10) of MT2 DNA mixed with DNA pooled from 9 negative donors. Under the given experimental conditions, the pol HTLV-1/actin co-amplification, four of them were actin negative. actin positive.To carry the blind study, the samples were selected based on the suspicion of malignancy; the final diagnostic was seen after the nPCR was conducted. A total of 410 samples were analyzed using the n-PCR with pol and actin amplifications were observed in the positive control sample (MT2) and in the positive case (line 16). The remaining samples in lines 1–14, 17 and 18 showed only the actin gene amplification. In line 15, amplification of actin was barely detected and it was consider as negative for actin amplification. A second molecular diagnosis of this case was performed using the same DNA extracted from tissue in 2011 and another sample of DNA extracted from the peripheral blood in 2014 (A positive case for HTLV-1 (0.25%) was detected in a sample corresponding to a T-cell lymphoma from 2011. In  in 2014 .The prevalence of HTLV-1/2 in Germany is low  even in drug users and the HTLV-1 screening of blood and organ donors is consequently not mandatory in this country –22. Nonepol gene amplification in DNA samples extracted from tissues in 2011 and blood in 2014. The corresponded medical diagnostic for those years are T-cell lymphoma and a HTLV-1 lymphoma, respectively. The prevalence of HTLV-1 in this population was 0.25% (1/406), but if we consider only the patients with lymphocytic disorders the prevalence was 1.4% (1/67). Since this prevalence is relatively high compared to previous reports from Germany, we believe that in order to reach a more accurate picture of the real impact of HTLV-1 infection in this country, more studies need to be conducted.In this retrospective study, we detected one HTLV-1 positive case among 406 selected samples stored in the Pathology Department at Freiburg UniKlinik during 2008–2012. The HTLV-1 infection was confirmed by the Public Health—European Commission [The implementation of mandatory HTLV-1/2 testing may not be cost-effective, but applying perhaps a more detailed, HTLV-1/2-risk-factors-assessing interview to blood and organ donors should be considered as it has been recommended by the mmission . Otherwimmission , 26.As it was mentioned in the introduction, we believe that molecularly diagnosing HTLV is a logical and economic approach, especially in endemic regions –19. In aactin and the pol gene, samples can be analyzed faster, cheaper and with an increased efficacy. Furthermore, we were able to adapt the n-PCR for analyses of 10 samples in one reaction, which makes this ‘pooling n-PCR’ time-saving even though the PCR for actin amplification needs to be run separately. In particular, for this study, we considered that the 410 samples selected were a small number and decided to test them only by co-amplification nPCR, and not by doing pools. Nevertheless, we were capable of validating the high sensitivity of this procedure even when analyzing 10 samples at the same time and illustrate its potential to study the HTLV involvement in distinct disorders. Consequently, employing this strategy may a) increase the safety of blood and organ donations, b) diminish the risk of losing donors due to false-positive screening results and c) help establish definitive diagnoses of diseases known to be causatively associated with HTLV-1. In conclusion, this virus is circulating in the population of Germany although the low prevalence, probably due to the greater number of immigrants in recent years. Physicians should bear HTLV-1 infection in mind and suspect it regardless the patient's residence.A number of reviews and research articles have provided a detailed description of the key parameters that may influence the performance of n-PCR. Fewer publications discuss multiplex PCR . Other s