Fertility desire for a second child has been a lively topic since the implementation of the two-child policy in China. Chinese researchers have explored various factors influencing the fertility desire for a second child. However, there have not been studies on the individual differences in the relative fertility costs and fertility benefits and their effects on fertility desire for a second child.A total of 396 participants rated four kinds of relative fertility costs, four kinds of fertility benefits and their fertility desire for a second child. Latent profile analysis (LPA) was used to explore the individual differences in the relative fertility costs and fertility benefits and their effects on fertility desire for a second child.Stepwise regression analysis showed that parenting joy, health risks, mutual care among siblings, the flourishing of family, and time pressure and opportunity cost significantly predicted the fertility desire for the second child. According to the latent profile analysis, the participants were classified into four classes. Participants in the lowest-cost/lowest-benefit and high-cost/medium-benefit classes had low fertility desire for a second child, while those in the low-cost/high-benefit and highest-cost/highest-benefit classes had high fertility desire.Fertility benefits have a stronger effect on the fertility desire for a second child than relative fertility costs. Fertility benefits should be paid more attention to than relative fertility costs. Fertility desire for a second child has been a hot topic since the implementation of the two-child policy in China. All kinds of factors affecting the fertility desire for a second child have been explored. However, no studies have been conducted on the individual differences in the relative fertility costs and fertility benefits and their effects on fertility desire for a second child. A total of 396 participants scored four kinds of relative fertility costs, four kinds of fertility benefits and their fertility desire for a second child. Stepwise regression analysis showed that parenting joy, health risks, mutual care among siblings, the flourishing of family, and time pressure and opportunity cost significantly predicted the fertility desire for the second child. According to the latent profile analysis, the participants were classified into four classes. Participants in the lowest-cost/lowest-benefit and high-cost/medium-benefit classes had low fertility desire for a second child, while those in the low-cost/high-benefit and highest-cost/highest-benefit classes had high fertility desire. The results showed that fertility benefits have a more important influence on the fertility desire for a second child than relative fertility costs. It is suggested that the government should implement favourable policies to decrease women’s time pressure and opportunity cost. We foresee that the fertility benefits that might gradually become apparent over time would encourage more people to bear one more children.The one-child policy was first implemented in 1980 to control the population size in China. However, given the ageing society, the one-child policy was phased out, and the two-child policy has been gradually implemented. Couples in which both the husband and wife were single children were allowed to bear two children as of November 2011. Couples in which one partner was a single child were permitted to bear two children as of December 2013. As of October 2015, all couples are allowed to bear two children. Fertility desire is an important predictor of fertility behaviour. When people want to give birth to a child, they take actions to become pregnant; otherwise, they take contraceptive actions. Therefore, fertility desire and its influencing factors have become a significant issue that both the public and government are closely concerned about since the implementation of two-child policy in China. Fertility desire for a second child has been a heated topic of conversation when people get together. A great many studies have explored the fertility desire for a second child in China and its influencing factors.N = 7810 participants from 29 Chinese provinces) carried by Survey and Data Center of China from Renmin University of China indicated that 68.8% Chinese residents wanted to give birth to their second child [N = 4194) showed that 81.7% of rural residents wanted to bear their second child [Researchers have investigated fertility desire for a second child in China. The Chinese General Social Survey . LPA is a multivariate approach which identifies groups based on individual’s observed response patterns to categorize the participants into optimal groups . BesidesThe data collection procedure included the following steps. First, we obtained ethical approval from the Research Ethics Committee of the host university. Second, the questionnaire was uploaded to the website of “Questionnaire Star”, which was the most widely used questionnaire website in China. Third, the electronic questionnaire forwarded by the social software WeChat and QQ. The principles of voluntariness and confidentiality were underlined in the instruction section of the questionnaire. Participants were reminded that their participation was voluntary and that they could discontinue it at any time. They were assured that their responses would be kept confidential. They were also asked if they would like their test results to be used as data sources for this study and published. The survey was conducted from October 2017 to December 2017.It was required that the participants be between 18 and 45 years old and not have two children. A total of 396 valid questionnaires were obtained from 52 administrative cities in China . The mean age of the sample was 31.4 years (SD = 7.5), and it comprised 4 primary school graduates, 162 middle school graduates, 177 college graduates, 48 master’s degree graduates and 5 doctoral degree graduates. A total of 305 were junior employees, 82 were mid-level employees, and 9 were senior employees. Two hundred and forty-four participants lived in urban districts, 61 in counties, 66 in towns and 25 in rural areas. One hundred and five people rented apartments, 203 owned one flat, 66 owned two, 8 owned three or more, and 14 persons did not indicate their living condition. One hundred and fifty-seven were single children, and 239 were not. One hundred and ninety-one had no children, and 205 had one child.α = 0.834.The three kinds of relative fertility costs of economic pressure, time pressure and opportunity cost, conflict among siblings were measured by the 15-item Fertility Costs Subscale in the Fertility Cost-Utility Questionnaire . Exampleα = 0.872.The four kinds of fertility benefits  were measured by the 16-item Fertility Utility Subscale in the Fertility Cost-Utility Questionnaire . ExampleThe fertility desire for a second child was assessed by one question, which asked participants to indicate their fertility desire for a second child on a 5-point scale ranging from 1 (strongly reluctant) to 5 (strongly voluntary).p value for a three-class model indicates that the two-class model fits better than the three-class model. Finally, entropy value was also used to evaluate classification accuracy. Values closer to 1.0 indicate better classification accuracy. While, values greater than 0.80 can be considered to have adequate classification accuracy [LPA was conducted with Mplus 7.0 software. Four criteria were used for deciding the best fitting model. The first criterion was the adjusted Bayesian information criterion (aBIC) which is the most reliable indicators of true model fit . Additioaccuracy .In addition, the effect size of the mean difference test was measured with Cohen’s d. The effect size was small when 0.2 ≤ d < 0.5, medium when 0.5 ≤ d < 0.8 and large when d > 0.8 , 38.The means, standard deviations, and correlations among relative fertility costs, fertility benefits and fertility desire for a second child are shown in Table R2) of 8.5% among the five predictors. The health risks, mutual care among siblings, the flourishing of family accounted for 4.2, 3.9, 2.1% of the variance of the fertility desire, respectively. Time pressure and opportunity cost accounted for the least variance of the fertility desire with the determination coefficients (△R2) of 0.8%. Totally, the fertility benefit accounted for 14.5% variance of the fertility desire for a second child, whereas the fertility cost accounted for 5.0% variance of the fertility desire.Stepwise regression analysis was performed to analyse the prediction of the four kinds of relative fertility costs and the four sorts of fertility benefits on fertility desire for a second child. Multicollinearity diagnosis was conducted. The least tolerance was 0.667 (bigger than 0.1) and the maximum VIF was 1.500 (less than 10), which indicated that there was not serious multicollinearity problem. It could be seen from Table p value for the four-class solution was significant, whereas the LMR p value for the five-class solution were not significant. These indicate that the four-class model was significantly better than the three-class model but not significantly better than the five-class model. Finally, the entropy value was larger than 0.80.In order to determine the optimal number of classes, LPA was conducted. Specifically, we included eight indicators in this analysis: economic pressure, time pressure and opportunity cost, conflict among siblings, health risks, parenting joy, mutual care among siblings, elderly care, and the flourishing of family. The fit indices of models withtwo to five class solutions are summarized in Table n = 7) participants of the sample. The conditional means of the four kinds of relative fertility costs and four kinds of fertility benefits were between 1.5–2.3. The values in this profile were the lowest among the four classes; therefore, we label this class as lowest-cost/lowest-benefit. Besides, Class 2 comprised 51.5% (n = 204) participants in this sample. The conditional means of the four kinds of relative fertility costs were between 2.5–3.0, and those of the four kinds of fertility benefits were between 3.0–3.8; therefore, this classwas named the low-cost/high-benefit. Class 3 comprised 28.0% (n = 111) participants in this sample. The conditional means of the four kinds of relative fertility costs were between 3.1–4.0, and those of the four kinds of fertility benefits were between 2.0–3.5; therefore, this class was labeled as the high-cost/medium-benefit class. Class 4 comprised 18.7% (n = 74) of the sample. The conditional means of the four kinds of relative fertility costs and four kinds of fertility benefits were all above 3.5. The values in this class were the highest among the four classes; therefore, this class was labeled as the highest-cost/highest-benefit.The conditional means, standard deviations (SD), and latent class probabilities for the four-class model are showed in Table t = − 0.154, p > 0.05). There was also little difference in the fertility desire between the low-cost/high-benefit and highest-cost/highest-benefit classes. The mean difference test showed that there was no significant difference between the two classes . In other words, there were two levels of fertility desire. The fertility desire for a second child was relatively low for lowest-cost/lowest-benefit and high-cost/medium-benefit classes (merged as the low-desire class), whereas the fertility desire for a second child was relatively high for low-cost/high-benefit and highest-cost/highest-benefit classes (merged as the high-desire class). According to the latent class probability in Table Table The difference of fertility desire between low-desire class and high-desire class is presented in Table The first purpose of this study was to investigate the effect size of different relative fertility costs and fertility benefits on the fertility desire for a second child. The results of stepwise regression analysis showed that three kinds of fertility benefits significantly have influence on fertility desire. Parenting joy accounted for the most variance in the desire to bear an additional child. The possible reason is that most Chinese women are still family oriented, and their most important values are still to help their husbands and teach their children. Additionally, with the development of society, many Chinese men have begun to pay more attention to fostering their children, and they view the growth of their children as their accomplishment and as a source of well-being. Mutual care among siblings is the second fertility benefit influencing the childbearing desire. The one-child policy was implemented for more than 30 years. Social relationships are indifferent in cities. Many single children often felt lonely because they had no siblings and kept limited contact with other partners. Therefore, having grown up, they hope that their children can have one sibling to accompany them. The flourishing of family is the third fertility benefit having an effect on the desire to have a more child. Blood relationships are the most important relationship in China. Having a thriving family and plenty of children and grandchildren are the wishes of a great deal of Chinese. However, elderly care did not significantly predict the fertility desire for a second child, which could be explained in two ways. The importance of family support for the elderly has decreased with the development of social security. In addition, with the development of Chinese society, a large number of children leave their parents when they grow up, which causes many parents to reduce their expectation that their children will take care of them when they are old.Two kinds of relative fertility costs have significant effects on fertility desire. Health risks accounted for the most variance in the fertility desire for a second child, which could be explained in two ways. Many women over 35 years old have missed the best age for childbirth because of the one-child policy that was carried out for more than 30 years. Furthermore, more and more women get married at over 28 years old, and many of them worry about the adverse pregnancy outcome to bear their second child. The other fertility cost that has an impact on the childbearing desire is time pressure and opportunity cost. With the development of Chinese society, more and more Chinese women receive a higher education and enter the work force. They worry that giving birth to their second child would have negative impact on their career. Counter to our expectation, economic pressure did not significantly predict fertility desire. As the Chinese proverb says, “If you are rich, you raise your children in a prosperous way; however, if you are poor, you foster your children in an economical way.” Those parents who want to give birth to their second child will keep expenditures within the limits of income if they are not wealthy. However, those couples who do not want to bear their second child often use economic pressure as their excuse. Finally, conflict among siblings did not have a significant effect on the desire to have an additional child. The possible reason is that this problem is not the important influencing factor of fertility desire, though some parents might worry about the objection of the older child.The second purpose of this study is to explore the individual differences in relative fertility costs and benefits and their influence on fertility desire for a second child. The participants were classified into four classes by using latent profile analysis: lowest-cost/lowest-benefit, low-cost/high-benefit, high-cost/medium-benefit and highest-cost/highest-benefit. There were two levels of fertility desire for the four classes. Though the relative fertility costs of the lowest-cost/lowest-benefit and high-cost/medium-benefit classes were different, their fertility benefits were relatively low  to 128 or 158 days in many Chinese provinces in 2017. However, such preferential policy is far from what is needed. Extended maternity leave should be considered. In addition, it is predicted that the fertility benefits including parenting joy, mutual care among siblings and the flourishing of family might gradually show up over time, which would encourage more people to bear one more child.Despite its contributions, the study has several limitations that should be acknowledged. The first limitation is that the sample consisted of only 396 participants recruited on the basis of convenient sampling through the internet, which could not assure sample representativeness. Future research can enlarge the sample size on the basis of representative sampling. The second limitation is that we look into the fertility desire only of individuals and not couples, which could better predict fertility behaviour. Future research can investigate the fertility desire of couples to better predict fertility behaviour. Thirdly, the figures used to account for the relative economic cost in this study is assumed data on the basis of daily experience rather than real data from survey, which should be collected to enhance persuasion in the future. Finally, fertility costs and benefits are constantly changing. Future studies should keep inquiring into fertility costs and benefits and their influence on fertility desire and behaviour.Parenting joy, health risks, mutual care among siblings, the flourishing of family, and time pressure and opportunity cost significantly predicted the fertility desire for a second child. Participants could be classified into four classes according to relative fertility costs and benefits by using latent profile analysis: lowest-cost/lowest-benefit, low-cost/high-benefit, high-cost/medium-benefit and highest-cost/highest-benefit. Participants in the lowest-cost/lowest-benefit and high-cost/medium-benefit classes had low fertility desire for a second child, whereas those in the low-cost/high-benefit and highest-cost/highest-benefit classes had the high fertility desire. Fertility benefits have a stronger influence on fertility desire for a second child than relative fertility costs. It is suggested that the government should implement favourable policies to decrease women’s time pressure and opportunity cost. We predict that the fertility benefits that might gradually become apparent in future would encourage more people to have one more child.


Tax-orf II mRNA, a nuclear/nucleolar protein with both transcriptional and post-transcriptional activity. The p30 protein inhibits the productive replication cycle via nuclear retention of the mRNA that encodes for both the viral transcriptional trans-activator Tax, and the Rex proteins that regulate the transport of incompletely spliced viral mRNA to the cytoplasm. In myeloid cells, p30 inhibits the PU-1 transcription factor that regulates interferon expression and is a critical mediator of innate and adaptive immunity. Furthermore, p30 alters gene expression, cell cycle progression, and DNA damage responses in T-cells, raising the hypothesis that p30 may directly contribute to T cell transformation. By fine-tuning viral expression while also inhibiting host innate responses, p30 is likely essential for viral infection and persistence. This concept is supported by the finding that macaques, a natural host for the closely genetically related simian T-cell leukemia virus 1 (STLV-1), exposed to an HTLV-1 knockout for p30 expression by a single point mutation do not became infected unless reversion and selection of the wild type HTLV-1 genotype occurs. All together, these data suggest that inhibition of p30 may help to curb and eventually eradicate viral infection by exposing infected cells to an effective host immune response.The extraordinarily high prevalence of HTLV-1 subtype C (HTLV-1C) in some isolated indigenous communities in Oceania and the severity of the health conditions associated with the virus impress the great need for basic and translational research to prevent and treat HTLV-1 infection. The genome of the virus’s most common subtype, HTLV-1A, encodes structural, enzymatic, and regulatory proteins that contribute to viral persistence and pathogenesis. Among these is the p30 protein encoded by the doubly spliced  In 1977, an unusual cluster of adult T-cell leukemia/lymphoma (ATLL) reported in southwestern Japan suggested the presence of an infectious agent , and Hinorf-II) ..118].+ cells accumulate DNA breaks and arrest cells in the S phase. Moreover, immunofluorescence has shown the colocalization of gamma-H2AX, a specific marker of DNA double-strand breaks and DNA-PK, suggesting that the damaged DNA is preferentially repaired by NHEJ when in the presence of p30 [Overall, these findings demonstrate that p30 impairs HR repair. However, HTLV-1 infected cells are still able to repair damaged DNA by activating the error-prone system NHEJ. Indeed, a 40% increase in NHEJ activation was observed using an in vivo NHEJ-GFP assay in the presence of p30. These results were confirmed by using an NHEJ-specific inhibitor, Nu7026, that blocks the activity of DNA-dependent protein kinase (DNA-PK), an essential component of the DNA repair system. Following Nu7026 treatment, these HTLV-1e of p30 .In conclusion, p30 impairs the DNA damage response in HTLV-1 infected cells. Further, treatment with inhibitors that target the DNA repair pathway  were found to induce apoptosis not only in HTLV-1 infected cells, but also in ATL-derived cell lines , 120, suC-MYC locus gene amplification, and it is associated with poor prognosis [C-Myc is a well-known proto-oncogene involved in cancer initiation  and implrognosis , 126. ThP30 is reported to interact with c-Myc and transactivate its E-box enhancer elements within the cyclin D2 promoter. By using shRNA against TIP60 and TIP60 mutants, Romeo et al. demonstrated that in cell lines p30 stabilizes the recruitment of TIP60 to the p30/c-Myc nuclear complex, promoting the transcription of cyclin D2 . The notWT) and p30 defective molecular clones in immortalized primary T-cells, p30 was shown to inhibit apoptosis in the presence of genotoxic stress induced by camptothecin [Using wild type HTLV-1 -DNA-damaging chemotherapy drugs that are commonly used to treat ATLL by inhibiting apoptosis in the presence of genotoxic stress Fig. . The expAberrant activation of specific oncogenes results in metabolic toxicity, which might lead to the cellular transformation necessary for the development of neoplastic disease. The oncogene c-Myc is commonly upregulated in human malignancies , 128. ImRomeo et al. showed that p30 augments TP53-induced glycolysis and apoptosis regulator (TIGAR) expression in a p53-dependent manner . The p30+ SLB1 or MET-1 tumor lymphocytes. These animals consistently develop aggressive lymphoid tumors accompanied by enlargement of the liver and spleen. In this model, the high level of TIGAR expression was found to be associated with increased expression of the pro-angiogenic markers VEGF and HIF-1α. Hutchison et al. suggested that TIGAR could promote angiogenesis in HTLV-1 positive tumor cells in the xenograft model, which might play a role in aggressive metastasis and infiltration in secondary tissues of HTLV-1-infected cells [High TIGAR expression and c-Myc deregulation were observed together in NOD/SCID mice engrafted with the HTLV-1ed cells . Very liorf II encoding protein p30 in vivo. Ablation of p30 expression does not compromise HTLV-1 replication in primary human cells or its ability to immortalize T-cells in vitro [hbz open reading frame demonstrated reduced viral infectivity in rabbits. In an additional study in the same species, only two of six animals exposed to the HTLV-1 mutant seroconverted and had reversion to wild type, providing evidence of the coexistence of mutant and wild type viruses in animals that became infected [p30KO) where p30 expression was putatively ablated by a single amino acid mutation in the p30 initiation codon without affecting the expression of the other known HTLV-1 mRNAs [Different laboratories have tried to address the importance of the in vitro , 30. Howin vitro . An earlinfected . However-1 mRNAs .WT or the HTLV-1p30KO in rabbits resulted in similar infectivity, suggesting that p30 is not required for persistent infection in this species [p30KO clone [p30KO fully seroconverted. Two macaques recognized a single viral protein, and one failed to recognize any HTLV-1 antigen. The animals with partial or complete seroconversion were found to be positive for infection by DNA PCR and reversion of HTLV-1p30KO to HTLV-1WT was observed [Inoculation of irradiated cells producing HTLV-1 species . DNA isoKO clone . RabbitsKO clone . Interesobserved . These fobserved , 79 may The HTLV-1 C subtype common in central Australia is ancient, but it has recently attracted widespread attention due to its alarmingly high prevalence of nearly 30% infection among the region’s aboriginal population. Mortality at a young age is elevated in HTLV-1C patients, and this virus represents a medical emergency. Molecular studies have identified HTLV-1C as a highly divergent strain of the virus, with the highest divergence found in the 3′end of the viral genome –133. In orf-II in both subtypes using the sequences of 160 individuals infected with HTLV-1A  indicate gaps in the amino acid alignment, asterisks (*) represent stop codons, and periods (.) represent similarity. The multi-alignment was performed with the Mega7 program using default parameters.Additional file 2: Figure S2. Amino acid sequence analysis of p30 from HTLV-1A infected individuals. Alignment of p30 amino acid sequences of from 160 HTLV-1A patients was used to generate a consensus. Dashes (–) indicate gaps in the amino acid alignment, asterisks (*) represent stop codons, and periods (.) represent similarity. The multi-alignment was performed with the Mega7 program using default parameters. Amino acid sequence analysis of p30 from HTLV-1A infected individuals. Alignment of p30 amino acid sequences of from 160 HTLV-1A patients was used to generate a consensus. Dashes (–) indicate gaps in the amino acid alignment, asterisks (*) represent stop codons, and periods (.) represent similarity. The multi-alignment was performed with the Mega7 program using default parameters.


Difficulties in confirming and discriminating human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 infections by serological Western blot (WB) assays  have been reported in Brazil, mainly in HIV/AIDS patients, with a large number of WB-indeterminate and WB-positive but HTLV-untypeable results. Nonetheless, a line immunoassay (LIA)  provided enhanced specificity and sensitivity for confirming HTLV-1/2 infections. Difficulties in confirming and discriminating human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 infections by serological Western blot (WB) assays  have been reported in Brazil, mainly in HIV/AIDS patients, with a large number of WB-indeterminate and WB-positive but HTLV-untypeable results. Nonetheless, a line immunoassay (LIA)  provided enhanced specificity and sensitivity for confirming HTLV-1/2 infections. To add information concerning the improved ability of the LIA in relation to WB when applied to samples of individuals from different risk groups from Brazil, we performed the present study. Three groups were analyzed: group 1 (G1), with 62 samples from HIV/AIDS patients from São Paulo, SP (48 WB indeterminate and 14 HTLV untypeable); group 2 (G2), with 24 samples from patients with hepatitis B or hepatitis C from São Paulo ; and group 3 (G3), with 25 samples from an HTLV outpatient clinic in Salvador, Bahia . Overall, the LIA confirmed HTLV-1/2 infection  in 66.1% (G1), 83.3% (G2), and 76.0% (G3) of samples. Interestingly, the majority of WB-indeterminate results were confirmed by the LIA as being HTLV-2 positive in G1 and G2 but not in G3, in which the samples were defined as being HTLV-1 or HTLV positive. These results agree with the virus types that circulate in such patients of different regions in Brazil and emphasize that the LIA is the best serological test for confirming HTLV-1 and HTLV-2 infections, independently of being applied in HTLV-monoinfected or HTLV-coinfected individuals. Despite regional variations, Brazil is one of the largest areas of the world where human T-cell lymphotropic virus type 1 (HTLV-1) is endemic .pol, long terminal repeat [LTR], env, and tax) in peripheral blood mononuclear cells using nested PCR, PCR hybridization, and/or PCR-restriction fragment length polymorphism (RFLP) assays  (HTLV Blot 2.3 and 2.4) have been reported in Brazil, due to a large number of WB-indeterminate and HTLV-positive but untypeable results, mainly in patients truly infected with HTLV-2 and/or HIV 35. Conseq) assays –11. None assays –, 10, 11. assays ––15.In 1998, a new HTLV-1/2 serological confirmatory assay employed a line immunoassay (LIA) with a nylon membrane sensitized with the most relevant antigens, recombinant proteins, or synthetic peptides of HTLV-1 and HTLV-2 . This neIn Brazil, molecular biology laboratories are not widely available due to differences in socioeconomic conditions. Thus, the use of a confirmatory serological assay with high performance is essential and necessary for HTLV-1/2 diagnosis. Accordingly, the present study aimed to evaluate the use of INNO-LIA for clarifying WB-indeterminate and WB-HTLV-untypeable serum samples.The serum samples employed in the present study were obtained from the biorepositories of the HTLV Research Laboratory (LPHTLV), Department of Immunology, Adolfo Lutz Institute (IAL), located in São Paulo, Brazil, and the Integrated and Multidisciplinary HTLV Center (CHTLV), located at the Bahiana School of Medicine and Public Health (EBMSP) in Salvador, Bahia, Brazil. Briefly, the samples from São Paulo were collected between 2012 and 2016 in the course of previous studies designed to detect the prevalence of HTLV-1/2 in HIV-infected individuals as well as in patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection in the state of São Paulo, Brazil , 18–20. 18–Of the 145 samples with WB-inconclusive results, a volume sufficient for immunoassaying was present in 111; these samples were used to determine the ability of the LIA  to confirm and discriminate HTLV-1/2-specific antibodies. The evaluated samples originated from three groups of patients: group 1 (G1), with 62 samples (48 WB indeterminate and 14 HTLV untypeable) obtained in the course of routine HTLV diagnosis at the Adolfo Lutz Institute or from the São Paulo Sexually Transmitted Disease/AIDS Reference and Training Center (CRT DST/AIDS-São Paulo), all of which were obtained from individuals who were known to be HIV seropositive; group 2 (G2), with 24 samples (21 WB indeterminate and 3 HTLV untypeable) that were collected from patients who were initially seen at gastroenterology centers in São Paulo, 14 of whom had HBV and 10 of whom had HCV infection, with 17 of these being HIV seropositive (10 with HBV and 7 with HCV); and group 3 (G3), with 25 HTLV-monoinfected samples from patients seen at the CHTLV outpatient clinic in Salvador, Bahia (16 WB indeterminate and 9 HTLV untypeable).The samples from São Paulo were screened for the presence of HTLV-1/2 antibodies by two enzyme immunoassays (EIAs): Murex HTLV-I+II  and Gold ELISA (enzyme-linked immunosorbent assay) HTLV-1/2 . The samples from Salvador were initially screened by the Ortho HTLV-1/HTLV-2 Ab-Capture ELISA system  as well as by four other HTLV-1/2 screening tests commercially available in Brazil: three EIAs  and one chemiluminescence assay (CLIA) kit . All assays were performed according to the manufacturers’ instructions, which were also used to interpret results. Cutoff values and gray zones were calculated for each assay, and samples considered reactive or inconclusive in screening were submitted for a confirmatory assay.A WB assay  was used to confirm HTLV-1 and HTLV-2 infection for all the previous studies that generated the samples analyzed here, and these results were interpreted according to the stringent criteria provided by the manufacturer. Briefly, HTLV-1-positive serum samples were defined as having the presence of Gag (p19 with or without p24) and two Env (GD21 and rgp46-I) bands. HTLV-2-positive samples were defined as demonstrating reactivity to Gag (p24 with or without p19) and two Env (GD21 and rgp46-II) bands. Samples that showed the presence of antibodies to both Gag (p19 and p24) and Env (GD21) were defined as being HTLV positive but were considered untypeable. Any other pattern of bands was deemed to be indeterminate.The present study employed an LIA  in an attempt to confirm and/or discriminate samples with inconclusive results by WB . The strips used in the LIA contain antigens for validation, confirmation, and discrimination. For validation, the line marked by each sample was compared to the control line, and a score ranging from +/− to +3 was assigned. The confirmatory antigens included Gag p19-I/II, Gag p24-I/II, Env gp46-I/II, and Env gp21-I/II. No bands or the occurrence of a single band  denoted a negative result. The presence of one band (Env gp21-I/II) or two bands (except Env gp21-I/II) indicated indeterminate results, while two bands  indicated HTLV positivity. Three discriminatory bands  were considered as follows: HTLV-1 positivity was indicated by reactivity to Gag p19-I and/or Env gp46-I, while HTLV-2 positivity was found when samples showed Env gp46-II or a higher intensity of the Env gp46-II band than the Gag p19-I and Env gp46-I bands.P value of ≤0.05 were considered statistically significant.Differences in the numbers of males and females in each group were evaluated statistically using the chi-square test. GraphPad Prism software version 5.03  was used for age comparisons between groups using Kruskal-Wallis analysis of variance (ANOVA), complemented with Dunn’s multiple-comparison test. Results with a The present research protocol was approved by the Institutional Review Board of the IAL in São Paulo, Brazil  and by the Institutional Research Board (IRB) of the EBMSP in Salvador, Bahia, Brazil (protocol no. 464.286). All procedures were performed in accordance with the principles established in the Declaration of Helsinki and its subsequent revisions.P = 0.0048). A comparative analysis of age among the groups showed no significant differences, although the individuals in G2 were older overall. Concerning the distribution of WB-inconclusive samples, the three groups contained more WB-indeterminate than HTLV-untypeable samples: 77.4% in G1, 87.5% in G2, and 64.0% in G3.The characteristics of the patients (sex and age) and the distribution of WB-inconclusive results in each study group are presented in The LIA provided confirmation of HTLV-1/2 infection  in 66.1% (G1), 83.3% (G2), and 76.0% (G3) of the samples analyzed. Interestingly, most WB-indeterminate results in G1 and G2 were confirmed to be HTLV-2 positive by the LIA, but this was not the case in G3. In G3, only HTLV-1 (40.0%)- and HTLV (36.0%)-positive samples were detected among both WB-indeterminate and HTLV-untypeable samples .n = 111) and the patterns returned by the LIA are presented in Plasmodium falciparum in regions where malaria is endemic . Several attempts have been made to improve WB sensitivity and specificity, such as adding HTLV-1 and HTLV-2 recombinant envelope proteins and transmembrane protein to the HTLV-1 viral lysate. These include rgp46-I, rgp46-II, and GD21, the latter of which blocks the cross-reactivity of gp21 with  endemic , 23. In ndemic 4––17, 21. ic 4––12–, 24–27.Interestingly, the majority of WB-indeterminate results that were not confirmed by seroconversion or PCR assays were due to cross-reactivity with Gag antigens. For instance, the WB-indeterminate pattern exhibiting p19, p26, p28, p32, p36, and p53, termed the HTLV-1 Gag indeterminate profile (HGIP), has been detected in epidemiological studies, mostly in Cameroon and in the Caribbean, but was not associated with true HTLV-1 infection , 24. Thipol and PCR-RFLP analysis for tax]) and confirmed a series of more than 10 HTLV-1 and 10 HTLV-2 samples as being positive by both WB and LIA criteria, and they were concordant. In addition, we revised data for two samples that were HTLV-1 and -2 positive by WB, one of which was confirmed to be HTLV-1 and HTLV-2 positive by the LIA and molecular assays  have reported differing numbers of WB-indeterminate samples, which were subsequently confirmed to be positive by PCR –31. Also–n = 14) that tested HTLV untypeable by the LIA, PCR confirmed the presence of HTLV-1 in 5 samples and HTLV-2 in another 2 samples from G1 (data not shown), emphasizing the need for employing molecular assays to confirm an HTLV diagnosis in patients with HIV-HTLV coinfection.In corroboration with these findings, the majority of serum samples here that presented WB patterns of GD21 and/or rgp46-I or -II plus p19 or p24 were subsequently confirmed to be positive by the LIA. These types of WB patterns were observed in G1, and PCR assays demonstrated HTLV-1 or HTLV-2 positivity (data not shown) . In addiIn G2, the majority of WB-indeterminate patterns presented either GD21 alone or this protein in association with one Gag or envelope band. The LIA confirmed the presence of HTLV-2 in 11/21 (52.4%) of the WB-indeterminate samples. The high number of HTLV-2-positive samples in G2 leads us to suppose that these patients acquired HBV and HCV, as well HTLV-1/2 and HIV, at the same time, probably by the parenteral route and prior to the time when serological testing for HIV and HBV 1989), and subsequently for HTLV and HCV (1993), became mandatory in blood banks throughout Brazil; in addition, intravenous drug addiction was more frequent in this country, as previously described –20. Corr–9, and suConcerning the two WB-indeterminate samples in G2 with negative HTLV results by the LIA, both presented reactivity for GD21 in the WB analysis, and one of the samples showed a faint band. Curiously, the LIA demonstrates the best performance in this group of patients, with 20/24 (83.3%) of HTLV-positive samples being detected among the WB-inconclusive samples. Unfortunately, only plasma/serum samples from these patients were available for analysis, which did not allow the use of PCR to perform a comparative analysis of serological and molecular results. Nonetheless, associations between HTLV-1/2 and hepatitis B and C have been reported in several studies conducted in Brazil and elsewhere 18–, 34.The WB-inconclusive patterns in G3 were quite different from those in the other groups analyzed. Several of the HTLV-untypeable samples demonstrated the presence of almost all bands corresponding to the HTLV-1 viral lysate, without reactivity to rgp46-I, and six of nine were subsequently confirmed to be HTLV-1 positive by the LIA. Twelve out of the 25 WB-inconclusive samples that could be submitted for PCR (9 WB indeterminate and 3 HTLV indeterminate), 11 were confirmed to be HTLV-1 infected, 6 of which were HTLV untypeable by LIAs (data not shown). In addition, the serum samples that tested negative by LIAs presented three different WB-indeterminate patterns: (i) GD21, (ii) p24 (faint band), and (iii) rgp46-II (faint band). Only one of these samples could be tested by PCR and presented an HTLV-negative result (data not shown). In summary, the samples in G3 were confirmed to be positive for HTLV-1 or HTLV but not HTLV-2 infection. This finding could be partially related to the ethnic origin of the included individuals (African descendants), the lack of HIV infection in this group, and the characteristics of the patients seen at HTLV outpatient clinics in Salvador, BA .Of note, the reasons described above to explain WB-inconclusive results could also be applied to the PCR-negative results in truly HTLV-1/2-infected individuals, including the low proviral loads in HIV/AIDS patients undergoing antiretroviral therapy in G1 and, in some cases, in G2 –15; the –It is noteworthy that since the discovery of HTLV-3 and HTLV-4 in central Africa , 37, stu38–36–In fact, we could not rule out HTLV-3 and HTLV-4 infections in Brazil, since populations in central Africa migrated from Africa and Australia to the American continent before Asiatic population migration, and their descendants, such as the Amerindians, could maintain such viruses or spread them to the general population, which could justify the frequent presence of WB-indeterminate results in Amerindians, as previously described .In relation to the LIA, although it presented the best performance in detecting HTLV-1 and HTLV-2, we could not exclude misclassified positive results, as occurred in HTLV-3- and HTLV-4-infected individuals, who could be erroneously diagnosed as being infected with HTLV-2 –38.–Of note, despite the fact that the LIA demonstrated better performance than WB in the serological diagnosis of both HTLV-1 and HTLV-2, additional considerations are warranted for both assays. With respect to WB, we consider the lack of an ability to score the intensity of a positive band to be a problem, since it is not known when a faint band should be considered truly positive. The criteria (band profiles) established by the manufacturer to confirm HTLV-1 and HTLV-2 infections in WB assays are excessively stringent and deserve a review. Taking into account the results obtained here, we suggest that samples presenting only one Gag band (p19 or p24) plus GD21 and rgp46-I or rgp-46-II should be considered HTLV-1 and HTLV-2 positive, respectively, since samples that demonstrated p24, GD21, and rgp46-I bands were confirmed to be HTLV-1 positive by the LIA and PCR. In contrast, we detected true HTLV-2 positivity in samples that showed p19, GD21, and rgp46-II bands. In addition, when Gag bands were undetectable but both envelope bands (GD21 and rgp46-I or rgp46-II) were present, it was impossible to rule out true HTLV-1 or HTLV-2 infection, since seroconversion could be taking place. Indeed, when all bands showed reactivity to HTLV-1 viral lysate antigens, even in the absence of rgp46-I, it was possible to confirm HTLV-1 infection.In conclusion, the LIA was shown to be the best serological test for confirming HTLV-1 and HTLV-2 infections, regardless of whether individuals were HTLV monoinfected or coinfected. We further highlight the need to review some WB criteria based on our results and those reported previously by others. It remains to be determined whether the superior performance of the LIA was due to the less stringent criteria employed than for WB. Further studies are necessary to confirm these results in a variety of risk populations from Brazil and elsewhere.


The Rho family of small GTPases orchestrates fundamental biological processes such as cell cycle progression, cell migration, and actin cytoskeleton dynamics, and their aberrant signaling is linked to numerous human diseases and disorders. Traditionally, active Rho GTPase proteins were proposed to reside and function predominantly at the plasma membrane. While this view still holds true, it is emerging that active pool of multiple Rho GTPases are in part localized to endomembranes such as endosomes and the Golgi. In this review, we will focus on the intracellular pools and discuss how their local activation contributes to the shaping of various cellular processes. Our main focus will be on Rho signaling from the endosomes, Golgi, mitochondria and nucleus and how they regulate multiple cellular events such as receptor trafficking, cell proliferation and differentiation, cell migration and polarity. The founding member of the Rho GTPase, termed Rho for Ras homolog, was identified in 1985 . ShortlyThe switch I and switch II domains of Rho GTPases undergo conformational change upon GTP-binding. Once active, Rho GTPases associate with membranes and selectively interact with downstream effectors and other scaffolding proteins to mediate a myriad of biological processes including reorganization of the actin cytoskeleton, regulation of membrane trafficking, cell motility and polarity. A large body of literature deals with signaling initiated by Rho GTPases and their effectors at or around the plasma membrane although intracellular pool of Rho GTPases and their regulators are increasingly apparent. It is becoming clear that coordination of functionally active Rho GTPases, their GEFs and GAPs, as well as their effectors extend beyond the plasma membrane to multiple intracellular organelles. Understanding how cells decode these spatio-temporal signals to generate biologic outputs is a major area of investigation.Here, we review emerging knowledge on biological processes and signaling events mediated by intracellular pools of Rho GTPases, with special emphasis on signaling patterns that are triggered or initiated by endomembrane localized Rho GTPases. In particular, we summarize recent studies that provide direct evidence for endosomal, Golgi, mitochondrial and nuclear pools of Rho GTPase signaling.The endosomal system consists of pleomorphic membranous carriers that processes and transports a range of cargoes including active signaling receptors. The ability of endocytic organelles in generating compartmentalized signaling patterns by directional shuttling and/or retaining of signaling molecules into specific locations within the cell is well documented and has been extensively reviewed elsewhere . In thisFrom the Rho GTPase family, RhoB was among the first ones to be predominantly detected in the endocytic compartments . Today, en route to the plasma membrane  of RhoB appeared functionally distinct from the geranylgeranylated  pool in that treatment of cells with farnesyl-transferase inhibitors, which abolishes the farnesylated pool of RhoB, resulted in an increased recycling of endocytosed epidermal growth factor (EGF) receptor . In agremembrane . This tyronectin . The exaronectin  suggestironectin . DepletiSeveral Rho family members regulate some aspects of actin cytoskeleton dynamics, and accordingly, multiple studies have demonstrated the importance of endosomal pool of Rho GTPases for actin-based endocytic vesicle movement. For example, a role for RhoD in controlling the endocytic vesicle movement has been documented. It was shown that cells possessing active RhoD display reduced velocity of early endosome movement, thereby slowing down the membrane trafficking events . This eftrans-Golgi network  that promote its nuclear translocation . Reporteignaling . Alternaignaling . On the ignaling . Moreoveignaling . Finallyignaling .Despite lacking a canonical nuclear localization signal, RhoA has been reported to translocate to the nucleus . When quAnother Rho GTPase with potential nuclear effects, but no clear nuclear localization signal is RhoB, which was shown to localize to the nuclear envelope, and to interact with and regulate the transcription factor DB1 . The Rhoin vivo suggests that nuclear Cdc42 activity might also be relevant in cancer  is localized to the ER-mitochondrial contact sites . The molSP and HF researched the relevant literature, conceived, and prepared 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.


Glutathionylation, that is, the formation of mixed disulfides between protein cysteines and glutathione (GSH) cysteines, is a reversible post-translational modification catalyzed by different cellular oxidoreductases, by which the redox state of the cell modulates protein function. So far, most studies on the identification of glutathionylated proteins have focused on cellular proteins, including proteins involved in host response to infection, but there is a growing number of reports showing that microbial proteins also undergo glutathionylation, with modification of their characteristics and functions. In the present review, we highlight the signaling role of GSH through glutathionylation, particularly focusing on microbial  glutathionylated proteins (GSSPs) and host GSSPs involved in the immune/inflammatory response to infection; moreover, we discuss the biological role of the process in microbial infections and related host responses. It is mainly localized in the cytosol (up to 90%), but it is also present in the mitochondria (10%), the endoplasmic reticulum, and the nucleus in a small percentage [The tripeptide γ-rcentage .2O2 level, which is a well-known intracellular messenger in signal transduction  has been known for many years. Initially considered an effect of oxidative stress and damage [However, today it is well known that the essential role of GSH overcomes its scavenger activity and it plays a crucial role in cell fate, regulating processes such as proliferation, differentiation, and apoptosis, and several other cellular functions, ranging from metabolism to immune and inflammatory response ,4,5,6. I reviews ,11,12,13 reviews . As for one –SSR . A prered damage ,17, it td damage ,19,20. Gd damage , where rd damage ,23.(1) It is known that several proteins undergo glutathionylation, resulting in different biological effects ,24. MostTherefore, this review is an attempt to highlight the signaling role of GSH through glutathionylation, particularly focusing on microbial GSSPs and host GSSPs involved in the immune/inflammatory response to infection.It is well known that viruses alter the intracellular redox state to pro-oxidant conditions, which is an alteration that contributes to viral pathogenesis ,29,30,31The first evidence for viral protein glutathionylation was provided by Davis et al. in 1996 . In factThe cellular redox proteome responds through modifications, in particular of cysteines, to environmental challenges, such as viruses . GlutathFinally, it must be borne in mind that a significant proportion of the “immunopeptidome”, that is, the peptides stimulating immune cells, have been shown to contain glutathionylated cysteines, with consequences for antigen presentation/T-cell recognition. It has been reported that glutathionylation of a coronavirus peptide reduced CD8 T-cell recognition, so this could be a redox mechanism for the virus to negatively impact the immune response .Listeria monocytogenes and Streptococcus agalactiae), while other Gram-positive bacteria produce LMW thiols, such as bacillithiol (BSH) and mycothiol (MSH), with similar functions. These functions, as in eukaryotic cells, include detoxification of different reactive species to maintain the redox balance of the bacteria cytoplasm. Recently, however, the direct role of GSH in virulence has been described for different bacteria. For example, it has been shown that GSH activates virulence gene expression in the intracellular pathogen L. monocytogenes [Staphylococcus aureus [Mycobacterium tuberculosis during infection [Growing evidence highlights the importance of LMW thiols and GSH in bacteria ,68. GSH ytogenes . Similars aureus ,71, and nfection .Salmonella typhimurium, a top-down proteomic approach identified over 500 proteins with 1665 post-translational modifications, among which 25 were s-thiolation. Interestingly, nine s-glutathionylated proteins switched to s-cysteinylation under infection-like conditions, suggesting that this shift could be a novel redox mechanism of infection control [L. monocytogenes, is reversibly inhibited by glutathionylation. Interestingly, a mutant of LLO unable to undergo this post-translational modification was less virulent in vivo, suggesting that the modification could be important to restrict the pore-forming capability to an appropriate temporal and/or spatial compartment during infection.Thanks to advances in proteomic studies, a growing number of bacteria proteins have been identified to be S-glutathionylated, S-bacillithiolated, or S-mycothiolated, making it plausible that S-thiolation could be the mechanism of redox regulation for these proteins. In  control . Portman control  demonstrPseudomonas aeruginosa, has been shown to be glutathionylated after caspase-dependent cleavage of Grx1. Glutathionylation of Fas then resulted in an amplification of apoptosis [P. aeruginosa pneumonia, showing that the modification promoted bacteria clearance. Overall, these results indicate the importance of the Grx1/Fas glutathionylation axis in the resolution of the disease [Borrelia burgdorferi infection, possibly through glutathionylation. Moreover, GSH-related metabolites have been found to be altered in patients with early onset of Lyme disease and these alterations persist for months after primary infection. Thus, it has been suggested that GSH metabolism and glutathionylation could be important processes in the pathogenesis of the B. burgdorferi-induced Lyme disease and, potentially, other inflammatory diseases as well [Regarding cellular proteins involved in the response to bacterial infection, the death receptor Fas, known to be activated by several pathogens, including poptosis . In a su disease . Recentl as well .The final section of this review is dedicated to a brief analysis of glutathionylation in inflammation, focusing on nuclear factor kappa β transcription factor (NFκB)’s glutathionylation.2O2 also induced p65-glutathionylation and its retention in the cytoplasm, leading to an increase of the apoptotic rate of hepatocytes [NFκB is a transcription factor that plays an important role in the activation of proinflammatory response. Under physiological conditions, NFκB is sequestered in the cytoplasm in a latent form by inhibitor κB (IκB). After phosphorylation by IκB kinase (IKK), IκB undergoes degradation via proteasomes and NFκB is then free to move into the nucleus, where it induces transcription of proinflammatory genes. Various studies have shown that glutathionylation, through the bond of GSH to the DNA binding domain, is able to regulate the activity of many transcription factors, including NFκB ,79. In patocytes . Besidesatocytes .Moreover, IKK, the protein responsible for IκB phosphorylation and NFκB translocation to the nucleus, can be inactivated by S-glutathionylation of Cys-179 . So, theJones et al.  demonstrFinally, we have to mention that interleukin-1β, together with a group of cytokines, has been shown to undergo glutathionylation itself . In partIn this review, we underscored the role of glutathionylation in viral/bacterial infections and host response. It is well known that infections, particularly viral infections, cause a redox imbalance towards pro-oxidant conditions in the cell, and we believe that glutathionylation fits into these virus-induced changes to regulate its replication and/or cell response. However, based on the available knowledge, we may speculate that the glutathionylation/deglutathionylation process is not a mere consequence of the oxidative burst but a finer regulation of the different phases of viral replication. In particular, glutathionylation could inhibit the premature activation of some viral protein functions, as in the case of retroviral proteases, which would be harmful for the cells and the virus and promote their activation at the optimal timing for viral maturation and spread. In a similar way, the process could play a role in bacterial infections, representing a potential mechanism of virulence. At the same time, the inflammatory cell response to infections could be affected, for example, by the redox processes regarding NFκB activation. This nuclear transcription factor was among the first identified to be redox modulated and strictly involved in inflammation, viral replication, and cell proliferation/apoptosis. The transient glutathionylation of NFκB could also take part in the regulatory processes committed by infections.Overall, a deeper knowledge of the chemical and regulatory processes through which glutathionylation occurs could pave the way for the identification of novel targets to control microbial infections and related host responses.


We provide an analysis of the main sources of data used to estimate fertility schedules in developing countries, giving special attention to Brazil. In addition to the brief history of various data sources, we present several indirect demographic methods, commonly used to estimate fertility and assess the quality of data. From the methods used, the Synthetic Relational Gompertz model gives the most robust estimates of fertility, independent of the data source considered. We conclude that different demographic data sources and methods generate differing estimates of fertility and that the country should invest in quality of birth statistics.The online version of this article (10.1186/s41118-018-0035-9) contains supplementary material, which is available to authorized users. In less developed and developing countries, there is often a common concern among demographers about data quality, especially concerning the vital registration systems, and a strong consensus about the necessity to improve vital statistics  has a long history of data production about demographic and socioeconomic characteristics for Brazil and its regions. The most important data source is the population census, which occurs every 10 years. Censuses offer broad possibilities for population research and its demographic components, such as fertility, mortality, and internal migration.In Brazil, as in other Latin American countries, there is a long tradition in collecting information on fertility and mortality in population censuses, even before the year 1960. In the 1940 census, the question on total (and surviving) live births to women of age 15+ was included (since 1991 to women of age 10+). In 1970, the information on children born in the previous 12 months before the survey was included. Since then, the use of such data has increased, especially in combination with indirect techniques, based on data from “children ever born” information, which allow us to estimate average parity (P), and “the number of births in the 12 months previous to the census” that produces cumulated current fertility (F), and the relationship between these two measures was used to estimate the correct level of total fertility rates (TFR) Brass , as wellDespite being the main source of demographic information, there are many issues with census quality. For one thing, census coverage varies according to the year of the inquiry: for the four last censuses, for example, under-enumeration ranged from 1.8% in 1980 to 2.5% in 2010 IBGE .Household surveys are the most widespread tool for gathering social, economic, and demographic information on population. They are important tools to provide intercensal information. The current household survey system in Latin American countries dates back to the early 1960s, when many countries initiated programs designed to measure employment, unemployment, and other labor force characteristics CEPAL . Over tiAs living conditions require more detailed information and variables that are not strictly related to the labor market and economic activity, these surveys started to include more detailed issues such as housing conditions and demographic variables . In the case of Brazil, the National Household Surveys (PNAD) since 1972 have included questions related to month and year of birth of the last child born alive, and the number of children ever born, within and outside the household IBGE .Since these surveys are performed on a yearly basis, they can also serve as an up-to-date source of fertility information in the country, but a number of issues concerning sample design  are considered the gold standard for fertility estimation in many developing countries. They use structured questionnaires that contain detailed information of demographic events, but during the years, the range of information collected has broadened in scope to include gender issues (surveys increasingly include both men and women), HIV/AIDS, maternal mortality, violence against women, nutrition etc. In general, they characterize the female population in childbearing age and children under the age of five according to demographic, socioeconomic, and cultural factors. Patterns of marriage, parenting, and female reproduction are identified, as well as profiles of morbidity and mortality during the infancy and breastfeeding phases, and many other reproductive and health aspects DHS .Despite their limitations, the full birth histories have become the dominant source of estimates of fertility levels and trends for countries lacking complete birth registration  from a period with the average parity distribution (P), measurement of cohort fertility (United Nations P as the average parity (cumulated lifetime fertility) of a cohort of women up to a given age and F to be closely related to the cumulated current (period) fertility up to that age. The P/F ratio expresses relation of these two quantities for each age group  would be greater than cumulated current fertility (F). In this case (and in the absence of errors in the data), the P/F ratio would depart from unity systematically with increasing age of mothers. We would expect the P/F ratio to be fairly close to unity at the youngest ages, because even by women’s mid-twenties, one would not suppose any significant deviation of cumulated period fertility from cumulated lifetime cohort fertility, as most of the births to women in that cohort would have happened fairly recently.When fertility is falling, the cumulated lifetime fertility  model is a conceptual improvement of the Brass P/F method and an extension of the relational GompertzIn concrete terms, the Gompertz relation assumes that the proportion of accumulated fertility follows a linear distribution of the following equation:F (x) is the accumulated fertility up to age x, TFR the total fertility, and α and β are two parameters. This function can be reduced to a linear form based on the following log transformation:where α and β. In this model, α is an indicator of the location of the fertility pattern in relation to age, while β is interpreted as the determinant of the dispersion or degree of concentration of the fertility pattern. In short, we can say that a negative value of α means a curve that is more inclined to the right while a positive one indicates a more left-skewed curve. With respect to β, a value greater than 1 is represented by a narrower curve and a value smaller than 1 by a wider curve.In this sense, we use an equation that is quite similar to the Brass logit, because the purpose of both is to perform a linear transformation of a distribution so that it can be compared to a standard distribution using two constants, The relational Gompertz method seeks to correct the errors commonly found in fertility data associated with too few or too many births being reported in the reference period and the under-reporting of lifetime fertility and errors of age reporting among older women. In sum, the Gompertz method has the additional advantage of smoothing age-specific fertility rates, a major issue when dealing with sub-national populations, and reduces potential problems of reporting errors in parity by older women. One important aspect is that the method requires some knowledge of the demographic patterns in the study area  is a census- or survey-based reverse survival technique designed to estimate age-specific fertility for the period preceding the enumeration  to the respective mother (from 15 to 64 years of age) in the census data is based on the information about their relationship to the head of household, to other women in the household, and the mothers’ information on the number of children ever born and children surviving. Once the data were matched, we tabulated age of children by age of their mothers. In some cases, it was not possible to match children with their mothers. This occurs either because the information about the relationship to the head of the household is insufficient or because some children do not live in the same household as their mothers or because the mother has deceased. These children are so-called “other than own” or simply “not-own” children in the household. They are proportionally distributed by age of mother using the age distribution of the mothers with identified children of their own. Besides, this addition of “not-own” children, age misreporting, and age under-enumeration can bias the data as well. Finally, a survivorship function is applied to generate the number of births by age of mother for the years prior to the survey (Retherford and Cho www.fertilitydata.org). The HFC web page there gives series of fertility rates for Brazil as a whole, estimated for the years 1966 to 2010  State. Rio Grande do Norte is one of the smallest states in Brazil and has been going through rapid changes in fertility. We used this sub-population as example for two main reasons: first, because Rio Grande do Norte is located in the Northern region of the country, a part of the country historically known for having the worst data quality IBGE . Second,Further tabulations are also made for the country as a whole, but only for census years 2000 and 2010. We make use of birth registers publicly available by the Ministry of Health Database (DATASUS) and other official estimates provided by the National Statistical Office (IBGE), the Human Fertility Collection HFC  and PNDSFigure For the test of methods performance, each time we created two groups of estimates, one without correction, i.e., the observed data extracted directly from the censuses, and second with level correction in age-specific fertility rates, given by P/F ratio from the 20–24 age group. Apart from that, fertility schedules were estimated between censuses, using the Synthetic Relational Gompertz model, without and with P/F ratio adjustment.TFR 5.11 (1970) ⟹ 5.46 (1975) ⟹ 4.09 (1980) ⟹⟹Unadjusted dataTFR 7.18 ⟹⟹⟹⟹5.50 ⟹⟹⟹⟹ 4.72 ⟹⟹⟹⟹ P/F adjusted dataFrom 1970 to 1980, Fig. Taking into account that past fertility was considerably higher in the State of Rio Grande do Norte and falling rapidly, one can argue that P/F adjusted data is more plausible.In the second period, Fig. TFR 4.09 (1980) ⟹ 3.63(1986) ⟹ 2.45 (1991) ⟹⟹Unadjusted dataTFR 4.72 ⟹⟹⟹⟹ 3.63 ⟹⟹⟹ 2.83 ⟹⟹⟹⟹⟹P/F adjusted dataThe TFR estimated for the intercensal period by the SRG did not change in value (TFR of 3.63). The same can be said about the pattern of fertility schedules, which practically overlapped. Moreover, once again, we have two scenarios of fertility change to choose.Since we did not observe any changes in both pattern and fertility level estimated by SRG, we take this estimate (middle year between censuses) as a benchmark for comparison. In the first scenario, when we analyze the first 5-year period, fertility fell by almost 11% and by 33% from 1980 to 1991. The second scenario shows a fertility drop of 23% (between 1986 and 1986) and 22% (between 1986 and 1991). Despite small differences, it is more plausible that the biggest drop occurred in 1980–1986 (scenario 2), while we expected the strongest fertility declines to have taken place in the past, when fertility levels were higher.TFR 2.45 (1991) ⟹ 2.44 (1996) ⟹ 2.06 (2000) ⟹⟹Unadjusted dataTFR 2.83 ⟹⟹ ⟹ 2.44 ⟹⟹⟹ 2.30 ⟹⟹⟹⟹ P/F adjusted dataFrom 1991 to 2000, Fig. Both possibilities are plausible, but it is more likely to believe that from 1991 to 1996, fertility dropped instead of remaining stable. Therefore, we disregard the first scenario, without P/F ratio correction.TFR 2.06 (2000) ⟹ 1.80 (2005) ⟹ 1.53 (2010) ⟹⟹Unadjusted dataTFR 2.30 ⟹⟹ ⟹ 1.80 ⟹⟹⟹ 1.84 ⟹⟹⟹⟹ P/F adjusted dataFor the last period of analysis 2000 to 2010, Fig. Unlike in previous analyses, the second scenario with fertility correction in 2010 seems less plausible: first, because the likelihood that fertility has increased between 2005 and 2010 is rather low, and second because it is hard to believe that the Brass method is reliable in this case. As a first sign, the P/F of Brass seems to work fine even when the condition of stability is violated. However, if the decline of fertility affects younger ages, the method leads to misleading level correction. On the other hand, the SRG model gives more robust results, independent from the scenario chosen.In this section, we compare both methods with the estimates generated by the Own-children method, but this time using different data sources, i.e., information derived from the civil registration and censuses for Brazil as a whole. Table The fertility schedules differ slightly between the sources. However, the adjustment by the Brass method generates different estimates, according to the source used. The civil records always present lower fertility levels than census data, and the P/F ratio also points to small corrections necessary (4% in 2000 and 2% in 2010), to civil registration data  with civil records. However, when taking into account census data, there are differences in virtually all ages. The estimates for the OCM show a fertility peak at the age group 25–29, whereas estimates based on other sources still present the 20–24 age group at the highest reproductive level , and whether or not P/F corrections are applied to fertility levels, which may be an indication of the robustness of the method. In addition to that, the average parity, contained in the synthetic cohort and estimated by the SRG, seems to capture quite well the reproductive changes during the intercensal period, just as the model appropriately confines the pattern inherent in these rates.On the other hand, even with similarity in fertility level, the PNDS  shows a In this paper, we have provided an overview of the data sources and methods used to estimate fertility, level, and age-profile, in developing countries, using Brazil as a case study. Additionally, in the absence of high-quality vital registration systems, census data and household surveys, such as DHS, are the most common sources of information to investigate the evolution of fertility and its differentials.Vital registration systems are the most desirable source for demographic analysis, since they usually contain up-to-date information and have a broad geographical coverage. However, the majority of countries in Latin America, including Brazil, are still lacking a well-structured institutional system for collecting such information  P/F ratio of Brass, (2) Synthetic Relational Gompertz models, and (3) Own-children method of retrospective reconstruction of fertility schedules. We perform a series of analysis using these methods and investigate how they perform, considering different data sources and demographic circumstances. There is a wide variation in estimates as they require specific conditions, i.e., stable population, which cannot always be fulfilled, especially in the context of rapid fertility decline. We show and argue that Brass P/F potentially overestimates the country’s TFR in recent periods, as also observed by Castanheira and Kohler .Different data sources yield different results, giving a significant variability in TFRs and ASFRs. They can vary somewhat from above- to below-replacement level and present different age schedules, depending on the method and the data. Insofar, we conclude that these estimates can be very misleading, especially in the context of sub-replacement fertility that some countries in the region have been facing recently


Cardiac fibroblasts (CF) play a critical role in post-infarction remodeling which can ultimately lead to pathological fibrosis and heart failure. Recent evidence demonstrates that remote (non-infarct) territory fibrosis is a major mechanism for ventricular dysfunction and arrhythmogenesis. β-arrestins are important signaling molecules involved in β-adrenergic receptor (β-AR) desensitization and can also mediate signaling in a G protein independent fashion. Recent work has provided evidence that β-arrestin signaling in the heart may be beneficial, however, these studies have primarily focused on cardiac myocytes and their role in adult CF biology has not been well studied. In this study, we show that β-arrestins can regulate CF biology and contribute to pathological fibrosis. Adult male rats underwent LAD ligation to induce infarction and were studied by echocardiography. There was a significant decline in LV function at 2–12 weeks post-MI with increased infarct and remote territory fibrosis by histology consistent with maladaptive remodeling. Collagen synthesis was upregulated 2.9-fold in CF isolated at 8 and 12 weeks post-MI and β-arrestin expression was significantly increased. β-adrenergic signaling was uncoupled in the post-MI CF and β-agonist-mediated inhibition of collagen synthesis was lost. Knockdown of β-arrestin1 or 2 in the post-MI CF inhibited transformation to myofibroblasts as well as basal and TGF-β-stimulated collagen synthesis. These data suggest that β-arrestins can regulate CF biology and that targeted inhibition of these signaling molecules may represent a novel approach to prevent post-infarction pathological fibrosis and the transition to HF. Heart failure (HF) is a frequent complication of myocardial infarction (MI) that is associated with adverse ventricular remodeling . As a reCardiac fibroblasts (CF) make up 60–70% of the total cell number of the heart and play a critical role in regulating normal myocardial function as well as in the adverse remodeling that can occur post-MI and the transition to HF. CF are responsible for ECM deposition and create the scaffold for cardiac myocytes . Cardiacin vitro proline incorporation was measured according to the method of Swaney et al [3H]proline  for 48 hours in either 2.5% FBS with 10ng/mL TGF-β, 10μM ISO, or no drug. Cellular protein was precipitated overnight with 20% trichloroacetic acid (TCA) and washed 3 times with 1ml of 5% TCA plus 0.01% proline, then dissolved in 0.2 M NaOH. The activity of [3H]proline was determined by liquid scintillation counting.proline incorporation. Normal control CF demonstrated a low level of basal collagen synthesis and a robust increase in response to TGF-β stimulation  territories of the left ventricle. This is temporally associated with CF transformation to activated myofibroblasts and increased collagen synthesis and deposition. Specific inhibition of β-arrestin1 or β-arrestin2 expression using an siRNA approach led to inhibition or reversal of the myofibroblast phenotype and decreased collagen and fibronectin expression and collagen synthesis. These effects occurred under basal conditions where the post-MI CF had a very robust elevation in collagen expression compared to non-infarct control CF. In addition, knockdown of β-arrestins also inhibited TGF-β-stimulated α-SMA and collagen I expression and collagen synthesis. This is consistent with our previous work in isolated adult human CF showing that this is mediated through inhibition of MAPK/Smad signaling .Recent work has shown that cAMP can have an antifibrotic action in CF through multiple intracellular signaling pathways. One is the inhibition of Smad-mediated transcription via competition between cAMP response element binding protein (CREB) and Smad for key transcriptional co-activators . AnotherBased on recent work from our group and others, another important mechanism may be inhibition of β-arrestin signaling. We have shown that β-arrestin overexpression in normal adult human cardiac fibroblasts leads to increased basal collagen synthesis that is mediated through ERK and Smad signaling . This wa1-adrenergic receptor transactivation of the EGFR has been shown to be cardioprotective in mice subjected to chronic sympathetic stimulation [Although the role of β-arrestins in receptor uncoupling and internalization is well known, more recent work has revealed that β-arrestins are multifunctional scaffolding proteins that are involved in numerous aspects of G protein-coupled receptor signaling. β-arrestins can mediate signaling independent of classical G protein-mediated signaling, and this may be protective in the heart . β-arresmulation .Our data shows that β-arrestin1 and 2 expression in isolated rat CF is upregulated early post-MI and that knockdown of either subtype results in decreased α-SMA and collagen I expression. Baseline and TGF-β-stimulated collagen synthesis were also significantly decreased in these CF after β-arrestin knockdown. These findings suggest that chronically upregulated β-arrestin signaling in fibroblasts is maladaptive and that the beneficial effects seen in other studies may be more specific to cardiac myocytes. Our data provide additional evidence that inhibition of β-arrestins in fibroblasts can reverse the myofibroblast phenotype and inhibit collagen synthesis and fibrosis.in vivo fibroblast-specific inhibition of β-arrestins will provide further insight into the role of these signaling molecules in ECM remodeling and the progression to HF.In conclusion, this study provides support for the potential therapeutic role of inhibiting β-arrestin activity following injury in a CF-specific manner to prevent pathological fibrosis and remodeling. The primary mechanisms for inhibition of transformation or activation of quiescent fibroblasts to myofibroblasts and collagen synthesis/fibrosis appear to be restoration of β-AR signaling and intracellular cAMP as well as decreased β-arrestin signaling. Future studies with


Recently, extensive efforts have been made to understand the importance of the extra-renal uric acid (UA) excretion pathways and their contribution to UA-related diseases. However, the method typically used to measure UA concentrations in the intestinal lumen is difficult to real time and dynamic analysis. In this study, UA excretion in the rat intestinal lumen was measured in real time using an electrochemical method. A sensitive electrode to detect UA was constructed using a gold electrode modified with a mixed self-assembled monolayer. Excretion rate of UA in the intestine was calculated using time course data. A decrease in UA excretion rate was observed in the intestine after administration of serum UA-lowering drugs . Inhibition of ATP-binding cassette transporter G2 (ABCG2) which has been reported as an important exporter of UA was suggested by administration of these drugs. On the other hand, an increase in excretion rate of UA was observed in the intestine of 5/6 nephrectomy rats. Upregulation of mRNA expression of the UA transporter organic anion transporter OAT3, which is related to the secretion at the basal membrane, suggested an enhancement of UA excretion by ABCG2, a high-capacity UA exporter. Observed urate excretion dynamics and mRNA expression of UA transporters in the intestine upon administration of serum UA-lowering drugs and 5/6 nephrectomy improve our understanding of the underlying mechanisms of intestinal UA excretion. Uric acid (UA) is the end product of purine metabolism in humans. Serum UA levels are determined by the balance between UA production and UA excretion rates. High serum UA levels, which are considered to be higher than 7 mg/dL, are diagnosed as hyperuricemia. This condition induces gout and accelerates the progression of renal and cardiovascular diseases –3. On thRenal transport of urate  is known to be the main urate excretion route. A number of urate transporters have been identified in renal proximal tubule cells . Among tAbcg2-knockout mice showed a reduction in intestinal UA excretion and an increase in serum UA levels; however, the urinary UA excretion was significantly increased in these mice -UA in ABCG2-expressing membrane vesicles indicated that the general serum UA-lowering drugs topiroxostat, benzbromarone, and febuxostat drastically inhibited the UA transport activity of ABCG2.Serum UA-lowering drugs for hyperuricemia and gout treatment fall into two categories: uricosuric agents and UA production inhibitors. The effect of serum UA-lowering drugs on the urate transport activity of ABCG2 has been reported . The tra14C] urate transport ex vivo [Further investigation on the excretion of UA to the intestinal lumen is required in order to fully understand the contribution of this UA excretion pathway to UA-related diseases; however, the general method used to measure UA excretion in the intestine requires the resection of intestinal specimens from animals and measurement of [ ex vivo , 15. Eve ex vivo . In this6-Fc) and 6-Hydroxy-1-hexanethiol (HS-C6-OH) were purchased from Dojindo Laboratories . Febuxostat and benzbromarone were purchased from TCI chemicals , and Wako Pure Chemical Industries , respectively. Topiroxostat were kindly provided form Sanwa Kagaku Kenkyusho Co., LTD . All other chemicals were commercial products of reagent grade.6-Ferrocenyl-1-hexanethiol , but SAM surface compositions were not determined.Gold bead electrodes were prepared by melting φ = 0.2 mm gold wire 99.999 from Unique Medical Co., Ltd  in flame. The procedure for the preparation of clean gold bead electrodes has been previously reported . The cryIntegrated devise with 0.8 mm diameter which was fixed Pt wide and Ag/AgCl wire was constructed by Unique Medical Co., Ltd . Gold bead electrode was fixed into the integrated devise for a three electrode measurement. Prepared measurement device was fixed in intestinal closed loop.Male SD rat (10 week of age) and male SD 5/6 nephrectomy (Nx) (Severe) rat (10 week of age) were purchased from Japan SLC, Inc. . Surgical resection of the upper and lower thirds of the left kidney was performed at 8 week of age, then a right subcapsular nephrectomy was performed at 9 week of age. The animals were housed at room temperature (24 degree) with relative humidity (55 ± 5%). They were fed a standard chow  with free access to tap water and kept on a 12-h:12-h light-dark cycle. All studies were carried out in accordance with the Institutional animal care committee at the Tokyo University of Pharmacy and Life Sciences.Topiroxostat, febuxostat and benzbromarone were suspended in methylcellulose, because the solubility of these drugs are very low. The prepared UA lowering drug solution was orally administrated to rat (50 mg/kg). A group orally administrated only methylcellulose was used as sham group. In this study, to avoid data variation based on the administration, excess amount of drugs compared dose prescribed in each interview form were administrated to rats. After two hours of oral administration, closed loop was constructed in intestine detailed below, then UA excretion was measured with electrochemical method.Rats were anesthetized using pentobarbital (20 mg/Kg) with intraperitoneal administration and kept isoflurane with inhalation anesthesia. A midline abdominal incision was made. The intestinal segment was manipulated in order to minimize any intestinal blood supply disturbances. The 10 cm upper portion of the cecum was hemocliped with metal clip , then end of the cecum was cut. In order to remove all intestinal contents, the small intestine was flushed with a saline. To avoid the effect of other molecule than UA in intestine on the measurement, the appropriate amount of saline for flash was investigated by HPLC measurement (PU-980 Intelligent HPLC pump and UV-970M UV Ditector (Hitachi High-Tech Science corp.) with Supelcosil LC-18DB column (Sigma-Aldrich Co. LLC.). Potential molecules which affect a current change in electrochemical measurement, for example ascorbic acid, were confirmed that it did not exist. End of the cecum was hemocliped with a plastic disposable clip , and slight upstream was tie off with suture . A slight downstream from the inlet was cut and electrode devise was inserted into intestine and fixed with suture. The constructed closed loop at ileum with electrode device (about 3 cm length) was filled with 0.2 mM phosphate buffer (pH 7.8) containing 0.3 mM potassium oxonate.Cyclic voltammetry (CV) and amperometry were measured by ALS electrochemical analyzer . Calibration curve of the constructed measurement device was demonstrated each time with CV by changing UA concentration. Obtained amperometric data in intestine for 200–1400 sec were used for a regression expression. The correlation coefficient was used as change in current per second. Change in UA concentration per second was calculated by using calibration curve of each electrode.™ . Primers are shown in Supporting Information . Total RNA was extracted by using NucleoSpin RNA Plus . After measuring the total RNA concentration using NanoPhotometer P-330 , cDNA was generated from 500 ng total RNA with PrimeScript RT reagent kit  . Quantitative RT-PCR was performed using Thunderbird SYBR qPCR Mix  and StepOneormation . ExpressP < 0.05 was considered to be significant.Since data were not normally distributed, the values presented here correspond to the median for each group. Unpaired Student’s t-test was used to compare the control group and groups treated with drugs. A value of 6-Fc:HS-C6-OH = 1:3 showed the highest sensitivity of UA detection compared with SAM modified with HS-C6-Fc:HS-C6-OH = 1:1 and 3:1 (see 2) modified with mixed SAM (HS-C6-Fc:HS-C6-OH = 1:3) showed current change in 1.8 E-9 A by alteration of UA concentration in 0.1 μg/mL. Hereafter, the gold bead electrode modified with mixed SAM (HS-C6-Fc:HS-C6-OH = 1:3) was used for further investigation in this study.Gold bead electrodes modified with mixed SAM were set in the integrated device and current change based on UA concentration was measured in CV and amperometry.  3:1 see . The golBy fixing the electrode device in the closed intestinal loop of naïve rats, a current increase with time was observed  showed a clear amplification curve and were compared between groups  and from the serosa to the mucosa, the majority of the transporters responsible for such permeability, particularly those on the basal membrane, are still unknown . On the n groups . Relativ tubules , 25.Km of 8.2 mM related with the transport of UA [The excretion rate of UA in the rat intestine after administration of serum UA-lowering drugs  was decreased. Among these drugs, febuxostat was able to induce the most significant decrease (p<0.005). Febuxostat has shown to exert a strong inhibition of ABCG2, both in vivo and in vitro, and to induce an increase of the intestinal absorption of an ABCG2 substrate . Benzbrort of UA . Based oOat3 in the intestine showed a tendency to increase in the groups treated with UA-lowering drugs, especially topiroxostat, compared with the sham group. Oat3 is known to be abundantly expressed on the basal membrane and excrete a wide variety of drugs and exogenous toxins, including uremic toxins [Oat3 mRNA expression after administration of UA-lowering drugs might suggest an increase in the excretion of these drugs. On the other hand, no significant changes were observed in relative mRNA expression of the examined transporters located in the apical membrane, MRP4 and ABCG2. These have been reported as efflux transporters for drugs and other molecules [Moreover, relative mRNA expression of c toxins . Increasolecules , 27. ThuAbcg2 in the ileum measured by real time PCR has been reported [Abcg2 showed no significant changes. Probably, the portion of the intestine examined in our study presented different expression levels. Even in the literature, no significant difference in the expression of Abcg2 was observed, except in the ileum, duodenum, jejunum, and transverse colon of Nx rats. Similarly to Abcg2, Mrp4 showed no significant changes in mRNA expression in the intestine. MRP4 has been reported to be a transporter in UA excretion with a calculated Km of 1.5 mM [Mrp4 and Abcg2 suggests that the UA exporter capacity of MRP4 and ABCG2 was not saturated relatively to the UA concentration in epithelial cells. On the other hand, relative mRNA expression of Oat3 showed a tendency to increase in the Nx group (approximately 104 in median) compared with the sham group. OAT1 and OAT3 have been reported to be UA transporters contributing to its secretion at the basal membrane in proximal tubules [Km of OAT1 and OAT3 in the transport of UA were 0.9 mM and 2.9 mM, respectively [In the present study, excretion rate of UA showed a tendency to increase in the Nx group compared with that of the sham group, though without statistical significance. It has been previously reported a significant decrease in urinary excretion of UA in Nx rats, but no significant differences were observed in serum UA levels in this case . A compereported . In our f 1.5 mM . The lac tubules . Moreove tubules . The calectively , 29. IncIn conclusion, a dynamic analysis of UA concentration in the rat intestine was achieved with an electrochemical method. Observed excretion rates of UA in the intestine were in agreement with the in vitro results reported in literature. Furthermore, the observed excretion dynamics of UA and the mRNA expression of UA transporters improve our understanding of the underlying mechanisms of intestinal excretion of UA. However, we consider that further examination is necessary to confirm the suggested mechanisms.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.