Adsorbable organic halogens (AOX) are formed in pulp bleaching as a result of the reaction of residual lignin with chlorine dioxide. The natural structure of lignin is very complex and it tends to be damaged by various extraction methods. All the factors can affect the study about the mechanism of AOX formation in the reaction of lignin with chlorine dioxide. Lignin model compounds, with certain structures, can be used to study the role of different lignin structures on AOX formation. The effect of lignin structure on AOX formation was determined by reacting phenolic and non-phenolic lignin model compound with a chlorine dioxide solution. Vanillyl alcohol (VA) and veratryl alcohol (VE) were selected for the phenolic and non-phenolic lignin model compound, respectively. The pattern consumption of lignin model compounds suggests that both VA and VE began reacting with chlorine dioxide within 10 min and then gradually steadied. The volume of AOX produced by VE was significantly higher than that produced by VA for a given initial lignin model compound concentration. In a solution containing a combination of VA and VE in chlorine dioxide, VE was the dominant producer of AOX. This result indicates that the non-phenolic lignin structure was more easily chlorinated, while the phenolic lignin structure was mainly oxidized. In addition, AOX content produced in the combined experiments exceeded the total content of the two separate experiments. It suggested that the combination of phenolic and non-phenolic lignin structure can promote AOX formation. AOX het al. [et al. showed that the degradation of phenolic and non-phenolic lignin model compounds contains three independent parallel reactions. Hamzeh et al. [The simplest methods to research these structural changes employ lignin model compounds –18. Manyh et al.  indicateh et al. ,26. Howeh et al. . Theoreth et al. –30. It iIn this research, the effect of lignin structures on AOX formation was studied using phenolic and non-phenolic lignin model compound. 4-Hydroxy-3-methoxybenzyl alcohol  and 3,4-dimethoxybenzyl alcohol  were selected as the phenolic and non-phenolic lignin model compound, respectively. The effect of lignin structures on AOX formation can be studied easily using model compounds but would be difficult to elucidate using the natural lignin macromolecule. The aim of the present work was to assess the effect of the combination of different lignin structures on AOX formation by the reaction of phenolic and non-phenolic lignin model compound with chlorine dioxide in solution. These results provide insight into the mechanism of AOX formation in the reaction of lignin with chlorine dioxide in pulp bleaching.2.2.1.−1. All other reagents were analytical grade and purchased from Aladdin .Vanillyl alcohol (VA) and veratryl alcohol (VE) were purchased from Aladdin . Activated carbon and ceramic cotton were purchased from Analytic-Jena Instrument Company . Chlorine dioxide solution was procured from a paper mill located in Guangxi, China, and had an effective chlorine concentration of 18.5 g l2.2.Chlorine dioxide solution was transferred into a conical bottle and adjusted to pH 3 with sulfuric acid. Then, the respective lignin model compound was transferred to the chlorine dioxide solution and reacted at 70°C with magnetic stirring at 150 r.p.m. The reaction was terminated by the oxidation of sodium sulfite with residual chlorine dioxide. In addition, the reaction mixture was diluted immediately in deionized water to achieve reaction termination. The reaction mixture was stored in a refrigerator and analysed as soon as possible.2.3.The AOX content of the reacting solution was measured using a MultiX2500 AOX analyser . The general methods were as follows: the pH of the reaction solution was adjusted to 2 with nitric acid. Then, 100 ml of the diluted solution and 50 mg of activated carbon were transferred into a conical bottle and shaken for 60 min in a thermostatic oscillator at a speed of 150 r.p.m. at room temperature (25°C). Quartz filter cups were used to collect the activated carbon, and the adsorbed inorganic chloride was washed with sodium nitrate. The water was drained, and the quartz filter cup and activated carbon were then combusted in a furnace. The AOX content was quantified by the microcoulomb titration method ,32.2.4.−1, a temperature of 50°C and UV detection at 280 nm [The mass of the lignin model compounds was measured using high-performance liquid chromatography . Both model compounds (phenolic or non-phenolic) were analysed on a Diamonsil C18 column  using a mobile phase composed of acetonitrile–water (10/90) (vol) and 0.1% acetic acid, a flow rate of 0.3 ml mint 280 nm .2.5.The chemical composition of the reaction solution was quantitatively determined by GC–MS  with a VF-1701 MS column (30 m × 250 µm × 0.25 µm). The basic method and process were described by Yao and co-workers .3.3.1.−1. Sampling analysis was performed at 2, 4, 6, 8, 10, 15, 20, 30, 40, 50 and 60 min of reaction time.The AOX content and consumption of lignin model compounds were measured over the course of the reaction to compare the reaction of chlorine dioxide solution with phenolic and non-phenolic lignin model compounds. Each individual reaction was performed in a solution with an initial lignin model compound of 6.2 mmol and concentration of 124 mmol l−1 at 2 min to 232 mg l−1 at 10 min and then steadied at increased reaction times. One major reason for this trend is the rapid consumption of VA . VA containing phenolic structure has higher reactivity. Phenolic hydroxyl groups were converted into free radicals, for it was easily attacked by chlorine dioxide and then the oxidation fracture of the benzene ring. Most free radicals are further oxidized by chlorine dioxide to form chlorolipids. It was further decomposed into muconic acid, ortho-quinone, para-quinone and oxacyclopropane structures for its unstable structure. Oxidative fracture of aromatic structures was the main reaction of VA in chlorine dioxide solution. Therefore, the lower content of AOX was generated from VA. VE has lower reactivity due to the absence of phenolic hydroxyl group. The hydroxymethyl side chain was easily oxidized by chlorine dioxide. Then chlorine substitution occurred on the aromatic ring. More content of AOX was generated at a lower reaction rate. When the concentration is 248 mmol l−1, the AOX content formed by the reaction of VE with chlorine dioxide solution was 1345.6 mg l−1 and almost triple the quantity formed by VA with chlorine dioxide solution (466.4 mg l−1). Unsurprisingly, at identical initial model compound concentrations, the AOX content produced by the reaction of VE with chlorine dioxide solution was higher than that by the reaction of VA with chlorine dioxide solution. In addition, the gap in AOX formation between the two separate reactions widened with an increase in concentration, which indicates that the reaction of VE with chlorine dioxide is more easily influenced by concentration.The AOX content formed by the reaction of VE with chlorine dioxide solution was 60.9 mg l−1, and the molar ratio of VA and VE was 1 : 1.The effect of the total concentration of VA and VE on AOX formation in the combined reaction was studied. The combined initial concentrations of lignin model compounds were 12.4, 62, 124, 186 and 248 mmol l−1 for a total concentration of 124 mmol l−1, which is 10-fold higher than that at a total concentration of 12.4 mmol l−1 (73.6 mg l−1). This suggests that the effect of total concentration on AOX formation is larger at concentrations below 124 mmol l−1. However, the AOX content cannot increase significantly any more once the total concentration exceeds 124 mmol l−1.The comparison of the AOX content of separate and combined experiments of VA and VE with chlorine dioxide solution is presented in 3.3.−1. The molar ratio of VA and VE was 0/1, 0.1/0.9, 0.3/0.7, 0.5/0.5, 0.7/0.3, 0.9/0.1 and 1/0, respectively. The AOX formation was analysed after reacting for 60 min under the condition of bleaching.The effect of the molar ratio of phenolic and non-phenolic lignin model compounds on AOX formation was studied. The VA and VE had a total content of 3.1 mmol and concentration of 62 mmol l−1) as the proportion of VE is 100%. The AOX formation decreases slowly with an increase in VA as the proportion of VA falls below 50%, which decreases by 17.5% from 191.3 to 157.75 mg l−1. However, the AOX formation decreases significantly with the further increases in the proportion of VA. When VA reaches 100%, it decreases by 38.8% from 157.75 to 96.5 mg l−1. This result confirms that VE contributes more to AOX formation, and so, it is the VE content that more greatly influences AOX formation in the reaction of VA and VE with chlorine dioxide solution.3.4.The products of the reaction of VA with chlorine dioxide solution were separated and detected by GC–MS. The chlorinated products obtained are listed in The products of the reaction of VE with chlorine dioxide solution were separated and detected by GC–MS . The typThe chlorinated products of the combined reaction of VA and VE with chlorine dioxide solution are shown in 4.The AOX content produced by the reaction of VE with chlorine dioxide solution is higher than that of VA with chlorine dioxide solution, although the consumption of VE is smaller than that of VA. This indicates that the phenolic lignin model compound is more likely to react with chlorine dioxide solution even though the non-phenolic lignin model compound contributed more to the formation of AOX. In addition, the amount of AOX generated in the combined reaction of VA and VE with chlorine dioxide was higher than that in the separate reactions. This indicates that the interaction between VA, VE and the primary oxidized products tends to promote the formation of AOX.


Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus discovered. It is estimated that 10–20 million people are infected worldwide by this oncogenic virus. About 5–10% of HTLV-1-infected individuals are at risk of developing either a fatal malignancy, adult T-cell leukemia (ATL), or a chronic progressive neuroinflammatory disease, called HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Both diseases are incurable at present.Many issues concerning HTLV-1's life cycle and pathobiology are still unsolved or controversial, and new approaches for prognostic stratification of patients and eradication of HTLV-1 infection are in high demand  in vivo and that HBZ influences early stages of hematopoietic cell differentiation and induces infected progenitor cells to abnormally differentiate into Treg-like T-cells. The authors also highlight the ability of HBZ to affect cell turnover by inducing genomic instability through upregulation of miR-17 and miR-21, which inhibit the expression of OBFC2A, a single-stranded DNA-binding protein that protects genome stability. Using affinity-tagged protein and shotgun proteomics, Panfil et al. identified the UBR5 E3 ubiquitin ligase as a novel binding partner of HBZ. The authors show that this interaction results in HBZ ubiquitination. Interestingly, UBR5 was found to be overexpressed in T leukemia/lymphoma cell lines and during the later stage of T-cell transformation in vitro, while loss of UBR5 decreased cellular proliferation, suggesting a role for UBR5 in maintaining the proliferative phenotype of transformed T-cells. Gazon et al. discuss the effects of Tax and HBZ on the transcription factors AP-1, ATF/CREB, and Maf and the contribution of these pathways to HTLV-1-induced cellular transformation. The authors dissect the intricate mechanisms of AP-1 pathway activation and propose that AP-1 is activated in HTLV-1-infected T-cells through Tax-dependent and Tax-independent mechanisms. Experimental evidence suggests that HBZ interacts with all three members of the Jun family: while sequestering c-Jun and JunB in HBZ-containing nuclear bodies, it cooperates with JunD to enhance transcription of the 3′LTR and hTERT. Several aspects of NF-κB pathway deregulation mediated by Tax and HBZ are reviewed by Fochi et al. By focusing on differences in the abilities of Tax-1/HBZ and their HTLV-2 homologs Tax-2/APH-2 to interact with host factors, the authors summarize the distinct impacts of Tax and HBZ on NF-κB deregulation and their effects in cell survival and proliferation. In an investigation of the influence of the microRNA network on the turnover of HTLV-1 infected cells, Sharma et al. provide evidence that HTLV-1 infection induces upregulation of miR-34a. Based on their findings, the authors propose that high miR-34a levels may provide a selective advantage to HTLV-1 infected cells in vivo that persists during the transformation process. Recent studies have shown that the p8 accessory protein of HTLV-1 induces T-cell conjugates and cellular conduits that may facilitate virus transmission. Donhauser et al. have developed a novel method based on flow cytometry for the automated quantitation of p8 transfer between cells. Using this method in time course experiments the authors show that p8 is rapidly transferred between Jurkat T-cells and that actin polymerization affects this process. Cavallari et al. present a review of the interactions between mitochondria and proteins coded by HTLV-1 and the human tumor viruses, Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis viruses B and C (HBV and HCV), and human papillomavirus (HPV). The authors discuss how these interactions may contribute to viral replication, persistence and transformation and how these functions are connected to the crucial role of mitochondria in cellular bioenergetics, apoptosis, innate immunity, and redox balance.Yoshida, . By analTagaya and Gallo provide an insightful comparison of the oncogenicity of HTLV-1, other human oncoviruses and Helicobacter pylori. Interestingly, the lifetime risk to develop a malignancy is markedly higher in patients infected with HTLV-1—compared to EBV, KSHV, HBV, HCV, HPV, and H. pylori. Furthermore, the oncogenic mechanism of HTLV-1 is more direct. These striking features of HTLV-1 are often overlooked, possibly because of the low prevalence of the virus in the US and Europe. Based on these considerations the authors affirm the notion that this most carcinogenic virus deserves the “leukemia” name. Based its high oncogenic potential, Gallo et al. thus recommend to return to the virus' original name, “Human T-cell leukemia virus” in place of the more generic “Human T-cell lymphotropic virus.”Pasquier et al. discuss the difficulties in treating HTLV-1 patients and the current therapies available, in particular for ATL, and provide a schematic representation of the two modes of HTLV-1 transmission, i.e., through de novo by virus particles and clonal expansion of cells harboring the provirus. Using a novel in vitro assay, the authors screened an impressive number of drugs, and showed for the first time that the acyclic nucleoside phosphonates Adefovir dipivoxil and Tenofovir disporoxil are much more effective at blocking HTLV-1 transmission compared to the conventional nucleoside reverse transcriptase inhibitor AZT. In their review, Marino-Merlo et al. provide an up-to-date overview of recent efforts to understand the mechanisms involved in current therapeutic regimens for ATL. Targeted biological therapies for ATL are thoroughly described in the prospective to find novel molecular targets in ATL therapy. Based on the accumulated evidence, the authors point out that the control of virus spread is a crucial aspect in ATL therapy.Yamagishi et al. provide an overview of the multiple genetic and epigenetic aberrations that characterize in vivo HTLV-1 infection in its initial stages, when both Tax and HBZ are at work. The authors highlight the crucial, and apparent transient role of Tax in shaping the host cell genome and epigenome with the active, persistent role of HBZ, which further potentiates initiation and progression to ATL and HTLV-1 associated diseases. Queiroz et al. investigated the inflammatory response in HTLV-1 associated diseases and analyzed the relation between the IFNG+874A/T polymorphism and the progression of HTLV-1 infection to symptomatic disease. Patients carrying the polymorphic allele showed significantly higher levels of IFN-gamma than those carrying the wild-type allele, suggesting that asymptomatic carriers with the T allele of IFNG+874A/T and a high proviral load have a high probability of developing HTLV-1 associated inflammatory diseases. Moodad et al. provide a comprehensive review of the mouse models of ATL and HTLV-I infection. Transgenic animals for Tax and HBZ, as well as knock-outs for key cellular regulators provide useful models to understand the role of viral and host genes in the development of ATL. In addition, models based on xenografts and humanized immune-deficient mice provide a valuable platform to test new therapies against ATL.Vicario et al.. The authors describe experiments in which they injected an HTLV-1-immortalized T-cell line either alone or together with normal primary fibroblasts into NGS mice, and demonstrate that fibroblasts strongly enhance HTLV-1 the growth of this cell line, which partly recapitulates the in vivo pattern of the lymphoma variant of ATL. This model may be useful to test novel therapeutic interventions for the aggressive lymphoma subtype of ATL.An interesting new murine preclinical ATL model is presented in the original paper by Enose-Akahata et al. review the role of Tax and HBZ in the pathogenesis of HAM/TSP. They describe the infiltration of HTLV-1-infected lymphocytes into the CNS and their chronic release of Tax, which is capable of producing a neurotoxic effect on the long axons of corticospinal tracts involved in progressive neurological disease. It appears that HBZ expression is able to confer a pro-inflammatory phenotype to infected T-cells, a property that would position HBZ as an important factor in the HAM/TSP disease process.Sato et al. describe an analysis of 453 HAM/TSP patients enrolled in the Japanese “AAM-net” registry. By measuring eight candidate biomarkers in PBMCs, serum and cerebrospinal fluid (CSF) samples, they demonstrate that the levels of neopterin and CXCL10 in CSF represent valuable markers to discriminate among rapid, slow and very slow progressors. Based on these results, the authors propose new classification criteria for disease activity which will aid in optimizing treatment.ex vivo approach, Leal et al. show that antiretroviral genes and HAM/TSP cluster in two distinct “proviral/antiviral” classes, of which the TRIM5a/TRIM22/BST2 antiviral subset is selectively up-regulated by IFN-beta signaling in HAM/TSP. These findings provide mechanistic evidence for the in vivo immunovirological effect of IFN-β treatment in HAM/TSP and identify novel biomarkers as well as possible therapeutic targets.Using an integrated Nearly 40 years of research have yielded important insights into diverse aspects of the biology of HTLV-1 infection and pathogenesis. Nevertheless, we are still quite far from deciphering the complexity of its associated diseases. We hope that the broad range of issues addressed in this Research Topic will provide new perspectives and groundwork for future studies aimed at unraveling HTLV-1 pathogenesis and lead to the development of novel tools to eradicate the virus and its diseases.All authors UB, VC, and MGR contributed equally to the writing of 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.


The future pace of fertility decline in sub-Saharan Africa is the main determinant of future world population growth and will have massive implications for Africa and the rest of the world, not least through international migration pressure and difficulties in meeting the sustainable development goals. In this context, there have been concerns about recent stalls in the fertility decline in some African countries. Our findings suggest that these stalls are in part explained by earlier stalls in female education and that less-educated women are more vulnerable to adverse period conditions. This has important implications for setting policy priorities. Population projections for sub-Saharan Africa have, over the past decade, been corrected upwards because in a number of countries, the earlier declining trends in fertility stalled around 2000. While most studies so far have focused on economic, political, or other factors around 2000, here we suggest that in addition to those period effects, the phenomenon also matched up with disruptions in the cohort trends of educational attainment of women after the postindependence economic and political turmoil. Disruptions likely resulted in a higher proportion of poorly educated women of childbearing age in the late 1990s and early 2000s than there would have been otherwise. In addition to the direct effects of education on lowering fertility, these less-educated female cohorts were also more vulnerable to adverse period effects around 2000. To explore this hypothesis, we combine individual-level data from Demographic and Health Surveys for 18 African countries with and without fertility stalls, thus creating a pooled dataset of more than two million births to some 670,000 women born from 1950 to 1995 by level of education. Statistical analyses indicate clear discontinuities in the improvement of educational attainment of subsequent cohorts of women and stronger sensitivity of less-educated women to period effects. We assess the magnitude of the effect of educational discontinuity through a comparison of the actual trends with counterfactual trends based on the assumption of no education stalls, resulting in up to half a child per woman less in 2010 and 13 million fewer live births over the 1995–2010 period. All human populations have entered the process of demographic transition in which first, death rates start to fall due to socioeconomic development and improved public health, and then after some time lag, birth rates start to decline. During the period when death rates are already low and birth rates are still high, populations grow rapidly. This was the case in Europe and North America around 1900, and the process subsequently spread to Latin America and eastern Asia, and then to southern and western Asia. In most of these regions, fertility rates have already fallen to low levels, even when the population still continues to grow due to the age-structural momentum in which larger cohorts of young women still enter the reproductive ages and death rates continue to fall.Sub-Saharan Africa has been the last world region to enter this demographic transition. It was only in the 1980s that birth rates started to fall in most countries, but these declines have been uneven and have stalled at times. Particularly in the late 1990s and early 2000s, some sub-Saharan African countries have experienced a leveling off of their fertility decline and, in some cases, even saw a reversal, leading to an increase  is consistent with the higher proportion of poorly educated women of childbearing age in the late 1990s and early 2000s than there would have been without these education disruptions. This phenomenon coupled with the relatively higher vulnerability of less-educated women to period effects has likely contributed to the stalls in the period fertility declines.The postindependence period was a time of great expectations in sub-Saharan Africa, as most countries engaged in a process of expansion of social services . HoweverIn the literature so far, fertility trends in sub-Saharan Africa have hardly been linked to the slow-down in education progress. The two phenomena are, indeed, almost two decades apart, which may have seemed too long for any direct causal effect from, for example, reduced reproductive health spending on fertility. This 20-y lag is, however, precisely the timing that would be expected for an effect on fertility operating through female education: declining primary school enrolment rates for girls during the 1980s would result in lower education, and hence higher fertility, for women in their prime childbearing ages around 2000. Given the strong differentials in fertility by level of female education in all African countries and the extensive body of literature that explains the causal mechanisms behind the pervasive negative association between the two –14, it sIntroduction. Mixed results are coming out of studies trying to assess the impact of various types of crises on fertility, especially in the African context in which increasing economic hardship may not necessarily lead to lower fertility (as it does in countries further advanced in demographic transition), because there are still strongly pronatalist norms and children are typically seen as a way to diversify risk. Also, increased fertility enhances the probability of having a certain number of surviving children in times of mortality crisis. But there is also evidence for opposite effects of crises on lowering fertility in certain cases, such as short-term economic crisis, extreme weather events, or epidemic diseases (In addition to this hypothesis or in combination with it (as we will show later), the fertility stalls in the early 2000s could also be linked to some period effects affecting the women of childbearing age, as mentioned in the diseases . DiffereThe purpose of this study is to systematically assess the pattern of period and cohort changes in fertility and educational attainment, as well as other possible drivers of fertility, using the broadest available datasets. We investigate the link between longitudinal cohort education trends and longitudinal cohort specific fertility trends from consecutive Demographic and Health Surveys (DHSs) conducted in the region. We also apply multivariate statistical analyses, adding national-level indicators to microlevel data, to explore alternative explanations and explore the relative impact of the different potential determinants of fertility.This study is primarily based on a pooled microdataset from a total of 72 DHSs collected in 18 sub-Saharan African countries over the years 1990–2016. Within each country, the surveys made use of a two-stage cluster sampling technique to collect comparable, reliable, and nationally representative data on living conditions and demographic characteristics of households. The 18 sub-Saharan African countries represent about 66% of the population of the region in 2015 (SI Appendix).DHS individual and household files were used to construct fertility histories by age and educational attainment of the mother by single-year birth cohorts. Since DHSs are sample surveys, the information gathered at different points in time for the same national-level cohorts of women is not necessarily identical. Hence, considerable effort was invested in reconstructing consistent series of age- and cohort-specific data for the period 1985–2010 covering the cohorts of women born between 1950 and 1995. Because of these limited time windows of data availability, we will only consider fertility rates between the ages of 15 and 35 y, which do cover most of the reproduction time over the life cycle. In the case that different surveys provided different information on fertility for the same cohorts, a weighted average of the different surveys was used  the fact that without the education stall, more women would be in the educated category and, since these women would have lower fertility levels, overall fertility would be lower; and (ii) the fact that—as shown in This combination of a stall or even reversal in the cohort educational attainment and the above-described pronounced differences in fertility by education suggests a link between education and fertility stalls. It is not trivial, however, to estimate the size of this education-related cohort effect in relation to some also-evident period effects around the time of the stall. We do not choose a simple decomposition analysis because we should capture the joint implication of two independent forces:  per capita as the most consistently available and most frequently used such indicator. We operationalized GDP changes over time in such a way that average growth rates over 5-y intervals were categorized as normal when they stayed within the band of ±2%, as negative when they fell below this band, and as positive when they fell above this band. In addition, the models include three levels of educational attainment assessed at the level of each individual woman by age 25 y, as well as the 5-y birth cohorts to which the women belong, to capture the above-described cohort effects. All of the results of this multivariate analysis based on different models are given in The results of these multivariate models over the reproductive experience of over 670,000 women in sub-Saharan Africa clearly support the above findings based on more descriptive analysis and aggregate-level simulations. Mothers’ education clearly comes out as the most significant determinant of individual-level fertility, with the estimated odds ratio indicating that women with completed primary education have, on average, 64% of the level of fertility of uneducated women, even after controlling for cohort membership and period changes in GDP.Due to the great momentum of population changes, the prospects for future population growth in Africa and consequently for the world as a whole greatly depend of the fertility trajectory in Africa over the coming years. For assessing the likely future fertility trends of Africa, it greatly matters what general approach is taken to population projections and, in particular, whether heterogeneity of the population with respect to its changing education composition is explicitly taken into account. The projections produced by the United Nations Population Division only consider the age and sex structure of the population and base their assumptions about future fertility trends on an extrapolative statistical model of the overall TFR, which is sensitive to recent trends in the TFR. The medium scenario of the most recent assessment of 2015 projects that the number of people on the planet will rise to 9.8 billion in 2050 and that sub-Saharan Africa will be responsible for more than half of the world’s population growth over the next 35 y . CompareIn the above-given analysis, we have found strong empirical support for the hypothesis that this fertility stall aligned in part with a temporary stall in the education of female cohorts born in the late 1970s and 1980s. We have suggested that for most of the countries experiencing fertility stalls around 2000, there have been stalls in the education improvement of the female cohorts that entered the prime childbearing ages around that time. Detailed analyses of cohort-specific patterns and multivariate models including possible macrolevel period effects also indicate that the exceptional education experiences of the cohorts born around 1980 could indeed be associated with the observed fertility stalls. Because the more recent cohorts of young women have again picked up in terms of education, this finding suggests that in the future, we may expect an acceleration of the fertility decline as the subsequent better-educated cohorts of women move into the main childbearing ages. This finding is also relevant for the ongoing discussion as to whether population projections should be carried out by breaking down only by age and sex or whether educational attainment should be routinely included as a third demographic dimension , 19. TheThe most recent findings from the Kenya DHS are a point in case. The TFR in Kenya in the late 1970s was around 8.1 children—considered to be the highest in the world. After an initial decline, it remained stagnant for about a decade around 2000 at a level of 4.6 to 4.9 children. But most recently, the TFR experienced a rather steep dive to 3.9 children for the 3 y preceding the 2014 DHS and even 3.7 in the 2015 Malaria Indicator Survey (While the evidence for this education–fertility link at the cohort level seems to be robust, evidence for directly blaming the IMF-initiated SAPs for the education stalls seems less certain, despite the fact that many authors draw clear and strong connections between these programs and worsening health and education outcomes in the countries affected –26. WhilAfrica’s future population growth will be relevant for the rest of the world. Whether it will “only” increase to two billion as shown by optimistic scenarios that assume successful implementation of the sustainable development goals , 28, or Supplementary FileSupplementary File


The CORE Group Polio Project (CGPP) has contributed to polio eradication by successfully engaging civil society, particularly the non-governmental organization (NGO) community. This engagement, which began with a grant from the U.S. Agency for International Development in 1999, has contributed to improvements in routine immunization programs, polio campaign quality, and surveillance for acute flaccid paralysis in many challenging geographic areas. The CGPP has worked closely with polio eradication partners in a collaborative and supportive role. The CGPP has focused largely on high-risk areas with marginalized or hard-to-reach populations where health systems and immunization programs have also been weak and where transmission of poliovirus had not been stopped. The CGPP has engaged local civic leaders and communities in ways to complement top-down vertical efforts of ministries of health and other partners in the Global Polio Eradication Initiative. The CGPP has developed innovative strategies to detect cases using community-based surveillance, promoted independent campaign monitoring, established cross-border initiatives, and developed a strong and creative cadre of community mobilizers to track missed children and deliver behavior change education. Many of the innovations and approaches that the CGPP helped to develop are now being replicated by governments and international agencies to tackle other public health priorities in underserved and marginalized communities around the world. This article is the first in a series of articles describing the work of the CGPP. Because the article describes the work of more than 40 NGOs in 11 countries over 20 years, it provides only an overview, leaving many important details and variations of the CGPP’s work to be described elsewhere, including in other articles included in this series. Thanks in large part to the urgings of the Pan American Health Organization and Rotary International, the World Health Assembly of the WHO launched the Global Polio Eradication Initiative (GPEI) in 1988 when it was estimated that 350,000 children were still being paralyzed by polio each year. There are between 200 and 2,000 asymptomatic infections for every symptomatic case of paralytic polio. Accordingly, in 1988, there were probably at least 70 million cases of wild poliovirus (WPV) infection being transmitted annually throughout the world, and 125 countries were considered to have ongoing circulation of indigenous WPV. A decade later, in 1998, the number of clinically recognizable cases of polio had dropped by 95%, the region of the Americas was polio-free, and the Western Pacific and European regions were almost polio-free. Type 2 WPV was eliminated globally the following year in 1999.1 leaving the remainder of cases primarily in sub-Saharan Africa and parts of Asia. To accelerate the eradication of WPV in Africa and Asia, mass vaccination campaigns were initiated in the mid-1990s. At that time, the standard four doses of oral polio vaccine (OPV) given at birth and at 6, 10, and 14 weeks of age were considered sufficient to ensure immunity, and expert opinion led to the strategy of adding a few doses per year in mass campaigns in places where routine immunization coverage was low. This would presumably create the herd immunity needed to provide protection against transmission of WPV. These mass campaigns also had the added benefit of supplanting the endemic WPV with the live attenuated vaccine virus in the general population, thereby providing both direct and indirect exposure to the attenuated vaccine virus. The strategy was both simple and visionary, and it succeeded in interrupting the transmission of WPV in most countries—but not all. A great many non-governmental organizations (NGOs), including CORE Group members, participated in these early campaigns in the 1990s by providing logistical and mobilization support at the provincial, district, and community levels where they were already actively implementing health projects unrelated to polio.Pan American Health Organization led the way by achieving polio-free status for the Americas in 1994,1. Strengthening of routine immunization services and expansion of immunization coverage in the population, including coverage with at least three doses of OPV2. Supplemental immunization with OPV to build and sustain population/herd immunity3. Outbreak response vaccination campaigns4. Surveillance for acute flaccid paralysis (AFP)By 1999, high levels of polio immunization coverage had led to the elimination of polio from all but a few areas of the world. At that time, a “Four Pillars Strategy” was developed that has continued to guide the GPEI to the present day, consisting of the following:However, at that same time in 1999, “pockets” of polio transmission had developed where there was a critical mass of children who had consistently remained insufficiently immunized and who provided a reservoir for WPV transmission. To completely interrupt transmission, an intensive effort was required to reach every child in these “pockets” multiple times. It had become clear that these “pockets” of WPV transmission were in mobile populations, slums, politically insecure areas, communities inhabited by religious or ethnic minorities, densely populated areas, and underserved populations that experience difficulty accessing health services.The remaining cases were almost exclusively arising in these subpopulations. Furthermore, in socially marginalized populations, community resistance due to many years of frequent polio immunization campaigns became yet another obstacle to reaching these subpopulations. Anti-vaccine messaging led to increased refusals in some places, and lack of confidence and distrust contributed to hidden refusals. By 2001, there was a growing awareness that annual or even semiannual national mass immunization campaigns were insufficient to achieve the high levels of immunization required to interrupt transmission in these high-risk, hard-to-reach populations.The interruption of WPV circulation ultimately depends on sufficiently high vaccination coverage to establish herd immunity. Although it was widely thought that herd immunity could be achieved with routine vaccination coverage rates of around 80%, experiences with polio eradication demonstrated that both the number of doses and the coverage rates necessary to interrupt transmission in high-density populations with poor sanitation and high levels of malnutrition and diarrhea had to be much higher. To succeed, a higher percentage of children needed more doses. Achieving this goal was additionally challenged by civil war, civil unrest, and insurgencies in countries such as Angola, Nigeria, South Sudan, Somalia, Afghanistan, and Pakistan. The quality of routine and campaign vaccination coverage—namely, the percentage of children vaccinated and the ability to detect cases through high-quality surveillance—has led to the elimination of WPV transmission throughout most of the world. Where the coverage is of high quality (meaning the percentage of the population being immunized is high), the WPV will not find enough susceptible children to sustain transmission. And, where case-based and environmental surveillance is strong, any remaining cases or silent transmission will be identified.Consequently, the U.S. Agency for International Development (USAID) along with other major international donors and technical support groups such as the United Nations Children’s Fund (UNICEF) and the WHO came to recognize at that time the critical importance of identifying high-risk areas, developing focused social mobilization activities to increase the coverage of polio immunization (as well as the coverage of other basic immunizations), and implementing high-quality surveillance to identify new outbreaks. High-risk areas were defined as areas with low polio immunization coverage in combination with one or more of the following characteristics: 1) proximity to geographic areas with active WPV transmission; 2) a history of previous outbreaks in the area; 3) the presence of nomadic, mobile, or socially marginalized subpopulations; 4) inadequately functioning AFP surveillance systems; 5) the presence of insecurity, civil unrest, or armed conflict; or 6) a limited capacity to respond to outbreaks.It was simply not enough to get the vaccine to the doorstep if the families refused to participate. Similarly, quality programming was difficult to achieve without reliable campaign coverage data. Both these factors underscored the importance of interpersonal communication and evidence-based communication strategies. Furthermore, numerous instances arose in which incorrect immunization campaign coverage figures were produced through official channels. Thus, it became critically important to develop independent campaign monitoring to measure the quality of coverage during campaigns. To stop polio transmission in these “pockets,” high-quality, current, and localized immunization coverage data were needed along with high-quality AFP surveillance to identify new outbreaks.2 The CORE Group evolved in the 1990s as a group of NGO health practitioners who were sharing best practices and ideas on how to implement USAID Child Survival projects, including approaches to behavior change education targeting mothers and their children. The CORE Group became a formal entity in 1997.The CORE Group  is an association of NGOs working with communities around the world to improve health. Its mission is to “improve and expand community health practices for underserved populations, especially women and children, through collaborative action and learning,” and it envisions “communities where everyone can attain health and well-being.”3 The CORE Group also hosts the International Community Health Network, a large global network of civil society organizations. CORE Group’s members include international NGOs with a long history of collaborating with civil society organizations and with marginalized and hard-to-reach communities around the world. The CORE Group Polio Project (CGPP) was established in 1999 to promote greater NGO engagement in community health, making it possible for USAID to channel grants to them to work on polio eradication in high-risk areas.Since that time, the CORE Group has convened thousands of practitioners working in global community health to share knowledge, evidence, and best practices so that they can translate these into programs that can achieve a demonstrable impact on health.In 1998, USAID met with the CORE Group board of directors to discuss NGO engagement in polio eradication. Based on these discussions, World Vision submitted a proposal to USAID on behalf of the CORE Group NGO members. In 1999, USAID gave the CORE Group a US$25 million award for 5 years, and a committee worked to search for the first project director. The CORE Group and USAID established guiding principles for the CGPP, including the need for the NGOs to work cooperatively in high-risk places that would benefit the most from the CGPP’s engagement. The first project director traveled to potential target countries to assess the need and feasibility of launching the CGPP. The CGPP issued an initial request for proposals to CORE Group members to work in the polio-priority countries of Angola, India, Ethiopia, Uganda, Bangladesh, and Nepal and invited the Ministry of Health (MOH), the WHO, UNICEF, Rotary International, donors, and NGOs to participate in a preliminary partners workshop to discuss the polio eradication context in that country, the current roles of various partners, and the potential contributions that NGOs might make. These workshops provided the opportunity to present the CGPP in an open and transparent manner, allowing for debate and discussion across a range of partners, organizations, and interests to ensure that what evolved was understood and supported by the larger polio eradication community.1. There should be a full description of the proposed project.2. The project should be built around NGO collaboration.3. One NGO should host the in-country secretariat, through which the NGOs in country would collaborate.4. The MOH and other implementing partners should provide a letter of support.The initial CGPP headquarters team worked with the secretariats to develop a strategy for how the NGOs should plan and design a polio project. The basic requirements for funding project proposals were as follows:4 and the CGPP closed its programs there. In each country, the CGPP collaborated closely with the USAID mission and the in-country Interagency Coordinating Committee (ICC) for polio eradication.The first countries to start a national CGPP were Uganda, Angola, India, Nepal, Bangladesh, and, later, Ethiopia. Endemic transmission of WPV ended in Uganda (1996), Nepal (2000), and Bangladesh (2000),This early experience set the stage for later CGPP project launches in Nigeria, Kenya, Somalia, South Sudan, and Afghanistan. Although the initial launch of CGPP in a new country was often met with a degree of resistance and skepticism from other in-country GPEI partners, over time the CGPP developed strong working relationships with other partners, including the MOH, WHO, UNICEF, Rotary International, and donors, contributing jointly to improved polio programming.With financial support from USAID, the CGPP launched social mobilization down to the household level and provided technical support for grassroots programming in geographic areas at high risk of WPV transmission in Angola, Bangladesh, Ethiopia, India, Nepal, and Uganda. A CGPP secretariat was established in Angola, Ethiopia, and India to coordinate the in-country work. These secretariats became members of the in-country ICC.Various technical partners worked together to define high-risk areas of the country based on population immunity, previous campaign performance, known polio reservoirs, silent areas (where no reporting of AFP is presently occurring), refusals, insecurity, geographic inaccessibility, areas with border communities , or the presence of large mobile or displaced populations. The WHO, UNICEF, and the CGPP secretariat coordinated, at the request of the MOH, to develop joint strategies for strengthening polio eradication activities in these selected high-risk areas and provide technical oversight, monitoring, and evaluation of activities of the CGPP’s NGO partners. From the start, the CGPP focused on improving the quality of campaigns through the collection of reliable coverage data, better micro-planning and logistical support, and strong community engagement to promote positive participation and acceptance on the part of the community members. NGO organization partners also carried out household surveys, analysis of survey data, and corrective programming.Supplemental Appendix II) in polio eradication and between various levels of engagement from the community to the district, provincial, national, regional, and global levels.Although NGO health projects often have a significant community-level impact in limited geographic areas addressing specific health concerns, their efforts can be somewhat isolated or balkanized and, therefore, not conducive to the achievement of large-scale global initiatives such as polio eradication. The CGPP used a secretariat model to establish better collaboration and coordination between numerous NGO partners  for Polio, the Polio Partners Group (PPG), various regional ICC meetings, and various Technical Advisory Groups (TAGs) for immunization and polio eradication. The global secretariat, based in Washington, DC, consists of a director, a deputy director , a senior technical advisor for monitoring and evaluation, a senior technical advisor for communications, a project officer, a finance manager, and a contracts manager. The senior management functions are handled by the global director and the global deputy director/technical lead who select, supervise, and support the national secretariat directors, review and approve all sub-grants, maintain strong relations with CGPP donors (USAID and the Gates Foundation), and contribute to global planning and policy. From the start, the global secretariat was established as a virtual team, with staff coordinating from multiple cities and paid by different NGOs. This significantly reduced the headquarters operating costs and fostered ownership among partners.At the country level, the CGPP secretariat coordinates CGPP activities through contracts with international NGOs who are members of the CORE Group and are already working in-country. These international NGOs then contract with local or national NGOs in high-risk areas to carry out social mobilization and other activities to achieve the goals of polio eradication. The international and national NGOs that are interested in collaborating on polio eradication submit a bundled proposal in advance of receiving USAID funding. The bundled proposal describes where the NGOs will work, their implementation plan, and how they will collaborate with other partners. They also provide letters of support from the government, the WHO, and UNICEF, as well as letters of concurrence from the in-country USAID mission. The U.S.-based senior management team of the CGPP coordinates with USAID, World Vision , the CORE Group, international NGOs, the WHO, UNICEF, Rotary International, the CDC, donors, and the country CGPP secretariats.Examples of specific in-country activities of the CGPP that were carried out between 1999 and 2008 are shown in From the start, one of the key strategies of the CGPP has been the utilization of community health workers, also sometimes called community mobilizers, to educate mothers and youth about the reasons for eradicating WPV and how transmission of poliovirus can be stopped. This strategy was a natural application and adaptation of the standard approach of NGOs to maternal and child health programming. In many cases, community health workers were already working on child survival projects managed by CORE Group member NGOs, conducting behavior change education in the community. Thus, adding polio messages to their existing activities required minimal adaptation. In Angola and Ethiopia, the community health workers were part-time volunteers, whereas in India, they received an honorarium. The decision to pay the community mobilizers in India was a joint decision with UNICEF. In Angola and Ethiopia, the NGOs chose not to pay the community health workers for fear of creating a non-sustainable expectation. CGPP health workers, NGO field staff, and government health workers sat together and studied district-level health data to determine where to canvas households and direct their mobilization efforts. The community health workers in some localities were also taught to conduct community-based AFP surveillance. NGO staff and community health workers were creative and persistent in putting into practice the commitment to reach every unvaccinated child in communities where there was endemic WPV or very low immunization coverage to decrease the build-up of susceptible cases and stop transmission of WPV.The GPEI technical coordinating committees defined the districts and segments of districts in which the CGPP would work, and these areas were subject to change on short notice based on surveillance and immunization data and/or the presence of special high-risk and inaccessible populations. The international partner NGOs were already in-country, carrying out other projects. The polio eradication functions were integrated into their other ongoing field programs. Although international NGOs did not always have programs in the areas at high risk of polio transmission, they had the in-country expertise to identify local NGOs operating in these areas and to provide needed technical and administrative support. Local NGOs were the key to identifying local civic and religious leaders for cooperation and coordination.By 2009, the CGPP had closed successful programs in Uganda, Nepal, and Bangladesh, although in 2018 the government of Uganda asked the CGPP to return and focus on refugees in the border areas with South Sudan. CORE Group Polio Project activities were ongoing in districts or portions of districts in Angola, Ethiopia, and India that contained a total population of 82 million people, including 27 million children aged 15 years or less. Among these were 3.8 million children in Angola, two million in Ethiopia, and 21.4 million in India. Between 1999 and 2008, the CGPP had trained 250,000 mobilizers  to 1) provide essential, culturally relevant information about polio to mothers and caretakers; 2) promote good child immunization practices; 3) follow-up defaulters and track unimmunized children; 4) mobilize communities to support immunization campaigns; and 5) correct false information and dispel rumors about immunization campaigns. During this period from 1999 to 2008, the number of identified polio cases declined from 131 to three in Ethiopia, and from more than 1,000 to around 500 in Uttar Pradesh, India. Angola went from more than 1,000 in 1999 to zero from 2001 to 2005 and then had a reimportation of WPV from India in 2006, which lasted until 2011.In addition to the specific activities noted earlier, there were several less tangible, less quantifiable results achieved by the CGPP by 2009. These included such actions as bringing together communities and elected leaders to address chronic problems in addition to time-sensitive polio eradication activities, a holistic approach to the newborn (such as the India “Butterfly Booklet” described below), micro-censuses and better population data , innovative approaches to nomadic and mobile populations, and engagement of religious leaders. Linking local community surveillance activities with the national MOH programs had a significant value as well.In India, each new polio case would be a blot on administrative and health officials’ records. This compelled them to seek out catalysts such as CGPP mobilizers to start conversing with communities and their leaders, which further led to building bridges that went beyond acceptance of polio immunization to better access to government-provided rations, road repairs, and health camps.The CGPP in India developed a “Butterfly Booklet” see  that wasworedas (districts). The use of community members for surveillance significantly increased the capacity for early detection of cases by expanding surveillance outside of health facilities. During the period from 2005 to 2008, a total of 119 suspected cases of AFP and 135 suspected cases of measles were detected and reported by CGPP community volunteers from 41 woredas.In Ethiopia, the CGPP developed and introduced community-based surveillance for AFP, measles, and neonatal tetanus in 2003 in collaboration with its partners, thereby filling gaps in identifying and reporting of these diseases. At that time, more than 2,000 community volunteers received training and were deployed to their respective A major contribution of the CGPP has been to expand program ownership to include the community by engaging community leaders and local influencers at the grassroots level. Before the onset of the CGPP, many CORE Group NGOs were already using community health workers to encourage behavior change in various maternal and child health projects. The CORE Group NGO partners who received USAID funding to assist in the GPEI applied this strategy, thereby creating synergies with their new polio projects and also adding polio messages to the other maternal and child health messages already present in their other projects.In India, the CGPP collaborated with UNICEF to jointly establish the SMNet in the state of Uttar Pradesh to guide the coordination and collaboration of thousands of CORE and UNICEF community mobilization coordinators who had a very significant impact on community participation in polio campaigns through a well-designed behavior change education program. The success of this strategy in India promoted its adoption in other countries such as Nigeria, Pakistan, and Afghanistan.India’s polio eradication program started as a booth-based mass campaign. Although it was successful, it still missed too many children. The government shifted to a house-to-house approach that reached more children, but this also led to increased suspicion about the government’s motives, given the unfavorable national experience with target-oriented family planning programs in the 1970s. CORE Group Polio Project’s observations and community feedback contributed to a more collaborative approach that emphasized child health more broadly and thereby served to increase transparency, build trust, and thus maintain national momentum toward polio eradication.One of the traditional and often controversial roles of civil society that the CORE Group has been able to play in polio eradication has been to challenge the status quo and push for greater transparency and innovation. One example of this was the push that the CGPP made for better data and independent polio immunization campaign monitoring to reliably measure the quality of individual campaigns and the population coverage achieved in Angola. At the start of the CGPP in Angola in 1999, the country suffered a major polio outbreak with more than 1,000 documented cases. Despite obvious evidence of poor vaccination coverage, the administrative data  to estimate campaign coverage were routinely used to monitor campaigns, leading to official declarations that coverage had been more than 100%. In 2000, the CGPP advocated for and jointly implemented with the WHO, UNICEF, and the MOH the first rapid independent campaign monitoring survey (based on surveys of randomly selected households) and found that campaign vaccination coverage in Luanda  was only 71%. Although this finding was initially met with skepticism and resistance, in the long run, the importance of independent campaign monitoring was accepted. This contributed to the interruption of WPV circulation in Angola in 2001 by promoting much greater campaign quality.5 of the first decade of CGPP activities concluded that the CGPP had made a strong contribution to global polio eradication efforts through its local community presence in high-risk areas, its training of local mobilizers to assist in polio eradication efforts, and its participation in polio eradication programming . Through its strong field presence, the CGPP was able to provide feedback to higher levels regarding the reality on the ground. The flexibility of CGPP field activities made it possible to quickly shift activities to new locations as the need arose. This greatly enhanced the CGPP’s effectiveness. The evaluation also found that the CGPP was increasingly valued over this period by other partners and stakeholders at the national level in polio eradication activities. The evaluation recommended that the country secretariats have a stronger funding base and more authority so that they could provide stronger oversight and technical support for field activities. Finally, the evaluation of that first decade made the following observation:5It is becoming clear to many who are working at top influential levels that there are huge benefits to have collaborating partners located within many small communities, a partner who is trusted, able to take the pulse of communities, learn citizen reaction to program efforts, knows the map of the community, [and] can head off rumors and diminish resistance to vaccines or child health care services.An evaluationThe second phase of the CGPP began in 2009 with financial support provided by USAID and supplemental funding provided by the Gates Foundation, which started in 2011. The main objectives in this second decade were to 1) build effective partnerships between NGOs, governments, and agencies involved in polio eradication; 2) support NGO efforts to strengthen immunization systems; 3) support NGO involvement in polio vaccination campaigns; 4) support NGO efforts to strengthen AFP case detection and surveillance for measles and neonatal tetanus; 5) support the timely documentation and use of information; 6) organize immunization activities in response to cases of vaccine-preventable disease identified through surveillance; and 7) support NGO participation in polio eradication certification activities.Supplemental funding from the Gates Foundation was provided to the CGPP for the following specific objectives to 1) support campaign quality, routine immunization, community-based AFP surveillance, and independent campaign monitoring (in South Sudan); 2) decrease resistance to polio vaccination among caregivers of children aged 0–59 months ; 3) reduce the pool of susceptible or under-immunized children aged 0–59 months (in Angola); and 4) increase the availability of data to facilitate evidence-based decision-making that will strengthen polio eradication programs .6The Gates Foundation funding to decrease resistance to polio vaccination in Uttar Pradesh (India) was specifically for providing other services beyond polio immunization among underserved populations, where basic health services were also weak. These populations had been growing weary of repeated polio campaigns with no apparent value to them while their basic health needs remained unmet.7Global, regional, and national levels of the GPEI came to recognize that local community support had become a critical ingredient for polio eradication in high-risk areas. This had proven difficult to achieve in areas where other health problems were far more pressing, where there was suspicion about the motives of the GPEI, and where there was often resentment about lack of access to and ineffectiveness of government health services. Thus, Gates Foundation funding supported health camps in India and Nigeria for targeted high-risk groups where not only polio immunizations were provided but basic maternal and child health-care services, such as antenatal care, immunizations, and basic curative services, could also be provided. These camps also provided communities a chance to get acquainted with their health service delivery staff, and thus access to them was facilitated.In addition, analysis of surveillance of AFP for detection of possible polio cases showed geographic areas that were “silent” . In other areas, there were delays in case identification and investigation of AFP cases as parents of paralyzed children sought help from traditional or faith healers before making their way to a health facility. Lack of awareness of the need for reporting suspected AFP cases was common and a process for reporting cases directly from the community was not in place. The importance of community-based surveillance as an adjunct to the traditional facility-based AFP surveillance began to emerge as a priority around 2010.§The Gates Foundation funding for India and Angola stopped in 2012 and 2014, respectively, but Gates funding continued to be the primary source of CGPP funding in South Sudan through 2018, where there is also a focus on community-based surveillance in the three difficult-to-access conflict states of Jonglei, Upper Nile, and Unity. In these settings, the CGPP reports most of the AFP cases.Supplemental Figures 1–3 provide illustrations of the field work carried out in Angola, Ethiopia, and Uttar Pradesh, India.The work of the CGPP during the second decade (2009–2018) was designed to be flexible so that international NGOs and their local NGO partners could “follow the virus” and respond to changing needs on the ground for support of polio eradication as defined by requests from the MOH and the Interagency Coordinating Committee. The CGPP gave greater emphasis to monitoring in Ethiopia and Angola by developing new quarterly monitoring forms and by conducting follow-up household surveys in 2013, 2015, and 2017. During this period, the CGPP demonstrated an ability to be flexible. It closed programs in Nepal, Bangladesh, and Angola following sustained interruption of WPV, and it launched new initiatives in Nigeria, South Sudan, Kenya, Somalia, Uganda, and Afghanistan in response to the needs of the GPEI. Although the CGPP discussed the continued use of the community-level workers with the governments as programs closed, CGPP’s programs were not designed for sustainability but rather for a targeted, time-limited surge for polio sustainability. Unfortunately, the networks of volunteer workers were not sustainable without supervision, funds, and oversight provided by the CGPP. There was also a concern about new outbreaks, but to date, WPV has not returned to any of the closed CGPP countries.,8 The CGPP secretariat model of collaboration and response has been adopted by other projects such as the Humanitarian Pandemic Preparedness project (2008–2011), sponsored by USAID, the CORE Group, the International Federation of the Red Cross, and FHI 360.The CGPP also took on a much more visible global and regional role through participation in numerous global and regional meetings, such as the PPG in Geneva, the IMB, the Transition IMB, the Africa Regional Immunization TAG, the Horn of Africa TAG, the Strategic Advisory Group of Experts, the Global Alliance for Vaccines and Immunization, the civil society constituency, and various other groups as well. In addition, the CGPP generated technical reports, a polio toolkit, and peer-reviewed articles.‖9 A later analysis demonstrated that by 2011, AFP surveillance in most of the CGPP catchment areas in Ethiopia had met international standards of quality.10Published evaluations of the CGPP’s work in Ethiopia have demonstrated that the number of non-polio AFP cases reported annually in the CGPP catchment areas achieved the expected number within 3 years after initiating surveillance in 2004, and the Ethiopia CGPP has been able to maintain that level since.11 The evaluations also found that in previously low-coverage, hard-to-reach, and resistant areas where SMNet was operating, the coverage was as high as or higher than in non-SMNet areas.12 In addition, the percentage of fully immunized children in the CGPP catchment areas increased from 48% to 63% between 2008 and 2011, whereas the percentage of fully immunized children in the entire state of Uttar Pradesh in 2011 was only 45%.13 A separate analysis also demonstrated that immunization coverage for the third dose of diphtheria, pertussis, and tetanus (DPT3) in the CGPP catchment areas in western Uttar Pradesh was substantially higher than that for the state of Uttar Pradesh as a whole.7Published findings of evaluations of the CGPP activities in Uttar Pradesh, India, have documented the importance of mosque announcements in improved campaign performance in high-risk areas.14 Articles published in the peer-reviewed literature also describe in detail the community-engagement activities of the CGPP in India13 and Ethiopia.15Analysis of household survey data from the CGPP catchment areas in Uttar Pradesh, India, and in Ethiopia demonstrated that children in homes that were visited by health workers had higher levels of routine polio immunization coverage than children whose homes had not been visited.16 They were the primary source of knowledge about polio and polio immunization campaigns in CGPP implementation areas, and their credibility and influence have grown over the past decade.Household surveys carried out in 2017 in CGPP implementation areas in India, Nigeria, Somalia, and Ethiopia revealed that community health workers trained and supported by NGOs were uniformly well-respected, knowledgeable, and influential in convincing parents to seek immunization, and that they were the top source of health information in hard-to-reach communities.16These surveys also revealed that routine immunization coverage, including that for the third dose of OPV, had increased in the CGPP target areas and had exceeded the national level. The increases in the percentage of children aged 12–23 months who were fully immunized were particularly notable in Nigeria (from 33% at baseline to 57%) and in Ethiopia (from 25% at baseline to 44%). The tracking of newborns and education of parents about the importance of the birth dose of OPV made it possible to achieve an OPV birth dose coverage of 56% in Ethiopia and 79% in India.The CGPP has supported supplemental immunization activities through monitoring, planning, social mobilization, and logistical support, with activities varying by country. The percentage of children younger than 5 years who were missed in each supplemental immunization activity in Nigeria decreased from 4.5% to 1.5%. In India, the percentage of houses missed decreased from 5.9% to 4.4%.Community-based AFP surveillance carried out by the CGPP in South Sudan, Ethiopia, Kenya, Somalia, and Nigeria has contributed to the goal of identifying at least two cases of non-polio AFP per 100,000 children younger than 15 years within 14 days of the onset of paralysis. Early detection of AFP cases by community informants has also contributed to improving stool sample adequacy in remote areas in the Horn of Africa. Despite challenges of long distances, lack of roads, and civil unrest, South Sudan achieved a stool adequacy rate of 66% following the CGPP’s introduction of community-based surveillance.17 the CGPP has established a timely, accurate, and robust community-based surveillance system, and the post-campaign monitoring conducted by the CGPP is the primary tool for measuring the quality of campaigns there. In 2014, the Southeast Asia region which includes India was declared polio-free despite deeply entrenched social resistance to immunization campaigns. The secretariat developed innovative behavior change communication strategies for social mobilization efforts. The use of child registries provided solid, up-to-date data to improve and track routine immunization coverage rates and facilitated improvement in the timing and coverage of the OPV birth dose timing and tracking of immunization defaulters. These strategies have been used by the GPEI in areas where the CGPP is not working.In South Sudan, as described further in this series,18 These authors go on to say:For decades leading up to eradication, building social support for vaccination has begun with a comprehensive and wide-reaching approach to generate mass public support for polio eradication. As vaccination rates increased and the proportion of missed children became increasingly confined to discrete social and socioeconomic groups, communication and social mobilization strategies were refined and targeted to reach the most vulnerable families…. Through this process of mobilizing communities large and small, the polio program has developed the expertise to overcome the logistic, geographic, social, political, cultural, ethnic, gender and other barriers to working with the most-marginalized, most-deprived, and, often, most-security-compromised children and communities.18According to several technical advisors for the GPEI, the most important lesson learned from the Initiative is the importance of communications and community engagement to mobilize social and community support for vaccination, and that this is one of the important legacies of the GPEI for future global health work.19 the GPEI’s endgame strategic plan for 2013–2018 had, more than any other global health program in history, accessed the “chronically unreached, marginalized and most vulnerable populations in the world.” The characteristics and innovations developed to build social support for vaccination that were highlighted by this WHO technical group include 1) the relentless pursuit of the missed child; 2) the identification of individuals, themes, and social pillars that can unify and motivate diverse population groups for a common goal; 3) the mobilization of communities house-by-house on a grand scale not only for polio immunization but also for other discrete health interventions, such as vitamin A supplementation, measles vaccination, anti-helminthic administration, and distribution of soap, bed nets, and oral rehydration solution packets; 4) the creation of detailed local neighborhood vaccination team “micro-plans,” maps, and identification of locally influential people to assist in addressing those who are hesitant or resistant to immunization; 5) the tracking and counseling of pregnant mothers and follow-up of newborns for postnatal polio vaccination and routine immunization; 6) the collection and analysis of social data at the most local level to understand and engage effectively with the local population; 7) the tracking of mobile and migrant groups and communicating with these groups while they are in transit; 8) the engagement with groups while they are away from home during campaign days, such as with those attending social, cultural, or religious events ; 9) the use of traditional, religious, community, and civil society leaders and structures for community mobilization; 10) improvements in interpersonal skills, management, and motivation of frontline health workers; 11) the development of evidence-based approaches to guide social mobilization and community engagement through ongoing, rigorous monitoring and evaluation; 12) the capacity to respond to community demands for additional services beyond polio immunization; and 13) assistance with vaccine distribution and maintenance of the cold chain.According to the WHO,20All of these elements described above are key elements of the CGPP, and the CGPP has played a key role in developing and operationalizing these elements. But perhaps even more importantly, these innovations and approaches are now a global resource that can (and should) be applied to other global health priorities among underserved and marginalized groups around the world. This theme is explored further in a companion article in this series.Although the contributions of the NGOs varied from country to country, they brought a few critical components to the polio eradication mix: the capacity to produce high-quality immunization coverage data to evaluate campaign quality, the introduction of community-based surveillance to enhance facility-based surveillance, and improved behavior change education through the deployment of community social mobilizers. This can all largely be attributed to four strengths of NGOs: 1) they are present and active at the community level; 2) they are innovative and willing to take on new activities; 3) they have a commitment to producing and sharing high-quality, reliable data, even if it conflicts with data obtained through official governmental channels; and 4) they are willing to be accountable to their donors and to their communities.The CGPP has contributed to polio eradication by successfully engaging civil society, particularly the NGO community. This engagement has led to improvements in polio immunization campaign coverage, campaign monitoring data, and surveillance for AFP in many challenging geographic areas. The CGPP has collaborated with the international NGO community and local NGOs in high-risk areas to support campaigns, to strengthen routine immunization services, and to carry out AFP surveillance. These high-risk areas have almost always been in marginalized or hard-to-reach populations where health systems and their immunization programs have also been weak. The CGPP has engaged local civic leaders and communities in ways that complement top-down vertical efforts of MOH and other partners in the GPEI. Many of the innovations and approaches that the CGPP helped to develop are now being replicated by governments and international agencies to tackle other public health priorities in underserved and marginalized communities around the world.Supplemental materials


Endometrial carcinogenesis is involved in several signaling pathways and it comprises multiple steps. The four major signaling pathways—PI3K/AKT, Ras/Raf/MEK/ERK, WNT/β-catenin, and vascular endothelial growth factor (VEGF)—are involved in tumor cell metabolism, growth, proliferation, survival, and angiogenesis. The genetic mutation and germline mitochondrial DNA mutations also impair cell proliferation, anti-apoptosis signaling, and epithelial–mesenchymal transition by several transcription factors, leading to endometrial carcinogenesis and distant metastasis. The PI3K/AKT pathway activates the ransforming growth factor beta (TGF-β)-mediated endothelial-to-mesenchymal transition (EMT) and it interacts with downstream signals to upregulate EMT-associated factors. Estrogen and progesterone signaling in EMT also play key roles in the prognosis of endometrial carcinogenesis. In this review article, we summarize the current clinical and basic research efforts regarding the detailed molecular regulation in endometrial carcinogenesis, especially in EMT, to provide novel targets for further anti-carcinogenesis treatment. Endometrial cancer is the most common neoplasm of the female genital tract. Its incidence and mortality rates are increasing. Endometrial carcinogenesis is a complex and multi-step process that features a slow progression from hyperplasia to endometrial cancer ,2. SeverPTEN, CTNNB1, PIK3CA, ARID1A, KRAS, and ARID5B were detected at high frequencies by array and sequencing analyses. PTEN and KRAS mutations in endometrial carcinoma may trigger the PI3K/AKT and mitogen-activated protein kinase/extracellular signal-activated kinase (MAPK/ERK) pathway. PIK3CA and PIK3R1 mutations frequently occur with the PTEN mutation. KRAS and CTNNB1 mutations are also involved in WNT signaling in endometrial carcinogenesis. Chang et al. [MAPT, IL24, MCM6, TSC1, BIRC2, CIITA, DST, CASP8, and NOTCH2) and 21 potential passenger genes  and impaired cell functions that included cell proliferation, cell cycling, and death, via the mTOR, Wnt, MAPK, and vascular endothelial growth factor (VEGF) pathways. Gibson et al. [NRIP1, which is an obligate cofactor of the estrogen receptor, accounted for 12.5% of the mutations.Genetic mutations also introduce several functional abnormalities and increased stress, which promote carcinogenesis. Kandoth et al.  collecteg et al.  investigHowever, detailed pathophysiology of endometrial cancer has remained unclear in clinical or basic studies. Many studies have focused on the molecular and cell biology on endometrial cancer, including the immune escape, local inflammation, mitochondria dysfunction, tumor cell proliferation, and cell death. These studies have informed novel approaches for the therapeutic strategies of endometrial cancer . In thisEndometrial cancer is the seventh most common cancer globally, with increasing rates of incidence rate and mortality. An incidence of 24.7 per 100,000 women has been reported in Flanders and similar rates have been reported in other western European countries ,10. The Transvaginal ultrasound (TVU) is an effective and noninvasive method in assessing the thickness and the characteristics of endometrium. The sensitivity and specificity of TVU with a cut-off of 5 mm were reported as 80.5% and 85.7% ,18. CompPTEN, KRAS, ARID1A, PIK3CA, and CTNNB1 and microsatellite instability have been investigated [According to the clinicopathologic features, endometrial cancer is divided into two major types. Type 1 endometrial carcinomas include low-grade endometrioid endometrial carcinomas (FIGO grades 1 and 2), which accounted for the majority of cases (80% in one study ). Estrogstigated . These mstigated . Type 2 stigated ,30,31. TEndometrial carcinogenesis involves several signaling pathways that promote cell proliferation and facilitate the escape from the immune system and apoptosis signaling ,33,34. SHypoxia is an important microenvironment in tumor growth and malignant progression. Hypoxia triggers the transcription of key genes for cell adaptation, including those that are important in angiogenesis and neovascularization, to increase oxygen availability, local invasion, and distant metastasis to escape from hypoxia and for metabolic reprogramming to adapt to hypoxia ,36. HypoVEGF is a key cell growth factor that stimulates tumor blood vessel growth . EndometAngiogenesis plays a vital role in the pathological development of tumors. Due to the sustained action of VEGF, a large number of new blood vessels can be produced in the progression of endometrial cancer. The new blood vessels not only provide the tumor tissue with the nutrients necessary for growth, but they also remove metabolic products ,51. As aThe PI3K/AKT signaling pathway is a central regulator in endometrial carcinogenesis. The pathway connects intracellular and extracellular signaling . The PI3K-RAS gene, which encodes a small GTPase superfamily protein, has been frequently observed in 20% of endometrial cancers [K-RAS was noted in endometrial hyperplasia, when compared to endometrial carcinomas. The K-RAS mutations were as early events and were significantly associated with endometrial carcinogenesis [Multiple steps comprise the Ras/Raf/MEK/ERK signaling pathway. The pathway regulates cell proliferation and differentiation . The sig cancers ,73. In aogenesis .The Wnt/β-catenin signaling pathway is divided into two major pathway types—the canonical (β-catenin dependent pathway) and the non-canonical pathway —which regulate several aspects of cell biology, including embryogenesis, cell differentiation, proliferation, and tumorigenesis . Along wInsulin/IGF-I activates the P13K/AKT signaling pathway through the tyrosine kinase receptor. This promotes downstream cyclinD1 expression and cell proliferation . The obsEMT is an important process, in which epithelial cells lose their cell–cell contacts and adopt a mesenchymal-like property that features cytoskeleton remodeling and migratory activity . EMT is Normal cells and cancer cells respond differently to transforming growth factor beta (TGF-β) in different ECMs . TGF-β rThe EMT pathway is triggered by several transcription factors in different signaling pathways. TGF-β is the major factor that leads to EMT via SMAD-mediated and non-SMAD signaling. Both endometrial cancer and stromal cells produce high levels of TGF-β and they may recruit TGF-β secreting cells, including macrophages and neutrophils . The relTGF-β signaling has two major functions—the activation of SMAD signaling and non-SMAD pathways. Several non-SMAD signaling pathways have been implicated in the response to the full EMT process, including the PI3K/AKT, Ras/Raf/MEK/ERK, and Wnt/β-catenin signaling pathways  116,117117. TGF-Estrogen signaling is an important prognostic marker that reflects the resistance to hormonal therapies in endometrial cancer. Non-genomic and genomic responses may trigger the estrogen signaling pathways . In the GPER is a protein that is encoded by the GPER gene. The protein binds to estradiol with high affinity as the third estrogen receptor . The binThe expression of PRs has been reported as a key factor that is associated with prognosis and drug-resistance of endometrial cancer ,128 The In the present article, we have summarized the clinical and basic research that has been focused on the detailed molecular regulation of endometrial carcinogenesis. The PI3K/AKT pathway mediates cell metabolism, growth, proliferation, survival, and angiogenesis. It also activates Ras/Raf/MEK/ERK signaling, which in turn regulates cell proliferation and differentiation. The Wnt/β-catenin signaling pathway is also involved in endometrial carcinogenesis by regulating cell proliferation. These major signaling pathways collectively promote EMT by triggering several transcription factors. The epithelial cells lose cell–cell contacts and adopt a mesenchymal-like property, with cytoskeleton remodeling and migratory activity via the EMT process, which promotes endometrial carcinogenesis and distant metastasis. The mutations of mtDNA, especially germline mutations, are associated with tumorigenesis. The detailed mechanism of mtDNA mutation-involved in endometrial cancer is still unclear. These concepts are worthy of investigation to provide novel targets in the development of efficacious therapeutic interventions.


ZmCPK genes were successfully cloned from all four phylogenetic groups in maize. qRT-PCR analysis showed the expression variation of these selected genes under abscisic acid (ABA) and calcium chloride (CaCl2) treatment. Due to the presence of N-myristoylation/palmitoylation sites, the selected ZmCPK members were localized in a plasma membrane. To clarify whether ZmCPK, a key player in calcium signaling, interacts with key players of ABA, protein phosphatase 2Cs (PP2Cs) and the SNF1-related protein kinase 2s (SnRK2s) and mitogen-activated protein kinase (MAPK) signaling pathways in maize, we examined the interaction between 9 CPKs, 8 PP2Cs, 5 SnRKs, and 20 members of the MPK family in maize by using yeast two-hybrid assay. Our results showed that three ZmCPKs interact with three different members of ZmSnRKs while four ZmCPK members had a positive interaction with 13 members of ZmMPKs in different combinations. These four ZmCPK proteins are from three different groups in maize. These findings of physical interactions between ZmCPKs, ZmSnRKs, and ZmMPKs suggested that these signaling pathways do not only have indirect influence but also have direct crosstalk that may involve the defense mechanism in maize. The present study may improve the understanding of signal transduction in plants.Calcium-dependent protein kinase (CPKs) is a key player in the calcium signaling pathway to decode calcium signals into various physiological responses. cDNA sequences of 9  Plants suffer from various environmental stresses, including drought, high salinity, and extreme temperatures ,2,3. To 2+- and ABA-mediating signaling pathways were independent of each other [AtCPK3, 6, 10, 21, and 23 of the Arabidopsis CPK family control stomatal closure by two signaling pathways of ABA and Ca2+ [Arabidopsis, ABA sensitivity was enhanced by the overexpression of ZmCPK4 [Arabidopsis [2+ signaling pathways [AtCPK4 and AtCPK11 catalyze the ABF1 and ABF4 (ABRE2), and meanwhile, ABF1 is also a target of SnRK2.2 and SnRK2.3, indicating that two kinases can share a single substrate. On the other hand, MAPK signaling cascade is another pathway which proceeds beside calcium signaling and is triggered by the same environmental stimuli. Most of the MAPK family members in Arabidopsis are related to the pathogen response, while AtCPKs were found to play their roles under abiotic stress as well as pathogen response [ZmMPK5 due to ZmCPK11 silencing, the role of ZmCPK11 upstream of ZmMPK5 has been proposed [OsMPK5 by OsCPK18 proposed the direct crosstalk between CPK and MAPK signaling pathways [Abscisic acid (ABA) signaling pathway regulate many processes of plant growth and development under abiotic stress conditions, such as dehydration or high salinity ,16,17. Ich other ,19, or tch other , but latch other ,22,23,24ch other ,25. AtCPand Ca2+ ,27,28,29f ZmCPK4 . The mutpathways . AtCPK4 response ,32. MAPKresponse ,34,35,36response ,38. Reporesponse ,39. In tresponse . With reproposed . Phosphopathways .2 treatment explained their sensitivity towards these treatments. Because of the importance of location of protein expression, we visualized subcellular localization of selected members in tobacco leaves. Yeast two-hybrid assay revealed the interaction between ZmCPKs, ZmSnRKs, and ZmMPKs.Although CPKs seem to play their role in ABA signaling, it is not clear whether there is a link between the CPK pathway and the core ABA signaling components in ABA signaling. Moreover, in addition to the classical MAPK cascade model, plants may have developed a calcium-mediated pathway for the activation of MAPK. Taking into account the importance of CPKs, we isolated nine CPKs distributed in four groups on the basis of phylogenetic analysis . ExpressIt has become clear that signaling pathways have to be envisaged as complex networks rather than as linear or branched signaling pathways.ZmCPKs were detected by qRT-PCR, and the results showed variations under ABA and CaCl2 as calcium treatments. The analysis with qRT–PCR showed that the expressions of these nine ZmCPK genes were regulated by these treatments. Under ABA and calcium treatment, we noticed that ZmCPK expressed differentially at different time points  showed possible interaction by growing on a selective QDO  medium, but had no effect of X-α-Gal application, which raised a question on the authenticity and strength of these interactions, which could be due to inaccessibility of prey protein to the UAS region that is related to Gal4 binding site in rare cases. In this case, we needed to confirm the interaction using additional techniques, e.g., coimmunoprecipitation (coIP) or Bimolecular fluorescence complementation (BiFC). To confirm the interaction between the pairs of possibly interacted proteins in yeast, we performed BiFC in tobacco leaves. The results of BiFC confirmed that the interaction showed by pairs in Y2H were false positives. We replicated the BiFC experiment three times to exclude any mistake and found negative results, indicating that there is no interaction between ZmCPKs and ZmPP2Cs in tobacco leaves.There were 72 yeast double-hybrid interactions between nine inations . Seven cZmSnRK2 subclass. In this study, yeast two-hybrid assay was carried out with five ZmSnRK2s and nine ZmCPKs of maize. Based on the predicted interactions of the BrCPK-BrSnRK superfamily in Chinese cabbage [There are 11 members in the  cabbage , we usedZmSnRK2 and nine ZmCPKs in 42 combinations. Out of all these combinations, only three combinations showed interactions. Those combinations wereZmCPK38 × ZmSnRK2.1, ZmCPK36 × ZmSnRK2.2, and ZmCPK17 × ZmSnRK2.5  and ascorbate peroxidase (APX) in maize protoplasts [ZmCPK26 and ZmCPK28 showed slight fluctuation under these treatments, indicating a possible role similar to their ortholog in rice, and suggesting that these two genes are not sensitive to calcium treatment or their affinity towards calcium is not enough for expression alterations as reported in Arabidopsis [An increasing body of evidence has shown that CPKs regulate ABA signal transduction in plants ,26,28. Zer study . ZmCPK14 AtCPK11 . Biphasitoplasts . ZmCPK26bidopsis ,47. This2, and these findings could be useful for researchers for detailed functional analysis.Given the expression pattern of the genes, all the genes have a heartbeat-like expression pattern at different time points. We can predict per expression levels of these genes that the first hour is the most important after abiotic stress treatments, i.e., ABA and CaClZmCPKs, we used translational fusions with (GFP) green fluorescence protein. All the selected ZmCPKs were located in a plasma membrane  and showed distribution throughout the cell, suggesting the importance of N-myristoylation and palmitoylation sites for CPKs’ plasma membrane localization. [AtCPK34 and AtCPK2 were localized on the plasma membrane, but after point mutation at myristoylation/palmitoylation sites, abolished the plasma membrane localization and caused cytoplasmic distribution [GmCPK3 and GmCPK31 had N-Myristoylation sites and were localized in the plasma membrane of Arabidopsis mesophyll protoplasts [ZmCPKs indicated their possible role in signal transduction or ion transportation in or out of the cell.In plants, CPKs are widely distributed, having ubiquitous expression in roots, flowers, leaves, and siliques, etc. Some CPKs are expressed in multiple places, while others are specific with respect to their expression. ,49,50 Thmembrane . Most ofargeting . NtCPK5 ization.  Supportiribution . In soybtoplasts . Anothertoplasts . As calctoplasts , the locIn plants, protein-protein interactions are influenced by many factors, such as tissue and organ specificity, stress response expression patterns, subcellular localization, protein-to-protein preference, and phosphorylation levels, etc. .ZmCPK, ZmPP2C, and ZmSnRK family members are numerous, their expression patterns and subcellular localizations are different, and interactions show variability in different crops, resulting in the complexity of the upstream calcium and ABA signal network of maize [Brassica napus and Arabidopsis, selected candidates of ZmCPK and ZmPP2C had no interaction in maize, suggesting that they may or may not be involved in other cellular functions [ZmSnRK2.1/2.2/2.3/2.8/2.10) and SnRK2b (ZmSnRK2.4/2.5/2.6/2.7/2.11) according to the degree of enrichment of its C-terminal amino acids [ZmSnRK2a and ZmSnRK2b subclasses were found to interact with ZmCPK. Subcellular localization of SnRK2s [ZmCPKs and ZmSnRKs will be needed to completely understand their specific role in the growth and development of maize.of maize . This diof maize , and in of maize . In thisunctions ,62. The no acids ,64,65. Ano acids ,67,68. Af SnRK2s  and our Arabidopsis and tobacco [ZmCPK 15, 17, 28 and 38 make direct contributions to the crosstalk between calcium and MAPK signaling mechanisms in rice as reported by [OsCPK18 (an ortholog of ZmCPK38) interacted with OsMPK5 (an ortholog of ZmMPK4) and was identified as activating OsMPK5 by phosphorylating two threonine residues without affecting the phosphorylation of conserved TXY (TEY & TDY) motifs [Plant protein interactions are controlled by many factors, including stress and tissue or organ specificity. Traditional MAPK cascade recognizes MAPK kinase as the only kinase for signal transmission in yeast, animal, and plant systems. Efforts have been made to study calcium/CPK and MAPK signaling in plants, but the crosstalk between these important kinase signaling pathways have not been studied. Previous studies suggest that these two pathways work parallel to each other without having direct crosstalk in  tobacco . In thisorted by . General) motifs . This sc2.The seeds of the model maize inbred line B73 were sterilized, germinated in petri dishes, and transplanted into 1/4 concentration Hoagland medium for hydroponic culture, and the nutrient solution was replaced once every 3–4 days. The culture temperature was 18–25 °C, the photoperiod was 12 h (light)/12 h (dark), and the light intensity was 400 μmol/s·m2 can significantly stimulate and mimic stress responses [2 by soaking roots in nutrient solution with 100 μM (±) ABA ) and 10 mM CaCl2. After 0 (Control), 6, 12, 24 and 48 h of ABA treatment and 0 (Control), 10, 20, 30, 60, 90, 120 and 240 min of calcium chloride treatment, roots and leaves were sampled and three biological replicates were set for each set of samples. Roots and leaves were sampled separately and frozen at −80 °C immediately after collection. RNAiso Plus  and the PrimeScript™II1st strand cDNA synthesis kit  were used for total RNA extraction and reverse transcription for cDNA synthesis, respectively.As described in the literature, 100uM ABA and 10 mM CaClesponses ,69,70,71http://www.ncbi.nlm.nih.gov/tools/primer-blast) was used to design gene-specific primers to amplify a 100–300 bp fragment of each selected member of the ZmCPK gene family  was used to normalize the differential gene expression among the multiple internal controls and the target genes [The Primer-BLAST tool  after 48 h of infiltration.Gene-specific primers were used to amplify the targeted coding sequences of nine  samples . AmplifiZmCPK family members from different phylogenetic groups, 8 ZmPP2C family, 5 genes of ZmSnRK family and 20 genes from the ZmMPK family . pGBKT7-53 and pGBKT7-lam were used as positive and negative controls, respectively. To exclude the possible autoactivation of ZmCPK members, a control experiment was carried out by co-transformation of loaded bait and prey plasmids with empty prey and bait plasmids, respectively. After screening on solid DDO (−Leu/−Trp) medium for 3 days at 30 °C, selected monoclones were inoculated in liquid DDO (−Leu/−Trp) medium until OD600 = 0.7−1.0. These cultures were inoculated on QDO  medium after ten-fold dilution. Results were observed after 4–5 days of incubation at 30 °C.The complete coding sequences of 9 K family  were ampK family . These aZmCPK15/36/38 and ZmPP2C3/4/5/7/9 were amplified by using two sets of primers .Coding sequences of  primers  and inseZmCPK family were amplified from different groups on the basis of phylogenetic analysis. qRT-PCR analysis showed that most of the ZmCPK genes are responsive to both ABA and CaCl2 treatments inferred by heat maps. Furthermore, subcellular localization of the candidate genes provided preliminary information for future investigation of the CPK family in maize. Moreover, three members of the ZmCPK family interacted with the members of ZmSnRK2 family that are active members in the core ABA signaling cascade. Four ZmCPK members were found to interact with thirteen ZmMPK members, depicting the complexity of signaling networks. The current investigation lays the groundwork for a functional characterization of the ZmCPK gene family to improve our understanding of complex signaling pathways.In this study, 9 members of the