Despite a significant number of studies on female fertility following childhood, adolescent, and young adult (CAYA) cancer, studies establishing precise (dose-related) estimates of treatment-related risks are still scarce. Previous studies have been underpowered, did not include detailed treatment information, or were based on self-report only without any hormonal assessments. More precise assessments of who is at risk for sub- or infertility are needed.The objective of our study is to describe the design and methods of 2 studies on female fertility (a cohort study and a nested case-control study) among female survivors of CAYA cancer performed within the European PanCareLIFE project.For the cohort study, which aims to evaluate the overall risk of fertility impairment, as well as the risk for specific subgroups of female CAYA cancer survivors, 13 institutions from 9 countries provide data on fertility impairment. Survivors are defined as being fertility impaired if they meet at least one of 8 different criteria based on self-reported and hormonal data. For the nested case-control study, which aims to identify specific treatment-related risk factors associated with fertility impairment in addition to possible dose-response relationships, cases (fertility impaired survivors) are selected from the cohort study and matched to controls (survivors without fertility impairment) on a 1:2 basis.Of the 10,964 survivors invited for the cohort study, data are available from 6619 survivors, either questionnaire-based only (n=4979), hormonal-based only (n=72), or both (n=1568). For the nested case-control study, a total of 450 cases and 882 controls are identified.Results of both PanCareLIFE fertility studies will provide detailed insight into the risk of fertility impairment following CAYA cancer and diagnostic- or treatment-related factors associated with an increased risk. This will help clinicians to adequately counsel both girls and young women, who are about to start anticancer treatment, as well as adult female CAYA cancer survivors, concerning future parenthood and to timely refer them for fertility preservation. Ultimately, we aim to empower patients and survivors and improve their quality of life.RR1-10.2196/10824 Advances in diagnosis and treatment of childhood cancer have led to major improvements in 10-year survival rate, which now exceeds 80% -4. AlkylDespite a significant number of studies on female fertility following childhood and adolescent cancer, studies establishing precise (dose-related) estimates of treatment-related risks are scarce. Previous studies have been underpowered ,8,9, didWe, therefore, initiated the PanCareLIFE project. This pan-European project, originating from the PanCare network, is a European Union funded project , coordinated by the University Medical Center Mainz (Germany), in which investigators from 10 countries provide data from over 15,000 CAYA cancer survivors . The proThe aim of the female fertility cohort study is to evaluate the overall prevalence of fertility impairment among female CAYA cancer survivors who are at least 5 years past diagnosis and alive at the time of study assessment. Moreover, it aims to assess the prevalence of fertility impairment for specific subgroups of female CAYA cancer survivors based on cancer diagnosis, type of treatment , age at treatment, and calendar period of treatment.In total, 13 institutions from 9 countries  collect cross-sectional data for the PanCareLIFE female fertility cohort study. These institutions, referred to as data providers, provide data from 16 different institutional cohorts in total. Two of these cohorts are registry-based cohorts (VIVE cohort and the Swiss Childhood Cancer Survivor Study cohort), whereas all other cohorts are hospital-based. Some of these data providers have previously collected their data as part of a local fertility study ,17-21, wThe eligibility criteria for the female fertility cohort study as well as the different survivor groups identified based on eligibility and type of response are described in Those who do not respond to the invitation as well as those who actively refuse to participate are categorized as nonparticipants. Participants are defined as those who agree to participate by providing either questionnaire data only, hormonal data only, or both. All local ethical committees have approved the use of the collected data from their institute for the PanCareLIFE project.For all women in the base cohort demographic, diagnostic and treatment-related data are collected from medical record files and registries. Basic demographic data include month and year of birth and of latest follow-up. Diagnostic data include type of diagnosis   and montData on fertility impairment are collected by questionnaire and hormonal assessments. A specific PanCareLIFE fertility questionnaire is developed for those data providers who collect questionnaire data on fertility issues during the PanCareLIFE project. This questionnaire evaluates sociodemographic and menstrual cycle characteristics, menopausal status, use of oral contraceptives and hormones, reproductive history, and smoking and alcohol behaviors. The questionnaire is translated from the original English into German, Czech, Italian, and Hebrew. All translated questionnaires are back-translated into English (by another translator) to check if the translation is performed properly.Questionnaire data from questionnaires used by data providers for previous local fertility studies address fertility issues using different questions at different levels of detail and with different answer categories. Therefore, a specific task for WP3 investigators is to recode the relevant data from these questionnaires for compatibility with the variables used in the PanCareLIFE fertility questionnaire in close collaboration with the relevant data provider to make them as compatible as possible.Hormonal measurements primarily involve the assessment of AMH levels. Study participants are asked to provide a blood sample during a clinic visit . Part of the sample is centrifuged and stored at −20°C. Subsequently, serum samples are transported in batches by courier to AUMC, where AMH levels are determined centrally in the endocrine laboratory. An ultrasensitive Elecsys AMH assay is used  with an intraassay coefficient of variation of 0.5%-1.8%, a limit of detection of 0.01 µg/L, and a limit of quantitation of 0.03 µg/L . FSH levData providers collect all data from their own survivor cohort, enter them into a local study database (under a unique PanCareLIFE-ID number), anonymize the data, check the quality of the data, and then send the data to the coordinating PanCareLIFE data center in Mainz. In this center, all subjects are assigned a new unique identification number. Subsequently, the data are compiled and sent to the WP3 investigators at AUMC, as seen in fertility impairment. However, given the fact that the fertility data come from different sources, it is difficult to apply one standardized outcome definition of fertility impairment to all participating cohorts in the cohort study. Therefore, fertility impairment is defined according to 8 criteria based on self-reported and hormonal data , hormonal data only (criterion 3), or on self-reported data only . These criteria were established by WP3 with a reproductive specialist . A survivor is classified as being fertility impaired if she meets at least one of the 8 criteria, as described in The primary outcome of the cohort study is Low AMH is defined as an AMH level <0.5 µg/L . For criCriterion 1: Primary amenorrhea (never had menses) in combination with a high follicle stimulating hormone (FSH) and/or a low anti-Müllerian hormone (AMH) level.Criterion 2: Secondary amenorrhea (no menses for >12 months before the age of 40) in combination with a high FSH and/or a low AMH level.Criterion 3: High FSH level in combination with a low AMH level, while being <40 years of age at time of study assessment.Criterion 4: Primary amenorrhea (without information on AMH or FSH level).Criterion 5: Secondary amenorrhea (without information on AMH or FSH level).Criterion 6: Low AMH level and <30 years of age at time of study assessment and not using exogenous reproductive hormones at time of blood sampling.Criterion 7: Use of artificial reproductive techniques  and being <40 years of age at time of study assessment.Criterion 8: Tried to conceive for at least 12 consecutive months without success and being <40 years of age at time of study assessment.The overall prevalence of fertility impairment will be defined as the number of participating survivors who are fertility impaired divided by the total number of participating survivors. The prevalence of fertility impairment will also be calculated for subgroups based on cancer diagnosis, type of treatment , age group at treatment, and calendar period at treatment. In addition, the prevalence of fertility impairment according to each of the different criteria will be calculated along with that of fertility impairment based on the criteria that evaluate ovarian function (criteria 1 to 6) and possible difficulties getting pregnant (criteria 7 and 8).Multivariable logistic regression analysis will be used to investigate which diagnostic- or treatment-related risk factors influence the probability of being fertility impaired. All analyses will be adjusted for possible confounders, such as age at the time of study assessment, time since diagnosis, smoking status, BMI, and use of hormonal contraception. Furthermore, to detect possible selection bias, descriptive statistics will be used to describe any differences in age at time of study assessment, age at diagnosis, cancer diagnosis, time since diagnosis, and type of treatment between participants, nonparticipants, and excluded women, as seen in The case-control study is nested within the cohort study, meaning that both cases and controls are selected from participants of the cohort study. However, only participants from institutions that are able to provide detailed treatment data are potential inclusions for the case-control study. This is the case for participants from 11 out of the 16 cohorts in the cohort study .The aims of the nested case-control study are to identify specific treatment-related factors associated with an increased risk of fertility impairment among CAYA cancer survivors and investigate possible dose-response relationships between cumulative dose of radiation from radiotherapy, cumulative dose of specific anticancer drugs, and the risk of fertility impairment.a priori. From a previous study, it was expected that 19% of female childhood cancer survivors exposed to potentially gonadotoxic treatment  have low AMH levels compared with 4% among survivors who did not receive such treatment [The minimal sample size to be included in the nested case-control study population is calculated reatment . Based oCases are defined as women who are fertility impaired, as assessed by the 8 criteria described in noncases) are actually not fertility impaired (true controls). Criterion 1 is considered to reflect fertility impairment with the highest certainty and criterion 8 with the least, considering the ability of each criterion to also identify “true controls.” First, women with self-reported (primary or secondary) amenorrhea validated by a high FSH and/or a low AMH level are selected as cases, followed by women with an established high FSH level together with a low AMH level. Subsequently, women who reportedly have amenorrhea (primary or secondary) with no further information on AMH levels are selected, followed by those with an established low AMH level while being younger than 30 years and not using any hormones. Finally, women who indicate by self-report to have ever used some type of artificial reproductive technique and subsequently those who have ever tried to become pregnant for at least one year without success are selected as cases. Ultimately, for the nested case-control study, each case is identified as a case based on one criterion only. The process of case accrual ends after 402 cases are selected.Prior to identifying the cases it is estimated that using these 8 criteria, substantially more than the 402 required cases will be identified. Therefore, to include the 402 cases that are most likely to actually be fertility impaired, the decision was made to hierarchically structure these 8 criteria. Moreover, in making this hierarchy, we also considered the certainty by which each criterion can establish whether the remainder of the participants  or for the PanCareLIFE female fertility study (n=2464) . In totaResults show that within the total group of 6619 participants, criterion 1 could be evaluated among 21.98% (1455/6619) of the participants, criterion 2 among 23.66% (1566/6619), criterion 3 among 18.24% (1207/6619), criterion 4 among 47.33% (3133/6619), criterion 5 among 88.55% (5861/6619), criterion 6 among 24.78% (1640/6619), criterion 7 among 41.68% (2759/ 6619), and criterion 8 among 76.30% (5050/6619) of the participants. However, data providers who collect their data during the course of PanCareLIFE collect data for all 8 criteria. This results in a total group of 464 participants for whom all 8 criteria can successfully be evaluated.All data are collected and entered into local electronic databases by data providers and sent to the coordinating data center in Mainz (WP1). These data are subsequently checked, merged, and cleaned by investigators from WP1 after which a final, aggregated dataset is sent to the investigators of WP3.The selection of cases and controls has been successfully performed using the hierarchically-ordered criteria of fertility impairment. However, ultimately, it appears that this hierarchy can be discarded since, after the application of the 8th criterion, a total of 504 cases have been identified from the total eligible cohort. Of these, 13 cases are excluded, because no treatment data are available, and 41 because no appropriate matching controls can be found. Therefore, ultimately, 450 cases are included in the case-control study. If the cases identified by the last criterion (criterion 8) are not included in the nested case-cohort study, this will lead to fewer than the required 402 cases.The 450 selected cases are matched to 882 controls. Some cohort cases cannot be matched to 2 controls owing to an insufficient number of controls in that cohort. However, because the Dutch Childhood Oncology Group - Long term Effects after Childhood Cancer cohort see  includesData analysis of both the PanCareLIFE cohort study and the case-control study is currently under way and the first results are expected to be submitted for publication in 2019.This paper describes the design and methods of 2 studies on female fertility within the PanCareLIFE project. Due to the large number of institutions collaborating within this project, these studies will encompass the largest group of CAYA cancer survivors among whom female fertility is investigated using both self-reported and hormonal data. Results will provide detailed insight into the prevalence of fertility impairment following CAYA cancer and the diagnostic- or treatment-related factors associated with an increased risk of fertility impairment. This will help clinicians to adequately counsel both girls or young women who are about to start anticancer treatments as well as adult female CAYA cancer survivors about issues concerning their remaining reproductive life span and the possible need for fertility preservation interventions. Moreover, knowledge gained from the 2 studies can be incorporated into existing evidence-based clinical guidelines on female fertility for CAYA cancer patients and survivors ,29.The 2 fertility studies conducted within PanCareLIFE have several strengths. First, the international collaboration, as achieved in PanCareLIFE, has resulted in an unprecedented number of female CAYA cancer survivors for whom data on fertility impairment are available. Large study populations are essential to achieve statistically and clinically meaningful results. Moreover, the large sample size in the PanCareLIFE fertility studies will allow many subgroup analyses. For these analyses, survivors whose former treatment is presumed not to negatively affect fertility  can serve as the reference group when calculating effect measures, such as relative risks or odds ratios. Second, within both PanCareLIFE fertility studies, a broad definition of fertility impairment has been employed using several criteria that have frequently been used in previous studies assessing fertility in female CAYA cancer survivors ,12,30-33fertility impaired when they meet at least one of 8 criteria. For the nested case-control study, however, a hierarchy is applied to these criteria, meaning that a survivor is defined as a case based on the criterion that established fertility impairment with the presumed highest level of certainty, considering the ability of this criterion to also identify “true controls” . The hierarchy applied to the 8 criteria is based on several considerations. Criteria 1 through 3 (primary or secondary amenorrhea combined with a high FSH and/or a low AMH and high FSH combined with a low AMH) are deemed strong indicators of fertility impairment because one marker of (in)fertility  is validated by another marker . AMH is currently considered the marker of choice when it comes to measuring ovarian reserve because it seems to be the most stable marker, it is randomly measurable throughout the menstrual cycle and it seems to reflect reduced ovarian function early in the sequence of events leading to menopause [For the cohort study, survivors are considered enopause ,36. Critenopause . Women lenopause . Howeverenopause , this crenopause ,40. Theynot fertility impaired, whereas in reality, they are), also possibly leading to an underestimation of the overall prevalence of fertility impairment. For future studies, it is of high importance that researchers achieve consensus concerning the assessment of fertility impairment among female CAYA cancer survivors in late effects studies.The fertility studies within PanCareLIFE have some limitations. First, due to missing information, some of the data on fertility impairment collected in previous local studies cannot be successfully recoded to make them compatible with the data that are collected with the PanCareLIFE questionnaire. As a consequence, data from some cohorts cannot be considered when calculating the overall prevalence of fertility impairment. Second, our studies may be subject to selection bias because from about 60% of the total invited group of subjects’ outcome data from are available for the cohort study and even less for the nested case-control study. This could impact the generalizability of our study results. To estimate the risk of selection bias, participants will be compared with nonparticipants relative to age at time of study assessment and disease-related characteristics. Third, no information is available on the fertility outcomes of women treated for CAYA cancer who died before the study (after having survived for at least 5 years). Because many of these women might have been treated with relatively high (gonado)toxic treatment regimens, they would most probably have met at least one of the 8 criteria of fertility impairment, were they still living. As a consequence, the risk of fertility impairment calculated based on this study results might be an underestimation of the “true” risk. Furthermore, for some (sub)cohorts, not all self-reported or hormonal data needed to evaluate each of the 8 criteria are available, because these data were not collected during the local fertility study in the past. As a result, survivors within certain cohorts can be evaluated by one or 2 criteria only. Hypothetically, these women could also have met one of the other criteria. However, because data for these other criteria are lacking, this group of women might be misclassified (ie, categorized as being In summary, the 2 fertility studies conducted within PanCareLIFE will generate evidence-based knowledge concerning risk factors for impaired fertility among female CAYA cancer survivors as well as valuable information regarding differences in the prevalence of fertility impairment using different criteria to define this impairment. These results will enhance clinical practice because they will help health care practitioners provide adequate counseling concerning future parenthood to CAYA cancer survivors as well as new patients and refer these individuals to a reproductive specialist for fertility preservation in a timely manner. The ultimate objective is to empower patients and survivors and improve their quality of life.


Census-based management information systems (CB-MISs) bring strength and power to public health programs by providing current information about everyone in the population covered by the program. Such a system has been developed by the CORE Group Polio Project (CGPP) in India. This article assesses the effectiveness of the CGPP CB-MIS in the management of social and behavioral change interventions. It also assesses the feasibility of the CB-MIS for vital events registration. We describe the procedures of the CB-MIS and measure the outcomes of the CGPP by observing the trends of vaccination coverage in CGPP catchment areas over time. We also compute vital statistics from births and deaths registered through the CGPP CB-MIS and compare them with the estimates from the Civil Registration System of India using statistics from India’s Sample Registration System in Uttar Pradesh as the “gold standard.” The CB-MIS has helped the CGPP to manage its social and behavior change communication interventions effectively, and it has contributed to the increase in polio vaccine coverage facilitated by the CGPP. We also estimate that the CGPP’s CB-MIS has registered 86% of births and 98% of infant deaths, a much higher level of registration than has been achieved by the Civil Registration System for the entire state of Uttar Pradesh. The CB-MIS has helped to make it possible for community-based health workers to make behavioral diagnoses of barriers to immunization and to overcome them. The CB-MIS also provides a robust platform for community-based health workers to register vital events. A CB-MIS can contribute to the effective planning and execution of any project or program that aims to engage with everyone in the population. Various types of CB-MISs have been used for different public health purposes, such as demographic surveillance, disease surveillance, vaccine safety assessments, epidemiological research, and clinical trials.Few studies in the peer-reviewed literature assess the contributions that a CB-MIS can make to the planning, monitoring, and evaluation of social and behavior change communication (SBCC) interventions. This article describes the effectiveness of the CORE Group Polio Project (CGPP) program to improve polio and routine immunization (RI) coverage in underserved populations, the contribution of its CB-MIS to achieving these results, the strength of the vital events registration system embodied within the CB-MIS, and the potential of this CB-MIS for contributing to the effectiveness of other public health programs in similar environments.7 With financial assistance from the U.S. Agency for International Development and the Bill & Melinda Gates Foundation, the CGPP currently supports polio eradication initiatives and related activities in high-risk geographic areas of eight countries .8 In India, the CGPP works in high-priority areas of 12 districts in the state of Uttar Pradesh with a total population of approximately three million people. Since 2018, the CGPP in India also initiated a block-level intervention in two blocks of Nuh district of Haryana state. Two other articles in this series provide further descriptions of the CGPP work in India.10The CORE Group is an association of nongovernmental organizations that engage with communities to help them improve their health.11 the SMNet has supported polio eradication through the following efforts: identifying high-risk areas and working with underserved communities in planning, implementing, and monitoring social mobilization and other immunization-related activities. The activities of the SMNet are carried out by community-based health workers called community mobilization coordinators (CMCs) at the community level, who are supervised by block mobilization coordinators (BMCs),The CGPP is a member of the Social Mobilization Network (SMNet) in India, along with the United Nations Children’s Fund (UNICEF), Rotary International, the government of India (GoI), and the WHO’s National Polio Surveillance Project. Since its formation in 2003,The CGPP/India has its own CB-MIS which contains information on all households and the individuals living in each household in the CGPP catchment areas. This CB-MIS tracks inputs, outputs, processes, and outcomes of its SBCC interventions. The CMCs are each responsible for 500 or so households and maintain registers about each of them, including the current vaccination status of every child. Although this information has been used since the CGPP began its work in India in 1999, the CB-MIS has become more comprehensive and robust over time.11 A network of 1,100 CMCs conducts social mobilization activities for social and behavior change related to polio vaccination and RI. Supplemental Appendix I contains details of the CGPP’s SBCC activities for polio Supplementary Immunization Activities (SIAs) and the CGPP’s activities for RIs, including routine polio immunization.The CGPP reaches every household and tracks the immunization status of children younger than 5 years by direct personal communication with families. When resistance is encountered, the CGPP facilitates engagement with informal and formal community leaders.The CB-MIS of the CGPP/India uses prospectively and retrospectively collected information for planning, implementation, monitoring, and evaluation of its SBCC activities. Key input, process, output, and outcome indicators of the project are tracked by internal and external assessments. In addition to routinely collecting data, the CGPP also conducts specific investigations using both quantitative and qualitative techniques, such as lot quality assurance sampling (LQAS) surveys and barrier analysis . The CB-MIS aims to support program implementation teams in the effective management  of SBCC interventions at various levels—from the household to the community to the block and to the district levels—for polio eradication in the CGPP catchment areas.Supplemental Appendix provides additional information about the registers used in the CB-MIS.The CB-MIS generates real-time information from the grassroots. All information is collected and consolidated according to a common protocol developed by the CGPP, which uses the same indicators as the SMNet and the RI system. At the community level, CMCs maintain a Child Tracking Register, an SIA Register, and a Planning and Reporting Register. The CB-MIS provides a structure for collecting, processing, and disaggregating information for tracking individual children and for planning SIAs and RI activities. The 14 We also use findings of a data validation study that was conducted by CGPP/India in 2014 to ensure completeness and accuracy of reported data for newborn registration and routine vaccination coverage. In this data validation exercise, which used a 30-cluster sample (with 10 households per cluster), selected CGPP staff visited households to collect information.This article uses data from October 2014 to September 2017 that were originally collected for the CB-MIS, including denominator populations, immunization status of children, and registration of pregnant women, newborns (births), and infant deaths. We also use 11 years of SIA coverage data (from 2007 to 2017) for areas adjacent to CGPP catchment areas that were compiled through the CB-MIS of the CGPP. These data were obtained as follows. After every SIA, BMCs collect block-level statistics regarding campaign coverage. The block-level data include aggregated information from both the CMC and non-CMC areas. Block mobilization coordinators compute statistics for non-CMC areas by subtracting CMC area data from the block-level data. In addition, vital statistics reported through the Civil Registration System and Sample Registration System (SRS) Bulletin of India were used for comparative analyses.We assessed the effectiveness of the CGPP program by using data from the CB-MIS and observing the trends over time. The following indicators were calculated:15 This intensive system provides accurate vital statistics at the country level and for the bigger states with more than 10 million population,16 but it is not feasible to implement such a system in all the blocks or districts of the country.The SRS is a vital events registration system maintained in sample districts by the GoI, which is widely considered to be the “gold standard” of vital events registration in India. The SRS is a dual registration system obtained by one worker (resident part-time enumerator of an SRS site–sampled area) who visits every house monthly to register new vital events since the previous visit (continuous enumeration) and by a different worker  who visits every house every 6 months to register vital events that occurred during the previous 6 months . Vital events missed by one system are likely to be identified by the other system.14 We then compared these rates of pregnancy, birth, and infant mortality produced by these two different systems of vital events registration.To assess the accuracy of the CB-MIS in registering vital events, we calculated the pregnancy registration rate, crude birth rate (CBR), and infant mortality rate (IMR) with data from the CB-MIS. We computed the level of vital events registration for CGPP work areas. We define the level of registration by dividing the number of vital events  in the CGPP work areas that were recorded by either the CGPP CB-MIS or the Civil Registration System by the number predicted by the SRS using locally derived SRS statistics, and then multiplying this by 100 to obtain a percentage. The levels of vital events registration during the period from October 2014 to September 2017 in the CGPP CB-MIS were compared with those of the Civil Registration System.We compared the data from the validation exercise in 2014 with the information in the CMC/BMC registers to assess the completeness and accuracy of the CGPP CB-MIS. Data accuracy in reporting of vaccination coverage was measured as a ratio between the vaccination coverage reported through the CB-MIS (CMC registers) and the vaccination coverage estimated through the household survey. The first systematic attempt to assess the quality of reported data was in 2014. Then, the CGPP conducted such exercises at least once a year.All the primary vaccines used in RI  were assessed for data accuracy using the following formula:X” households between the CMC and non-CMC areas is modest, it is important to keep in mind that the CMC areas contain high-risk and difficult-to-reach households, have pockets of resistance to SIAs, or have low levels of immunization coverage. Therefore, achieving a level of performance in the CMC area that is comparable to that of the non-CMC areas is a major achievement.In Uttar Pradesh, the outcome of a given polio SIA is assessed by calculating the percentage of eligible households that did not participate in the SIA. The high-risk areas where the CGPP implements community-level SBCC activities through the CMCs have maintained a slightly lower percentage of missed houses compared with the non-CMC area . AlthougIn 2006, SIA operations in Uttar Pradesh began to involve local influencers who were willing to help reduce resistance against polio vaccination. In the CGPP catchment areas, the birth dose coverage of OPV  more than doubled from 36% in 2010 to 78% in 2017. The OPV birth dose, also known as OPV0, is given to newborns younger than 15 days. Both the demand- and supply-side factors (including the SBCC interventions of the CGPP) contributed in the increase of OPV0 coverage. The OPV3 coverage provided through RI has been maintained at above 80%, and full immunization coverage increased from 48% in 2008 to 78% in 2017  and 5.17 Increased vaccination coverage is a result of continuous efforts of the GoI to improve the vaccination services. The GoI has taken various steps and adopted the learnings of the national polio program for RI to accelerate the increase in vaccination coverage. For instance, in December 2014, a special immunization drive, Mission Indradhanush (MI), was launched to strengthen health systems for addressing equity issues in access to immunization. Furthermore, in 2017, the GoI has also introduced “Intensified MI,” strategized to cover all left-outs and dropouts in select districts and urban areas with low RI coverage and achieve the target of more than 90% full immunization coverage in a specific time-frame.18In the past 15 years, RI coverage in India has increased steadily, although the rate of increase has varied by states and districts. National-level full immunization coverage had increased from 44% in 2005–06 to 62% in 2015–16. In Uttar Pradesh, about 51% of children aged 12–23 months were fully immunized in 2015–16, an increase of 28 percentage points from 2005 to 2006.21Compared with the national- and state-level data on “percent fully immunized children,” CGPP/India reports higher (78%) vaccination coverage. Increases in vaccination coverage in the CGPP catchment areas were made possible using local data of the CB-MIS to target local immunization activities.22 by the total number of households. The decline in the population of the CGPP catchment area is a result of the gradual withdrawal of the CGPP from 200 communities .The CB-MIS provides information about the target population for each CMC catchment area (number of households and number of children younger than 5 years) and newly identified pregnant women, births, and deaths during the first year of life . This inThe CGPP management team periodically performs desk reviews and physical verification (using an LQAS or household cluster-sampling method) to assess the data accuracy. The main parameters of data verification include SIA or RI coverage, whether SBCC field activities were performed, and perceptions of caregivers.During physical verification, an investigator personally visits mothers/caregivers and verifies with her whether the data in the CB-MIS  as reported by CMCs or BMCs are accurate. The observed variation between reported and verified data helps to assess the accuracy and completeness of the CB-MIS.23 The CGPP CB-MIS helps in reducing the MOI through the inclusion of provider prompts (such as defaulter tracking and due-list for vaccination) and client prompts . Based on our experience, we consider that the following strengths of the CB-MIS have contributed to these achievements:The CB-MIS makes it possible for the CMCs to create micro-plans based on solid household-level information at the community level.Complete information is available for each child identified by household, enabling CMCs to implement CGPP interventions better.The dual recordkeeping system of information compiled at CMC and BMC levels enables the use of information for both interventions at the household/community level (using disaggregated data) and management at the block level and higher (using aggregated data). Duplicate carbon copies of the monthly performance reports, which provide basic data such as number of live births and number of children vaccinated, are shared by CMCs/BMCs with their immediate supervisors. These data are not only used for reporting but are also used by the supervisors for validation.The information is collected, compiled, and analyzed by the frontline workers (CMCs and BMCs) with minimum technical support, empowering them to improve the effectiveness of their own work.The standardized templates for data compilation and analysis facilitate timely feedback to frontline workers and enable them to use this information for planning and implementation at their level.Following the introduction of the CB-MIS in the CGPP catchment areas composed of hard-to-reach and resistant populations, there has been an increase in polio campaign coverage, a reduction in the percentage of households that resist polio immunization, and a sustained high level of RI coverage for polio and other antigens. The GoI’s priority on immunization and polio has been a major enabler for expanding vaccination coverage along with two decades of uninterrupted donor funding that has enabled the CGPP to continue its initiatives. Vaccination programs of low- and middle-income countries face a challenge of about 32% prevalence of missed opportunities for immunization (MOI).For a health program that aims at universal coverage, information about every household and its participation in the program enables the program to determine which households are not participating, and then enables the program to target efforts on those households. In our case, the CB-MIS also provides the denominator for calculating the coverage indicators at each level of programming , which is crucial for monitoring the service coverage of the target population and for identifying households and individuals that are not covered at any given point of time. The timely aggregation of data at the local and higher levels, such as at the block and district levels, is useful for middle-level managers. For a behavior-specific communication program, it is necessary to collect information on the households where the desired behavior is not occurring and determine the underlying reasons. This capacity is not possible for an MIS that relies on sampled households.The CGPP in India has demonstrated that CMCs can make behavioral diagnoses and deal with negative behavior if they are provided with simple protocols and the necessary skills to use them. The CGPP has also demonstrated that there is potential and scope for monthly micro-planning for SBCC activities in the same way that the CB-MIS is used for micro-planning of immunization activities. The CGPP has demonstrated that its CB-MIS can be scaled up to cover a large population (3 million people). The CB-MIS has helped the CGPP in meticulous planning, execution, and monitoring of its intervention, applying the same approach for both the rural and urban areas. Frequent in- and out-migration in urban settings was a major issue that had to be dealt with by periodic updates of household information. Also, a specific mechanism had to be established to identify and track the children from areas with high rates of seasonal migration, such as those in nomadic families and those living at construction sites and brick kilns. The CB-MIS, originally designed for CGPP India, has been replicated in other CGPP countries. For instance, the CGPP in Nigeria has adopted the child tracking system to effectively execute polio campaigns and RI services. It could be adopted for other public health programs where case detection and follow-up are necessary, such as child survival programs or tuberculosis programs.24 appointed by the National Health Mission (NHM) in India who could benefit from the incorporation into their work of a CB-MIS similar to that developed by the CGPP. Accredited social health activists are the link between the community and service providers of the NHM. They assess the health needs of the community and maintain a record of health information for the assigned community in a Village Health and Index Register (VHIR). The VHIR is designed for ASHAs to register and track pregnancy, pregnancy outcomes, newborns, child vaccination, and other services that are offered by ASHAs. Accredited social health activists are also expected to maintain a record and report the vital events (births and deaths) in their areas to the authority responsible for maintaining the Civil Registration System.25 The existing system of vital events registration through ASHAs can be further strengthened by building the capacities of ASHA facilitators (supervisors of ASHAs) in monitoring and supportive supervision for maintenance and sharing of vital events with the Civil Registration System.CMCs appointed by the CGPP have the same educational qualifications and sociodemographic characteristics as accredited social health activists (ASHAs). There are over 870,000 ASHAsThe CGPP CB-MIS includes most of the programmatic indicators needed for monitoring program inputs, processes, outputs, and outcomes, and it has been crucial in the planning, implementation, monitoring, and evaluation of the interventions of the CGPP. The CB-MIS has been essential for achieving near-universal polio coverage of children in hard-to-reach and resistant populations because it provides the means for assessing the progress of the program on a monthly basis and for identifying priority geographic areas where intensified efforts are needed.22 In the 1980s and 1990s, similar approaches were developed for strengthening census-based programming for reducing child mortality in Pakistan and Bolivia.28 More recently, a census-based MIS has been implemented in the slums of Freetown, Sierra Leone, as a means for engaging communities in the use of local data to improve their local health services and health outcomes.29 However, more experience with and rigorous evaluation of these CB-MISs are needed.Census-based management information systems are not widely used, unfortunately, although their potential has been recognized since the 1970s and even before to guide the use of scarce program resources to optimize benefits for population health, including the detection of infectious disease outbreaks.30 Initially, CMCs felt that it would be difficult to collect, compile, and analyze all the information required. However, CMCs now find the system to be manageable and can maintain their registers with minimal errors. The CGPP management team conducts capacity-building activities for CMCs, BMCs, and supervisors. These activities improve the ability of the field staff to accurately record and report information in a timely fashion and, ultimately, help the CGPP achieve its goals.The CGPP’s CB-MIS has evolved over time from one with only several indicators to a full-fledged system with many indicators built into a log frame. The CGPP log frame provides a summary of interventions, expected results, and a monitoring and evaluation plan for each of the project’s objectives. Initially, the system used loose sheets of paper instead of uniform registers. Currently, the system uses standardized, printed registers.Social and behavior change communication activities reliant on epidemiological and social science data are crucial for the polio eradication program because it identifies every eligible child and the vaccination history of every eligible child. A unique aspect of CGPP strategies is the efficient utilization of the CB-MIS in the implementation of SBCC strategies. As the CGPP in India mainly supports polio and RI activities, the CB-MIS contributes to the design and implementation of SBCC activities for increasing immunization coverage at the time of polio SIAs and RI sessions. The same CB-MIS could be readily adapted to support programs addressed at other public health priorities. The CB-MIS helps CMCs track all primary beneficiaries (children younger than 5 years) for participation in SIAs and in RI sessions. The CMCs mark the location of every eligible child on their area maps. Before every RI session, they prepare a “due-list,” specifying the name of each child and the type of immunization needed.1. “XR” houses—families who refuse an SIA vaccination2. “XL” houses—locked houses with no likely possibility of families returning within 6 weeks (so marked after checking with neighbors)3. “XH” houses—houses with children who were away from home at the time of house visit of vaccinators4. “XS” houses—houses with sick children5. “XV” houses—houses with children who are “out of station” (houses with children who are not expected to return home before the end of the SIA)6. Houses that are converted from “X” to “P” are marked as “X-P” on the houses and in registers.The vaccination-related behavior of each household in the CGPP catchment area is tracked by specifying the SIA vaccination status of each child. During the SIAs, CMCs accompany vaccinator teams and record the vaccination status of all children younger than 5 years in each house. At the time of each SIA, all the houses of the CMC catchment areas are marked with either a “P” or an “X,” and houses that are converted from “X” to “P” are marked as “X-P.” This house-marking is universally used for polio SIAs in the entire state of Uttar Pradesh, and CMC registers follow a similar marking system. The designation “P” means either 1) all the children received a polio immunization at the time of that SIA or 2) there are no children in the household. The “X” designation signifies that either 1) children are present who were missed during the SIA or 2) the SIA vaccination status of the children is not known. The CMC registers further classify “X” houses into the following six categories:Although there was some opposition to marking homes at the outset, families generally appreciate the importance of this system, and few families remove the mark. The same information is also maintained in the CMC record.fatwa.§The CMCs identify households that have the potential for resistance or are known to be resistant to having their child vaccinated against polio during SIAs and at the time of RI sessions. The potentially resistant households may include families with a history of an adverse event following immunization, families belonging to an area where a severe adverse event was reported, or families from an area where an anti-vaccination group has actively disseminated messages in the form of Community mobilization coordinators investigate the reason(s) behind the refusal by having personal interactions during home visits to each resistant family.The CB-MIS maintains a CMC area-wide list of potential local influencers who are recognized and respected in the community, such as village heads, religious leaders, doctors, ration dealers , and schoolteachers.Having identified resistant or potentially resistant families and appropriate influencers, CGPP field staff  and influencers visit the families repeatedly, if necessary.The CB-MIS supports program implementation teams in the effective management  of SBCC interventions at various operational levels (from the household to the community level to the block level and above). The capacity-building efforts of the CGPP’s management team are a driving force behind the success of the CB-MIS in supporting the interventions. Periodic training and on-the-ground support by supervisors not only build confidence among the field staff but also improve the completeness and accuracy of recording and reporting.Although the CB-MIS was not primarily designed for the registration of vital events, it performs better than the government’s Civil Registration System in Uttar Pradesh. We estimate that the CGPP CB-MIS registered 85% of births and 98% of infant deaths. These results demonstrate the feasibility of the CB-MIS for guiding local program activities and improving the vital events registry. CORE Group Polio Project CMCs along with other frontline workers could have a formal link with the Civil Registration System to register the events there and at the same time generate awareness about the value of birth and death registration. The CGPP CMCs encourage communities to register all births with the civil authorities.32 A robust civil registration and vital statistics system plays a critical role in addressing health inequities and strengthening global health and development efforts.31 Vital events registration systems vary by country and apply the principles of prospective and retrospective registration of vital events, such as births, deaths, and marriages.36 The lack of completeness of civil vital events registration systems is a concern in almost all low- and middle-income countries.38 The civil registration and vital statistics systems are usually managed by governments to record every birth, adoption, death, marriage, and divorce that occurs among a country’s population.31A CB-MIS primarily used for the management of a specific program can also be helpful in strengthening the vital events registry. Vital statistics are important indicators of health status, and they are essential for effective planning and implementation of policies and services at the population level.17 The Civil Registration System in India registers vital events  as the family members report them. The Civil Registration System should be the best source of information on vital events and the population-level rates of these events at all levels. It follows a unified process of recording of vital events that is supposed to be continuous, permanent, compulsory, and universal.14 Unfortunately, the birth and death registration rates in the Civil Registration System are quite low in most parts of India, a reflection of both supply-side and demand-side factors.38 Improvements in supply-side factors, such as overcoming political, administrative, economic, and legislative barriers,38 and improvements in demand-side factors, such as improving knowledge about and the value of registration in the general population, are needed to improve the completeness of vital events registration in India.In India, the Civil Registration System and the SRS are the two major sources of vital statistics estimation.Vital Statistics of India, 2015 states that 88% of births nationally were registered through the Civil Registration System. However, a significant gap between the expected number of births and the actual number of registered births has been observed for some states in India, including Uttar Pradesh.14 According to our estimate, only 67% of births and 9% of infant deaths are recorded in the Civil Registration System in Uttar Pradesh. The potential of a strong CB-MIS with its own vital events registration component as part of a health or development program is not often recognized as a contributor to strengthening the Civil Registration System.The report on The CB-MIS developed by the CGPP in India has enabled the CGPP to improve its performance in hard-to-reach and resistant populations at high risk for polio transmission. Registration of pregnancies and births makes it possible to track all young children, target newborns for administration of the birth dose for polio immunization, and identify children in need of RIs. The CB-MIS vital events registration system appears to be almost as accurate as the “gold standard,” the GoI’s SRS. It also has the added advantage of being able to use the data to guide local management and field activities. This CB-MIS could be used for other purposes beyond polio eradication and RI, such as child survival and tuberculosis (TB) programming.The case-tracking system identifies immunization defaulters and is helpful in delivering the case-specific personalized inputs required to improve program outcomes. Monthly aggregate reporting helps middle-level managers to monitor progress. The CMCs, who are local community-based health workers responsible for about 500 households, can make behavioral diagnoses if they are provided with simple protocols and the skills necessary to use them. The CGPP has been able to achieve a high level of birth and infant death registration in a population of three million people during the period from October 2014 to September 2017, thereby demonstrating the potential of the CB-MIS for strengthening vital events registration at scale. In conclusion, the CGPP CB-MIS has been instrumental for polio eradication and RI activities in high-risk areas of Uttar Pradesh where hard-to-reach and resistant groups are located. The CB-MIS could be adapted for use in improving the civil registration of vital events and in addressing other public health priorities in underserved populations with a high disease burden, including childhood survival and TB programs, for which case detection and follow-up are necessary.Supplemental materials


Peroxisomes are ubiquitous organelles with well-defined functions in lipid and reactive oxygen species metabolism, having a significant impact on a large number of important diseases. Growing evidence points to them, in concert with mitochondria, as important players within the antiviral response. In this review we summarize and discuss the recent findings concerning the relevance of peroxisomes within innate immunity. We not only emphasize their importance as platforms for cellular antiviral signaling but also review the current information concerning their role in the control of bacterial infections. We furthermore review the recent data that pinpoints peroxisomes as regulators of inflammatory processes. Peroxisomes are dynamic, multifunctional, and ubiquitous organelles present in almost all eukaryotic cells . They arPeroxisomal dysfunctions have been linked to severe metabolic disorders in humans ,8. In reThe innate immune system is responsible for identifying threats and initiating a sequence of responses that allow the elimination of potentially infectious pathogens . It invoIn this review, we summarize and discuss the role of peroxisomes within innate immunity. We not only highlight their importance for the cellular antiviral response but also discuss different reports that demonstrate their role in the control of infections by other microbes. We furthermore review the role of peroxisomes as regulators of inflammatory processes.Peroxisomes harbor the essential adaptor transmembrane protein of the retinoic-inducible gene-I (RIG-I)-like receptors (RLR) signaling, the mitochondrial antiviral signaling protein (MAVS) ,21 Figu. Upon inDixit et al. described key differences, concerning the signaling kinetics as well as the end products, between peroxisomal and mitochondrial antiviral signaling pathways. The authors observed that peroxisomal MAVS signaling induces a rapid, but transient, type I IFN-independent expression of ISGs, while mitochondrial MAVS signaling responds with a later type I IFN-dependent and long-lasting induction of defense factors, with autocrine and paracrine effects . The autThe MAVS-specific signaling from distinct organelles was more recently contested by another group . They haThe important role of peroxisomes as signaling platforms in RLR antiviral immunity is supported by numerous studies that report the specific evasion of peroxisome-dependent signaling by different viruses. It has been demonstrated that the human cytomegalovirus (a virus with a slow replication cycle that has developed highly sophisticated immune evasion strategies ) specifiDifferent groups have also demonstrated that the hepatitis C virus protein complex NS3-4A localizes at peroxisomes, cleaving MAVS at the organelle’s surface, and impairs the production of ISGs, as it had previously been shown for mitochondria and MAMs ,28,30,39Dengue and West Nile viruses were also shown to impair peroxisome biogenesis and dampen the early innate immune signaling from peroxisomes, through PEX19 sequestration by their capsid proteins . Herpes simplex virus 1 was also observed to evade the peroxisomal MAVS-dependent signaling through the viral protein VP16, via a mechanism that has not yet been unveiled . pro from pestiviruses was reported to localize at peroxisomes, alongside with IRF3 and ubiquitin, inducing IRF3 degradation and inhibiting the downstream antiviral signaling [Additionally, Nignaling . PEX genes led to an increase of the mRNA levels of several innate immunity genes [Some other viruses have been described to interfere with the peroxisome-dependent antiviral signaling, although specific mechanisms of evasion have not yet been disclosed. Upon human immunodeficiency virus (HIV) infection, secondary structured HIV-derived RNA was detected at peroxisomes and induced IRF1 and IRF3 activation, as well as NF-кB, with apparently low expression of type I and III IFNs however . Additioty genes . Hepatitty genes . While the role of peroxisomes in innate immunity gained more visibility with the discovery of the localization of MAVS at this organelle, peroxisomes had already been implicated in other innate immunity processes. In 1979, Eguchi et al. proposed that, during phagocytosis in rat peritoneal macrophages, peroxisomes relocated to regions juxtaposed to phagosomes in order to discharge catalase . CatalasDrosophila and animal cells with impaired PEX5 and PEX7 revealed that peroxisomes are essential for the eradication of microbial infections. The impaired cells were incapable to react to microbial pathogens, presenting defects in immune signaling and reduced viability [Drosophila and murine macrophages have shown compromised phagocytosis due to defects on actin organization, as well as lysosome formation and/or maturation, which was shown to be associated with the accumulation of ROS andreactive nitrogen species (RNS). Moreover, treatment of macrophages with peroxisome-derived lipids enhanced the capacity of macrophages to engulf bacteria [Later, a study with iability . Both Drbacteria . Similarbacteria .Drosophila as an animal model, revealed that peroxisomes are essential platforms in the maintenance of enteric health and the functionality of the gut–microbe interface, efficiently coordinating different mechanisms such as stress, metabolic and immunity signaling pathways. Moreover, impaired metabolic signaling led to an increase of autophagy-induced epithelial cell death, and the reduced immune response led to a decrease in the reactivity to a subsequent immune challenge and early death [More recently, Di Cara et al., using ly death .Listeria monocytogenes , persruption .These results provide clear evidence that peroxisomes are not only essential for antiviral immunity but also for the elimination of other microbes such as bacteria.Inflammation comprises different mechanisms that allow the host to respond to infections and tissue damage, promoting pathogen destruction and wound healing. Upon infection, receptors of the innate immune system activate the production of a variety of proinflammatory mediators. These components, in turn, elicit a local inflammatory exudate, which consists of plasma proteins and leukocytes. At these sites, direct contact with pathogens or cytokines activates neutrophils, culminating with the release of toxic content of neutrophil granules, such as ROS and RNS, which have an unspecific targeting towards pathogens and the host (reviewed in ).Peroxisomes’ role on the elimination of ROS and RNS species establishes a connection between this organelle and inflammation, since catalase and peroxiredoxins, besides neutralizing ROS generated during β-oxidation of lipids, are also essential for maintaining cellular redox homeostasis ,54,55. IPeroxisomes were also shown to metabolize leukotrienes and prostaglandins, important modulators of inflammation ,60,61. TThe loss of peroxisomal functions has also been associated with an intensification of the inflammatory response that can be explained by an accumulation of arachidonic acid metabolites, observed in different models of pathology . For exaPeroxisomes are no longer considered mere metabolic organelles, and they are now widely recognized as signaling hubs and protective organelles with significant physiological functions and impacts on many important human diseases. Their emergence as regulators of the innate immune response against viral infections has raised the interest in this organelle, and a growing body of evidence demonstrates that different viruses have developed specific mechanisms to counteract the peroxisome-dependent antiviral response. However, the specificities of this organelle’s dynamics that influence these immune responses needs to be further clarified. Moreover, it is also unknown whether inter-organelle interactions are involved in the establishment of antiviral signaling pathways. Finally, it remains unclear whether other antiviral signaling pathways (besides the RIG-I/MAVS network) may also operate from peroxisomes. Further studies may reveal peroxisome- dependent host mechanisms that can be exploited not only to the discovery of specific viral combat strategies but also to the potential development of broad-spectrum antiviral therapeutics.As discussed above and summarized in A further understanding of the mechanisms involved in the role of peroxisomes in innate immunity and inflammation may not only disclose new targets for antiviral and/or antibacterial therapy, but it may also prove beneficial for therapeutic interventions in chronic inflammatory disorders.


Phospholipase C (PLC) is a family of enzymes that hydrolyze phospholipids, the most significant of which is phosphatidylinositol 4,5-bisphosphate [PIP2]. Cleavage at the internal phosphate ester generates two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), both of which mediate in diverse cellular functions including the inflammatory response. Recent studies have shown that some PLC isoforms are involved in multiple stages in TLR4-, CD14-, and FcγRs-mediated activation of nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and interferon regulatory factors (IRFs), all of which are associated with the regulation of the inflammatory response. Therefore, secondary signaling by PLC is implicated in the pathogenesis of numerous inflammatory diseases. This review provides an overview of our current knowledge on how PLC signaling regulates the macrophage-mediated inflammatory response.Macrophages are crucial members of the mononuclear phagocyte system essential to protect the host from invading pathogens and are central to the inflammatory response with their ability to acquire specialized phenotypes of inflammatory (M1) and anti-inflammatory (M2) and to produce a pool of inflammatory mediators. Equipped with a broad range of receptors, such as Toll-like receptor 4 (TLR4), CD14, and Fc gamma receptors (Fc Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or to molecular “irritants,” and is a protective response involving both cellular and molecular mediators , 2. Initγ, generate high levels of proinflammatory cytokines  and oxidative metabolites ; M2 macrophages stimulated by a variety of stimuli  are important in the resolution of inflammation , chemokine , and toxic molecules  [Stimuli, such as lipopolysaccharide (LPS) and cytokines, activate macrophages by ligation of corresponding receptors, such as Toll-like receptors (TLRs) . Upon acand ROS) , 18. Theand ROS) –21. For and ROS) , 22–24. and ROS) –27 and aβ, γ, and δ in macrophage-mediated inflammation has been extensively studied, and herein the corresponding mechanisms are summarized and discussed.Members of the phospholipase C (PLC) family are thus involved in intracellular and intercellular signal transduction. Accumulated evidence has demonstrated that the PLC signaling inhibitor U73122 attenuates both acute and chronic inflammation mediated by macrophages both in vivo and in vitro –30, link2+ from intracellular stores, and DAG mediates the activation of protein kinase C (PKC). The activation of PKC and calcium signaling in turn activate downstream signaling [PLC family enzymes are activated by numerous factors such as neurotransmitters, growth factors, histamine, and hormones, as reviewed by Nakamura and Fukami . PIignaling , 32. Conignaling .α, βI, βII, and γ), novel PKCs  , and atypical PKCs (PKC-ζ and λ/ι) [α, βI, βII, δ, ε, η, ζ, and λ) are expressed in macrophages [θ, its expression is upregulated in response to LPS/IFNγ stimulation [θ expression in macrophages is inducible by certain inflammatory stimuli. It has been known that PKC inhibitors reduce LPS-stimulated cytokine secretion by macrophages, linking PKC activation to TLR4 signaling. It has been further evidenced that PKCα, δ, ε, and ζ are directly involved in multiple steps in TLR4 pathways, as well as in the downstream activation of inflammation pertinent signaling, such as MAPK and NF-κB [θ and PKCε also activate NF-κB-dependent pathways in muscle cells to promote expression of proinflammatory cytokines and chemokine [ε regulates NF-κB-mediated NO production by macrophages in response to LPS stimulation [Protein kinase C (PKC) is a family of protein serine/threonine kinases that are involved in the phosphorylation of serine and threonine amino acid residues on other proteins, or other members of this family . The PKCand λ/ι) , 36. Accrophages . Though mulation , suggestnd NF-κB , 40. PKChemokine . PKCε remulation . Classicmulation . The rolβ, γ, δ, ε, η, and ζ. Each class of PLC is composed of many isotypes with distinct functions, domains, and regulatory mechanisms [β1–4, γ1-2, δ1, δ3-4, ε, ζ, and η1–2 [Currently, there are a total of 6 classes of PLC isoenzymes discovered in mammals including the PLCchanisms . Based oand η1–2 . The strand η1–2 , 45. PLCβ1–4, γ1-2, δ1, and η1-2 are expressed in unstimulated macrophages, PLCβ1–3, γ1-2, δ1 and 3, and η1-2 are expressed in M1 macrophages, and PLCβ1–3, γ1-2, δ3, and η1-2 are expressed in M2 macrophages. In addition, these PLC isoforms showed different subcellular localization in differently polarized macrophages [The spectrum of the expression of PLC isoforms in macrophages is phenotype-specific. It has been reported that in the case of human macrophages , PLCrophages . The disβ isoforms orchestrating the Ca2+ signaling [2+ signaling in macrophages is enhanced via PLCβ-4 signaling, but depressed by the PLCβ-3 signaling [2+ and Erk1/2 signaling play important roles in the regulation of inflammatory response. PLCβ is involved in the activation of Erk1/2 signaling in macrophages. It has been demonstrated that the glyceryl ester of prostaglandins activates Erk1/2 signaling in a dose-dependent manner through a pathway that requires PLCβ signaling [Macrophages express all the four PLCignaling , 48, forignaling . Ca2+ anignaling .β/PKC signaling cascade. Therefore, it is possible that PLCβ signaling may promote the differentiation of monocytes to macrophages via cell adhesion [Cell adhesion is required for monocyte differentiation into macrophages. In human cytomegalovirus- (HCMV-) infected monocytic THP-1 cells, the viral protein US28 promotes adhesion to the endothelial cells via the activation of PLCadhesion . U73122 adhesion . These tβ-2 and β-1 expression in macrophages in an MyD88-dependent manner, and the suppressed PLCβ-2 plays an important role in switching M1 macrophages into an M2-like state [β-2 signaling is closely involved in macrophage polarization.It has been reported that LPS suppresses PLCke state , 53, sugβ signaling broadly regulates the expression of proinflammatory cytokines or chemokines in diverse cell cultures. The binding of HIV-1 envelope glycoprotein gp120 to CCR5 leads to PLCβ-1 nuclear localization which promotes the release of chemokine CCL2 by macrophages [β-1 signaling stimulates the expression of CCL2 in macrophages. PLCβ-3 regulates IL-8 expression in bronchial epithelial cells via TLR-mediated activation of calcium signaling and NF-κB pathway [β-3 regulates cytokine expression in macrophages has not been reported.PLCrophages , suggest pathway . Howeverβ-1 signaling regulates the expression of CCL2, and PLCβ-2 signaling regulates cell polarization, while PLCβ-3 and PLCβ-4 signaling regulates Ca2+ signaling with opposite effect.In summary, in macrophages, PLCγ expressed in humans, PLCγ-1 and PLCγ-2, which regulate the development and functions of various hematopoietic cells [γ1 regulates T cell activation and development through interaction with T cell receptor (TCR), and PLCγ-2 regulates development and maturation of B cells via interaction with pre-B cell receptor (BCR), reviewed by Nakamura and Fukami [γ-1 and PLCγ-2 are activated downstream of receptor (RTK) and nonreceptor tyrosine kinases, with tyrosine phosphorylation of PLCγ as the major mechanism. However, there is a novel mechanism towards the activation of PLCγ-2, which depends not on protein tyrosine phosphorylation, but on Rac GTPases [γ-1 is mainly activated by growth factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) [γ-1 binds to the tyrosine-phosphorylated receptors of EGF via its SH2 domain and downstream proteins via the SH3 domain [α and IL-1β, as well as to influenza virus H1N1, leads to activation of PLCγ-1 in macrophages, which expands the spectrum of upstream stimulators for PLCγ-1 signaling [γ-1 signaling through EGR receptor (EGFR) in alveolar epithelial cell line (A549 cells) [γ-2, being predominantly expressed in hematopoietic cells, is activated by immune cell  receptors associated with multiprotein complexes [γ-1 and PLCγ-2 may be differentially activated to perform diverse functions.There are two main isoforms of PLCic cells , 57, ford Fukami . PLCγ-1  GTPases . Ubiquitor (FGF) . PLCγ-1 3 domain . We haveignaling . Influen9 cells) . But wheomplexes . So PLCγβ (TRIF), TRIF-related adaptor molecule (TRAM), and sterile α- and armadillo motif-containing protein (SARM) to form a signalosome, which activates downstream signals [κB and MAPK cascades and proinflammatory cytokine production [Upon stimulation by LPS, TLR4 signaling induces proinflammatory cytokine production. Generally, TLRs regulate TLR-specific gene expression through the recruitment of distinct combinations of TLR/IL1R (TIR) domain-containing adaptor proteins, such as myeloid differentiation primary response gene 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), TIR domain-containing adaptor inducing IFN- signals . TLR4 is signals –66, whicoduction , 68. Theoduction , 69. So γ signaling in TLR4-mediated inflammation has been well identified. Currently, it is clear that PIP2 plays an important role in TLR4 signaling. Mechanistically, TIRAP localizes to the plasma membrane by binding to PIP2; there it recruits TLR4 and MyD88 to PIP2-rich sites on the plasma membrane to form the TLR4 signalosome [κB signaling is activated. Subsequently, the TLR-4 is delivered to the endosome compartment where MyD88-independent IRF3 signaling is activated [γ which controls the metabolism of PIP2 [γ-2 have been established: IP3, the cleavage product of PIP2 by PLCγ-2, binding to IP3 receptors (IP3Rs) in the endoplasmic reticulum results in the release of Ca2+. The increased cytosolic Ca2+ is required for translocation of TLR4 from the plasma membrane to endosomes, where TRIF-dependent IRF3 activation takes place. In contrast, LPS-induced activation of NF-κB pathway did not require PLCγ2-IP3-Ca2+ cascade [The involvement of PLCnalosome . The disctivated . The criIP2 . Mechani cascade . Thus, sγ2-IP3-Ca2+ cascade. The increase in cytosolic Ca2+, released from intracellular calcium stores, promotes the translocation of TLR4 from the plasma membrane to endosomes and so results in the activation of downstream inflammatory signaling. In addition, the CD14-dependent endocytosis pathway is regulated by several cytosolic regulators. Among them, the tyrosine kinase Syk and its downstream effector PLCγ-2 have been identified. The stimulation of Syk/PLCγ-2 signaling by CD14 triggers an influx of Ca2+ from the extracellular environment, which promotes internalization of TLR4 [2+ originating either from intracellular calcium stores or the extracellular environment, which emphasizes the important role of Ca2+ in TLR4-mediated inflammation. In addition, these results support the idea that PLCγ-2 regulates the inflammatory response by controlling the cytosolic level of Ca2+. Apart from Ca2+, PKC signaling is also involved in TLR4 signaling in macrophages. It has been reported that the infection of both P. aeruginosa and K. pneumoniae activates TLR4/PLCγ cascades which in turn activates the PKCα/Jun N-terminal protein kinase (JNK)/NF-κB axis and eventually induces the production of proinflammatory cytokines [The LPS-binding protein CD14, together with TLR4 and MD-2, forms a multireceptor complex on the cell membrane . CD14 co of TLR4 , 74. So ytokines .γ-1/PKCα/Nox2 cascade and thereby stimulates expression of proinflammatory cytokines IL-1β, IL-6, and RANTES [γ-1 regulates inflammatory response by the activation of PKCα, which is different from the role of PLCγ-2-dependent regulation of cytosolic Ca2+. Interestingly, we have recently shown that influenza virus H1N1 infection activates PLCγ-1 signaling and triggers ROS expression in human macrophages dU937 cells, which can be blocked by the PLC inhibitor U73122 [γ signaling regulates the generation of an important messenger ROS.The generation of intracellular ROS in macrophages plays an important role in inflammation pertinent signaling transduction. The minimally oxidized LDL (mmLDL) stimulates ROS generation in macrophages through activation of NADPH oxidase 2 (Nox2), which is a suggested pathogenic mechanism for the development of atherosclerosis. It has been evidenced that mmLDL induces generation of ROS in macrophages through sequential activation of TLR4/Syk/PLCd RANTES , 77. Ther U73122 . Taken tα, IL-1β, and IL-12. The mechanism for FcγR-mediated phagocytosis has been extensively investigated. The ingestion of IgG-opsonized targets is initiated by the engagement and clustering of FcγRs, which induce receptor tyrosine phosphorylation and subsequent activation of multiple downstream signaling pathways to promote the development of the phagocytic cup and the extension of pseudopods. The sequential process including cup formation, phagosome internalization, and phagolysosome formation is critical steps in the process of phagocytosis [ε to phagosome is a critical step to regulate the rate of FcγR-dependent phagocytosis [γR-dependent phagocytosis is regulated by PLCγ signaling have been revealed, for example, PLCγ-1 is consistently concentrated at phagosomes and provides DAG to facilitate PKCε localization to the phagosome [γ-2 affects early and later stages of phagocytosis, respectively [Phagocytosis by macrophages is a process that involves engulfment and clearing of invading microbial pathogens, concomitantly stimulating an inflammatory response leading to upregulation of inflammatory genes, such as TNF-ocytosis . The traocytosis . Diversehagosome ; Syk-depectively .γRs are the key mediators of systemic inflammation in Gram-positive bacteria-induced sepsis [γR-mediated phagocytosis, which consequently leads to an inflammatory response [γR complex in macrophages is triggered by Ca2+ release from intracellular Ca2+ stores controlled by PLCγ-2 signaling [γ-2 is involved in IgG-FcγR-mediated phagocytosis and cytokine production.Peptidoglycan (PGN), the major cell wall component of Gram-positive bacteria, is able to stimulate proinflammatory cytokine production in macrophages. Normal human plasma from uninfected people contains low titer of anti-PGN IgG . The antd sepsis , 82. Theresponse . In thisignaling , suggestδ1-PH domain negatively regulates FcγRII-mediated cell spreading and phagocytosis through destabilizing PIP2 availability in macrophages [δ1 expression at both mRNA and protein levels, an effect which would allow upregulation of the TLR4-induced proinflammatory cytokine production and FcγR-mediated phagocytosis [δ1 negatively regulates TLR4/FcγR-mediated inflammatory response in macrophages. The roles of the other PKCδ isoforms including PKCδ3 and PKCδ4 in macrophage-mediated inflammation are not yet defined.The PLCrophages . In addiocytosis . These sε is involved in a variety of signaling pathways and controls different cellular functions. Its role in carcinogenesis has been documented. With a PLCε knockout mice model (PLCε−/−), PLCε has been identified as a novel tumor suppressor [in vivo was partially mediated by PLCε signaling [ε has also been convincingly demonstrated to regulate Ca2+ signaling in β cells and cardiomyocytes [ε is expressed in macrophages, as well as it is having any role in the macrophage-mediated inflammatory response, has not been identified.PLCppressor . Also wiignaling . The PLCmyocytes . However2+ levels regulate multiple signaling pathways. In addition, the PLC family contains at least 13 members with specific activity for each one. Diversity of PKC family and the versatile Ca2+ signaling networks confers PLC enzyme multiple functions in the regulation of inflammatory response. Therefore, PLC enzymes are promising targets for the development of novel anti-inflammatory drugs.Evidence accumulating from multiple studies has indicated that the PLC enzymes which functionally rely on the hydrolysis of PIP2 to produce IP3 and DAG with subsequent modulation of calcium and PKC signaling regulate macrophage-mediated inflammatory response. The macrophage inflammatory response, such as the expression of inflammation-related genes and endocytosis, is controlled by calcium and/or PKC signaling. The PKC family contains ten isoforms with individual regulatory mechanism (summarized in γRs, which have been identified as important upstream activators of PLC signaling (summarized in 2+ is increased by PLCβ-4 signaling, but decreased by PLCβ-3. These studies indicate the complexity of the PLC-dependent signaling in the inflammatory response, and further research on PLC-dependent functions will contribute towards our understanding of the underlying mechanism of some inflammatory diseases.Macrophages express various receptors, such as TLRs, CD14, and Fc


Macromolecular complexes are essential to intracellular signal transduction by creating signaling niches and enabling a chain of reactions that transmit external signals into various cellular responses. Analysis of SMYD3 interactome indicates this protein lysine methyltransferase might be involved in calcium dependent signaling pathways through forming complexes with the phospholipase PLCB3, calcium/calmodulin dependent kinase CAMK2B, or calcineurin inhibitor RCAN3. SMYD3 is well-known as a histone H3K4 methyltransferase involved in epigenetic transcriptional regulation; however, any roles SMYD3 may play in signaling transduction remain unknown. KEGG pathway enrichment analysis reveals the SMYD3 interacting proteins are overrepresented in several signaling pathways such as estrogen signaling pathway, NOD-like receptor signaling pathway, and WNT signaling pathway. Sequence motif scanning reveals a significant enrichment of PXLXP motif in SMYD3 interacting proteins. The MYND domain of SMYD3 is known to bind to the PXLXP motif. The enrichment of the PXLXP motif suggests that the MYND domain is likely to be a key interaction module that mediates formation of some SMYD3 complexes. The presence of the PXLXP motifs in PLCB3 and CAMK2B indicates the potential role of the MYND domain in mediating complex formation in signaling. The structural basis of SMYD3 MYND domain-mediated interactions is unknown. The only available MYND-peptide complex structure suggests the MYND domain-mediated interaction is likely transient and dynamic. The transient nature will make this domain well-suited to mediate signaling transduction processes where it may allow rapid responses to cellular perturbations and changes in environment. The N-tThe structure of SMYD3 in complex with a MAP3K2 peptide provided the structural basis for SMYD3-substrate complex formation . The weak interaction suggests the MYND-mediated interactions are transient in nature. Transient interactions usually show a fast bound-unbound equilibrium and can readily switch between different binding partners (Perkins et al., MYND domain-based interactions show relatively low binding affinities with KThe structural basis of SMYD MYND domains-mediated interactions is unknown. Based on the structural superposition , the pepIn contrast to binding specificity, MYND domains also show promiscuous binding. Many MYND domains are capable of binding to multiple targets, and single peptide motifs can be recognized by different MYND domains. The MYND domain of BS69 is able to bind to MGA, E1A, and EBNA2 proteins (Ansieau and Leutz, Motif scanning using ScanProsite (de Castro et al., The significant enrichment of PXLXP motif in SMYD3 interacting proteins suggests that the MYND domain is likely to be a key interaction module that mediates the formation of some SMYD3 complexes. The presence of PXLXP motifs in PLCB3 and CAMK2B indicates the potential role of the MYND domain in mediating complex formation in signaling including the aforementioned estrogen signaling pathway, GnRH signaling pathway, NOD-like receptor signaling pathway, and WNT signaling pathway. In PLCB3 there are two PXLXP motifs, PPLRP at residues 265–269 and PSLEP at residues 530–534 . The for2+ concentrations; as intracellular Ca2+ concentrations rise, the holoenzyme is released from the cytoskeleton and enters the cytosol (Swulius and Waxham, CAMK2B has only one PXLXP motif, PCLSP at residues 476–480. This motif is located at the variable segment of CAMK2B . The varPseudomonas aeruginosa-induced intracellular calcium signaling and regulates the activation of PKC and the nuclear transcription factor NF-κB in human bronchial epithelial cells (Bezzerri et al., Analysis of SMYD3 interactome reveals SMYD3 might be involved in signal transduction pathways in addition to epigenetic gene regulation. Most enriched KEGG pathways are associated with the signaling processes in cancer. Among them, the WNT signaling has been shown to affect the maintenance of stemness and metastasis of cancer cells (Zhan et al., Caenorhabditis elegans orthologs, Bra-1 and Bra-2, have been shown to participate in BMP and TGFβ signaling pathways (Kurozumi et al., The MYND domain might be a key complex-forming module in SMYD3 complexes. The PXLXP motif to which the MYND domain may bind is significantly enriched in SMYD3 interacting proteins. However, there is no significant difference in the enrichment between the interactors involved in signaling transduction pathways and epigenetic transcriptional regulation suggesting that the MYND domain does not have any particular binding preferences in either of these processes. No MYND domain has been directly linked to signaling, but some MYND domain-containing proteins such as BRAM1 as well as its SMYD3 interacting proteins were retrieved from four protein-protein interaction databases including GPS-Prot (Fahey et al., Pathway enrichment analysis of SMYD3 interactors was performed using STRING web-based analyzer (Szklarczyk et al., The presence of PXLXP motif in SMYD3 interacting proteins was scanned using ScanProsite (de Castro et al., All datasets generated for this study are included in the article/supplementary material.ZY and YZ contributed conception and design of the study, performed the database and statistical analyses, and prepared the figures. ZY wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neuromodulators modify intrinsic characteristics of the nervous system in order to reconfigure the functional properties of neural circuits. This reconfiguration is crucial for the flexibility of the nervous system to respond on an input-modulated basis. Such a functional rearrangement is realized by modification of intrinsic properties of the neural circuits including synaptic interactions. Dopamine is an important neuromodulator involved in motivation and stimulus-reward learning process, and adjusts synaptic dynamics in multiple time scales through different pathways. The modification of synaptic plasticity by dopamine underlies the change in synaptic transmission and integration mechanisms, which affects intrinsic properties of the neural system including membrane excitability, probability of neurotransmitters release, receptors’ response to neurotransmitters, protein trafficking, and gene transcription. Dopamine also plays a central role in behavioral control, whereas its malfunction can cause cognitive disorders. Impaired dopamine signaling is implicated in several neuropsychiatric disorders such as Parkinson’s disease, drug addiction, schizophrenia, attention-deficit/hyperactivity disorder, obsessive-compulsive disorder and Tourette’s syndrome. Therefore, dopamine plays a crucial role in the nervous system, where its proper modulation of neural circuits may enhance plasticity-related procedures, but disturbances in dopamine signaling might be involved in numerous neuropsychiatric disorders. In recent years, several computational models are proposed to formulate the involvement of dopamine in synaptic plasticity or neuropsychiatric disorders and address their connection based on the experimental findings. Dopamine signaling is one of the most important factors that affects the synaptic plasticity in the nervous system.Impaired dopamine signaling is thought to be involved in several neuropsychiatric disorders.Computational models incorporate dopamine as an additional factor to account for reward-related learning.Plastic neuronal networks in the nervous system are highly adaptive. In such networks, neuronal activity patterns shape and reshape the emerging connectivity patterns between interconnected neurons. The classical view of synaptic plasticity is mainly based on the stimulus-related learning that depends on the firing activity of pre- and post-synaptic neurons. However, recent experiments have revealed the crucial role of dopamine signaling in the reward-related learning. While proper signaling of dopamine has a wide variety of important effects on the function of the nervous system mediated by synaptic plasticity, interferences in its signaling are involved in several neuropsychiatric disorders. Here, we review theoretical and computational aspects of dopamine signaling in synaptic plasticity and its possible involvement in several brain diseases.1.The functional properties of neurons can be tuned based on the received input. The flexibility of the nervous system to adjust its function based on the input might even affect the structural connectivity patterns of the system, which is conceivable by the effect of synaptic plasticity on the global structures of neuronal networks . HoweverNeuromodulation is referred to the modulation of the intrinsic properties of nervous system that might affect the performance of cells . TherefoThe role of DA in the Central Nervous System (CNS) is approved by numerous studies , which iIn brain networks, the synaptic strengths can be modified based on the activity of neuronal populations that induce Long-Term Potentiation (LTP) and Long-Term Depression (LTD), which can also be affected by the action of several modulators such as DA. In dorsal striatum, DA signaling through specific pathways is required both for LTP and LTD .ExperimeWhile the appropriate function of DA signaling through nervous system leads to flawless synaptic plasticity and cognitive functions, malfunction of DA signaling can be potentially disadvantageous. DA’s dysfunction is engaged in several neuropsychiatric disorders such as Parkinson’s Disease (PD), drug addiction, schizophrenia, Attention-Deficit/Hyperactivity Disorder (ADHD), Obsessive-Compulsive Disorder (OCD), and Tourette’s Syndrome (TS) . PatientSeveral studies on patients with PD reveal that DA is involved in motivational processes . On the 2.The substantial source of DA generation in the mammalian CNS is characterized by dopaminergic neurons reside in the midbrain .The SubsTypically, these DA receptors are grouped into two subsets based on their intrinsic characteristics including D1-like and D2-like types. D1-like type of DA receptors consists of D1 and D5 receptors, while D2-like family comprises D2-D4 receptors. One of the most important DA pathway signaling is by BG circuitry that is crucial for stimulus-response learning . BG is iNeurons in direct pathway are influenced mostly by D1-like receptors, which boost desired actions in the ongoing state, while NoGo neurons are subjected to D2-like receptors and restrain actions that are inappropriate for the state . TherefoNeurons can modify their intrinsic membrane characteristics based on the received input that can adjust the membrane excitability and its responses to synaptic input, which is under modulatory signals . NeuromoDA signaling is also involved in the release process of neurotransmitters, which can change the release probability of both pre- and postsynaptic neurons by modifying their receptor signaling . However. On the other hand, DA signaling may reduce the Excitatory Postsynaptic Potential (EPSP) amplitude associated with AMPA- and NMDA-like synaptic currents  of pre- and postsynaptic neurons. However, introducing a new factor such as a neuromodulator defines a synaptic plasticity rule referred to as three-factor rule  that can follows :(2)F model , which meriments .It is sheriments :(4)δ(t)the form :(5)g˙(tise S(t) :(6)g˙ is described by In the TDRL model, learning ceases when the value function accurately estimates the reward, which produces no DA signal δ(t)=0. However, the enhancement of DA level due to the use of addictive drugs can be modeled by assuming that this mechanism induces a positive prediction error δ(t) that cannot be covered by changes in the corresponding value. In such a case, 4.3.Schizophrenia can be characterized by different cognitive symptoms including impaired attention and cognitive control, which may involve excessive levels of DA in the striatum, but reduced DA amounts in PFC . HoweverSome results from medical treatments indicate that schizophrenia can be due to excess activity of dopaminergic synapses ; howeverHoffman and McGlashan introduced a computational model called spurious attractor states that can explain some of the symptoms of schizophrenia by the reduction of connectivity patterns in an attractor network that partly detaches the attractor network. This can be related to the disconnection hypothesis of schizophrenia in which the connectivity of some parts of the brain becomes relatively detached .Another different approach called connectionist model simulates several cognitive impairments that occur in schizophrenia . It sugg4.4.ADHD is associated with abnormal hyperactivity and impulsivity behaviors that may be related to the dysfunction of PFC and its cortical units . The comTreatments of ADHD may include medication, therapy, or a combination of them, which can be a long-term approach. Symptoms of ADHD can often be treated with proper medications, which are typically based on the increased levels of DA. Conventional therapies are targeted at alleviating symptoms by regulating different neurotransmitters including DA . Althoug4.5.TS is indicated with abrupt and capricious movements referred to as tics. It is suggested that these tics may result from abnormal excitability or plasticity procedures in the direct (Go) and indirect (NoGo) pathways .This can5.The current review study considered the role of neuromodulator DA in one of the most important features of nervous system, i.e. the ability of neurons to tune their functional properties based on the required conditions of inputs. DA along with other modulatory signals regulates the performance of the synapses in order to function on a stimulus-reward learning basis, far from the classical view of stimulus-stimulus learning. Therefore, DA signaling in the learning process can regulate the efficiency of synaptic plasticity and learning procedures by modulating the cellular mechanisms that control the activity pattern of neuron.The impact of DA signaling on the synaptic plasticity at cellular level can be interpreted by understanding the molecular mechanisms that modify the intrinsic properties of cells. Such mechanisms are highly vulnerable to perturbations in their function, which can result in distorted circulation of DA signaling. In other words, the appropriate level of DA signaling across brain areas is crucial for proper function of the nervous system. However, the current review study summarized some important DA-induced computational models of synaptic plasticity associated with different learning rules. Finally, as it was mentioned earlier, disturbed DA signaling is associated with several important neuropsychiatric disorders. Although diverse disorders such as PD, drug addiction, schizophrenia, ADHD, and TS might seem to have little in common, they are all entangled with disturbances in DA’s function through nervous system.Experimental observations may provide a comprehensive, and in some cases controversial insights to the crucial role of neuromodulators such as DA in the nervous system, but mathematical formulation and computational modeling can be useful to understand their signaling machinery. The current review, however, summarized the significant findings regarding the understanding of computational role of DA in the brain through synaptic plasticity and its involvement in several neuropsychiatric disorders, which may shed light on the complicated task of DA in the nervous system.