In North East Nigeria, anti-immunization rumors and sentiments have negatively impacted the country’s polio eradication efforts. Since 2014, the CORE Group Partners Project (CGPP) has leveraged local-level strategies to help change prevailing attitudes and behaviors by improving immunization acceptability in some of the most difficult settlements in Nigeria’s states at highest risk for polio. The CGPP’s communication model in Nigeria, in part, emphasizes the need to counter suspicion and address myths and misunderstandings by convening community dialogs and compound meetings, both of which serve as safe spaces for open discussion primarily aimed at addressing non-compliance. In the communities in Kaduna, Katsina, Kano, Borno, and Yobe states located in the CGPP implementation areas, there has been a consistent reduction in the number of missed children and consistent improvement in polio immunization uptake, providing evidence of the effectiveness of the CGPP communication model. The last case of wild poliovirus in Nigeria was detected in August 2016. Since Nigeria has gone more than 3 years without a case of wild poliovirus, the CGPP communication model promises to remain highly relevant in sustaining the community’s awareness about immunizations that will be required to keep the population coverage of polio immunization high and, by extension, the herd immunity required to maintain zero transmission of poliovirus in Nigeria. This article describes the various strategies used to address noncompliance and provides examples of community engagement in Yobe state, which is one of the project’s largest implementation areas. The fight against polio in Nigeria has been marked by strong achievements and tested by significant setbacks. By 2015, Nigeria was on track to be polio free. However, this achievement was short lived, as two cases of indigenous wild poliovirus (WPV) were identified in August 2016. Unreachable and unimmunized children, unreliable coverage data, inadequate surveillance, vaccine myths and hesitancy, and violence from insurgencies all contributed to this failure.4 Over the same period, the Armed Conflict Location and Event Data Project, an independent non-governmental organization based at the University of Sussex, identified 3,346 incidents, in which 34,261 people were killed.4Perhaps, the greatest overarching challenge has been produced by Boko Haram, a fundamentalist Islamic group that has launched violent attacks in Northern Nigeria and in the neighboring countries of the Lake Chad basin region—Niger, Chad, and Cameroon. From June 2011 through June 2018, the Nigeria Security Tracker documented 2,021 incidents involving Boko Haram, in which 37,530 people were killed, nearly double the conventionally cited estimate of 20,000.6 Moreover, Northern Nigeria has historically experienced large-scale rejection of polio immunization initiatives because of widespread rumors, myths, and religious beliefs. The 2003–2004 polio immunization boycott in Northern Nigeria fueled further outbreaks of polio. In 2017, an outbreak of monkeypox was rumored to have been linked to the polio immunization program and led to resistance in some communities. In some areas of Northern Nigeria (as well as in Cameroon), some people have erroneously come to believe the polio immunization can produce sterility in women.7The consequences for the polio eradication initiative have been near-catastrophic. Boko Haram has targeted and killed polio workers, disrupted polio campaigns, and damaged or destroyed health facilities. In May 2013, the Nigerian President Goodluck Jonathan declared a state of emergency in Yobe, Borno, and Adamawa states because of the activities of Boko Haram.8 The CGPP in Nigeria substitutes the term “Partners” for “Polio” in its name because of the sensitivity of the term “polio” in the northern region of the country; the project in Nigeria is also referred to as CGPP as well. In Nigeria, the CGPP encountered several challenges at the onset of implementing the program: low awareness and low uptake of polio and routine immunizations; male heads of households disallowing mothers and caregivers from presenting their children for immunization; poor community participation in immunization activities (resulting in no volunteers being recruited from the communities to support immunization activities); myths and misconceptions about the polio vaccine and unavailability of reliable immunization data at the local government area (LGA) level.The CGPP is a multi-country, multi-partner initiative funded by the United States Agency for International Development to support Nigeria’s polio eradication initiative and routine immunization efforts in five states located in Northern Nigeria. These states are considered at high risk for polio transmission because of low routine immunization coverage, gaps in surveillance, and barriers related to access and security. A history of the global CORE Group Polio Project is available elsewhere in this series.9 analyzes the role of the volunteer community mobilizers (VCMs) in Nigeria, who made these transformations possible.To address the many challenges to immunization uptake in the North, the CGPP began implementing strong community-based engagement strategies to counter rumors, myths, and misconceptions that hindered polio immunization acceptance in the North East. Key approaches used by CGPP included community participatory dialogues, neighborhood meetings, training of male peer educators, and involvement of Muslim religious leaders to encourage fathers to support polio immunization acceptance. Engaging the community led to the acceptance of polio immunization among communities previously opposed to all immunizations. A separate article in this seriesThe CGPP’s contribution to the polio eradication initiative in Nigeria began in January 2014. Initially, the CGPP’s mandate called for it to focus program activities in North West Nigeria. Plans quickly changed once the National Polio Emergency Operations Center (referred to as the NEOC)10 The North East states were plagued by high levels of extreme and recurring violence, making it difficult to reach children with polio immunization and making the states particularly vulnerable to transmission of poliovirus.10When the CGPP launched its work in North East Nigeria, an assessment by the World Bank showed that more than 40% of the health facilities had been either damaged or destroyed, greatly limiting access to routine immunization services and increasing the risk of disease outbreaks.12 These findings indicate that at the time, there was a low awareness about and limited access to routine immunization, including polio immunization, as well as a health system that was ill-equipped to meet even the basic health needs of particularly vulnerable children. Immunization coverage has always been low in the North East Nigeria, and Yobe and Borno states have been the most affected by the Boko Haram insurgency. Yobe state was beleaguered with rampant violence, hard-to-reach populations, a dilapidated health system and, not surprisingly, low immunization coverage. The CGPP initiated full-fledged interventions in 2014 to reach as many children as possible with polio immunization in Yobe state.At the time the CGPP began its work in 2014 in Yobe state, the population coverage of the third dose of oral polio vaccine (OPV3) was 27.2%, Diphtheria, Pertussis, and Tetanus (DPT3) coverage was 11.0%, and the coverage of the third dose of pentavalent vaccineVolunteer community mobilizers are the foundation of the CGPP efforts in Nigeria. All VCMs are women, and they are selected by and from their local communities to serve as volunteers. They are trained and supervised by the CGPP and its partners. Volunteer community mobilizers are assigned households in their community and responsible for making frequent visits to speak with caregivers about polio immunization and routine immunization, aiming to dispel misconceptions. Volunteer community mobilizers in each settlement of the CGPP implementation areas regularly record child immunization, track pregnant women, and register births in their settlements to ensure that as many newborns as possible receive their birth immunization against polio (OPV0) as well as other routine immunization in a timely manner. They establish personal relationships with the families they serve, building trust and becoming valued sources of information. During immunization campaigns, VCMs mobilize the caregivers in their communities to immunize their children.suna) and other religious gatherings where they administer OPV0 to newborns. The CGPP has continued to build the capacity of VCMs to provide more information and support to their communities. They have been trained in breastfeeding, nutrition, and child health. In addition, they are equipped to refer families for other services when indicated.The VCMs go house to house to support families, resolve issues of non-compliance when they can, and record cases where noncompliance could not be resolved. Volunteer community mobilizers frequently attend naming ceremonies —in unique strategies. One successful example of male engagement is the IftarBefore the start of these efforts, VCMs create a list of children who did not receive polio immunization because of rejection by their caregiver along with the name of the mosque their fathers were likely to attend. Consultations with imams11 revealed that 57.2% of children 12–23 months of age had received their third dose of pentavalent vaccine in the whole of Nigeria. Although this indicates an improvement from 48.8% in 2015, the data were more troubling in the conflict-affected Yobe state. The survey showed that 48.4% of children aged 12–23 months had received the third dose of the pentavalent vaccine. Although this represented major progress from the 2014 coverage level of 7.8%, it was still almost 10 percentage points below the national average.The 2018 National Nutrition and Health SurveyIn the LGA of Karasuwa in Yobe state, the CGPP had been mobilizing communities for immunization acceptance through interpersonal communication. These efforts proved successful. The percentage of zero dose children (children who had never received a dose of polio vaccine) decreased from 1.0% to 0.2% in 2018, the lowest rate ever recorded. In addition, there were no areas in which acute flaccid paralysis (AFP) was not detected , meaning that cases of AFP were being detected through the surveillance system and that the surveillance system was quite sensitive.After every routine immunization session, the health provider extracts and shares with the CGPP VCM attached to the health facility a list of children who had defaulted. The VCM tracks down these defaulters to ensure that they are reconnected to the health facility to continue receiving their routine immunizations. In Yobe state, for example, one of the CGPP VCMs from Bursari LGA  was assigned to track defaulters from her settlement. Within 2 weeks, she was able to reconnect with all 21 caregivers who had a child younger than 1 year and who had defaulted. She was able to get all of these children caught up on their immunization schedule.una vaccination (the polio vaccination given during the mandatory naming ceremony that occurs 7 days after birth) has increased the number of children being immunized with the birth dose of OPV. Pregnant women are being tracked and monitored so that all deliveries can be documented and the VCM can vaccinate the baby during naming ceremony on the seventh day.In addition, the sIn April 2018, the CGPP joined the government and other polio eradication partners to launch the African Vaccination Week under the umbrella theme of “Vaccines work. Do your part!” Organized and led by the WHO, the annual event promotes increased awareness of the rights and needs of women and children to be protected from vaccine-preventable diseases through vaccination. The CGPP worked with the government and partners such as the United Nations Children’s Fund to pioneer the commemoration of African Vaccine Week at the LGA level as a means of making the local government more aware and accountable on immunization issues. The CGPP supported key components of African Vaccine Week such as message development for use in information, education, and communication materials, and it also generated awareness and understanding through advocacy and social mobilization activities.Several households in Anoma settlement (part of Karasuwa LGA in Yobe state) reacted to rumors that vaccines would sterilize their children and that they contained “impurities” that would taint Muslims, making them “unholy.” There had always been anti-vaccine rumors, which affected the uptake of immunization services. During this special week-long event, five households with nine children in the Anoma community were unwilling to accept the vaccine. After learning of the community’s rejection, the LGA government immunization team assigned the local CGPP team to meet the community members and address their concerns. Vaccine opposition had been a recurring issue in the LGA, prompting the NEOC to call upon the CGPP to change resisters into accepters.Duk lokacin da yaran mu ba su da lafiya, ba’a bamu Magani a Asibiti, ama kuna so ku ba wa yara masu lafiya alluran rigakafi bayan ba su bukatan shi.” The CGPP held compound (neighborhood) meetings, community dialogue, and face-to-face interactions using CGPP information, education, and communication materials. The CGPP was given permission to visit households. The CGPP team conducted a dialog with all the caregivers of the settlement. One reason given by the caregivers for rejecting polio vaccine was the following: “The CGPP team helped the mothers to understand the importance of vaccines by emphasizing that the free immunizations offered immunity from diseases that could sicken and possibly cause death in their children. After 90 minutes of lively discourse and engagement by the CGPP team,Just 1 month later (in March 2018), the CGPP worked to resolve another case of non-compliance in Yobe state. The Tsohuwar Kasuwa settlement in Gadaka/Shembire Ward has among the highest numbers of noncompliant households, according to the Ward review meetings that take place between immunization campaigns. To combat cases of immunization resistance during house-to-house campaigns in Fika LGA, the CGPP organized immunization teams to meet the members of resistant households to discuss immunizations and to listen to the concerns of non-compliant community members. The team successfully convinced seven noncompliant households to immunize all of the 28 children in these households. Efforts such as these led to increasing the number of immunized children and decreasing the number of noncompliant households at the local level.In 2014, the CGPP in Nigeria began with 647 VCMs and 106 volunteer ward supervisors across 23 LGAs in the five high-risk states of Kaduna, Kano, Katsina, Borno, and Yobe. These states had high rates of immunization rejection and noncompliance along with low rates of polio and routine immunization uptake. By September 30, 2018, the CGPP had grown to include 2,205 VCMs and 264 volunteer ward supervisors in 32 LGAs in the same five states, covering 598,781 households, an increase from 355,197 households in 2017. This expansion in coverage translates to more pregnant women identified, tracked, and referred for antenatal care along with higher levels of newborn tracking and immunization with OPV birth dose and routine immunizations. On average, 835 newborns and 7,165 children were tracked and immunized with OPV each month.The CGPP VCMs have continued to track the immunization status of children, newborns, and pregnant women. They have developed strong knowledge and interpersonal communication skills, enabling them to resolve most issues of immunization noncompliance. 13 According to the CGPP evaluation reports, OPV0 coverage increased from 54.8% in 2014 to 99.0% in 2018 and OPV3 coverage among children aged 12–23 months increased from 47.2% in 2014 to 62.3% in 2017 and 88.4% in 2018.14 Routine immunization coverage has also increased during CGPP implementation: the percentage of fully immunized children rose from 33.0% at the inception of the CGPP in 2014 to 57.0% at the end of 2017 and 67.0% in 2018.14The impact of these efforts is visible through reports provided by the VCMs in the CGPP implementation areas. Coverage of polio immunization and routine immunization has climbed steeply among children and families in CGPP implementation areas.14 The CGPP has also made significant contributions to the polio surveillance network in the five states where it is working, engaging 792 community informants in addition to the VCMs. By the end of 2018, despite not having a presence in all settlements of the LGAs where the CGPP was working, 18.3% (41 cases) of the total 224 identified AFP cases were reported by CGPP-supported VCMs and community informants.In addition, extensive community mobilization during campaigns has contributed to a marked increase in the number of children younger than 5 years reached during each supplemental immunization activity, increasing from 355,148 children in 2014 to 837,454 in 2018. The percentage of missed children during the supplemental immunization activities based on reports from VCMs has also gradually reduced in the CGPP areas from 4.5% in 2014 to 0.8% in 2018.Yobe state is currently one of the largest CGPP implementation areas. The CGPP works in 10 LGAs there, using 780 VCMs and 78 volunteer ward supervisors. According to the CGPP’s records, the VCMs visit an average of 59,000 households there each week (an average of 76 homes visited each week by each VCM). The VCMs also attend naming ceremonies to administer OPV0. At least 3,750 children younger than 5 years are immunized weekly against polio in the state across the 10 LGAs where the CGPP is active.9 describes further the contributions of VCMs along with the non-governmental and civil society organizations with which they worked that contributed to these achievements.The findings presented here suggest that the efforts of the CORE Group Polio Partners (CGPP) VCMs have contributed to improved immunization coverage in areas of CGPP work, especially in North East States such as Yobe. In most areas of Nigeria’s North East, the level of insecurity is such that only CGPP and UNICEF VCMs are presently mobilizing the communities. A companion article in this series15 In Yobe state, the CGPP is well recognized by the state Polio Emergency Operations Center and has made important contributions to improving the coverage of various vaccines by reaching children in difficult, hard-to-reach areas of the state. The CGPP has supported the National Polio Eradication Program in Nigeria to address longstanding misconceptions and myths that have resulted in immunization refusal or total immunization rejection. The CGPP has leveraged strong community connections and partnerships to generate positive results.Nigeria has now gone almost 3 years without detection of a case of WPV and is on track to possibly achieve a WPV-free-status in September 2019. However, the challenge of cVDPV remains a major hurdle to achieving complete polio eradication, and the cVDPV challenge highlights serious gaps in routine immunization that is mainly due to low coverage for inactivated polio vaccine. The CGPP and other Global Polio Eradication Initiative (GPEI) partners  have contributed to this important milestone of near attainment of WPV-free status in Nigeria by raising awareness regarding the importance of polio immunization in previously non-compliant communities. The CGPP currently works in five of the 11 states in Northern Nigeria that are at high risk for continued polio transmission. It has deployed VCMs in these areas to battle the myths and misconceptions about vaccines that have led to poor immunization uptake in these areas. With the support of the CGPP and other polio partners, a total of 4.5 million children younger than 5 years were immunized in 2018 alone.suna), where they administer OPV to the newborn. Local government area Coordinators and volunteer ward supervisors report their data weekly. The CGPP also provide supportive supervision on how to conduct activities and on-the-job mentorship to improve the efficiency and effectiveness of activities and reporting.A critical area of CGPP expertise is the development of context-specific messages in the local language that resonate with families. The solid knowledge of VCMs about polio, child health, and community needs along with the strong relationships that VCMs have with families help to make VCMs effective in community mobilization. Volunteer community mobilizers have raised awareness, dispelled myths, made referrals, and converted parents into acceptors of polio immunization. Furthermore, VCMs provide important information to mothers and caregivers concerning routine immunization, antenatal care, nutrition, and child health. Volunteer community mobilizers in each settlement of the CGPP implementation areas also regularly track the number of new births in their settlements and attend naming ceremonies (Nigeria is the last polio endemic country in the African region of the WHO. The polio eradication initiative in the country’s North East continues to be challenged by cases of immunization resistance rooted in myths and misconceptions. The CGPP in Nigeria contributed to overcoming resistance to polio eradication activities. The CGPP has evolved several innovative approaches to address high rates of non-compliance and immunization rejection in North East Nigeria, particularly in Yobe state. The CGPP has gained the access and trust of these communities by implementing strategies that contribute to stopping rumors before they start and quelling rumors rapidly once they begin to take shape. These achievements have been a key part of Nigeria’s overall progress in polio eradication since 2014. Similar strategies hold great promise for addressing other public health challenges in Nigeria and beyond, particularly for mothers and children.


Mitogen-activated protein kinases (MAPKs) are involved in signaling processes induced by various stimuli, such as growth factors, stress, or even autoantibodies. Sequential activation and phosphorylation of kinases upstream of the MAPKs result in activation of the MAPKs and phosphorylation of their downstream substrates that can be either nuclear  or cytoplasmic . MAPKs comprise three functional families, namely, extracellularly regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38. MAPK signaling thus provides a versatile signaling module that regulates cell proliferation, differentiation, homeostasis and even cell death. Individual proteins associated with the MAPK signaling cascades are regulated not only by phosphorylation but also by scaffolding proteins that can associate with several signaling proteins simultaneously and thus facilitate their regulation and interaction. Numerous scaffolding proteins are known to exist . In thiWhile MAPK pathways have homeostatic roles, the pathological mechanisms underpinning many disease processes involve signaling via one or more of the p38, JNK or ERK pathways. This Special Issue provides new insights into how MAPK signaling contributes to specific pathological processes across a range of conditions, including disorders of lung development, type 2 diabetes, cancer, proliferative skin diseases, cardiovascular disease and neurological disease. Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants characterized by interrupted lung development. Supplemental oxygen is critical for preterm infants with respiratory failure. However, this hyperoxic environment contributes to BPD pathogenesis. Using a newborn mouse model, Menon et al.  found thExcessive fibroblast growth factor receptor (FGFR) signaling has been shown to be involved in human diseases such as cancer and skeletal disorders . Thus, nScutellaria baicalensis Georgi and Coptis chinensis Franch, individually and in combination gave significant protection against hyperglycemia, hyperinsulinemia and lipid abnormalities in a rat model of type 2 diabetes. This was associated with reduced systemic and liver inflammation, with data suggestive of reduced JNK, NF-κB, and Akt signaling as the underlying protective mechanisms. Interestingly, the effects of these traditional medicines are comparable to that of metformin that acts via AMP-activated protein kinase signaling which inhibits JNK signaling [Activation of MAPKs, in particular JNKs, has been shown to inhibit insulin signaling and contribute to inflammation in type 2 diabetes . In thisignaling ,8.Activation of JNK signaling pathways is also associated with other pathological processes such as liver diseases. In this Special Issue, Win et al. discuss the implications of JNK signaling mode as a modulator of cell death . The autIn addition to MAPK, death-associated protein kinases (DAPKs) have been shown to be important regulators of cell death. In this Special Issue, Elbadawy et al. provide an overview of the functions of DAPKs in various diseases such as cancer, neuronal injury and cardiovascular disease . A speciInfantile myofibromatosis is a rare disorder of mesenchymal proliferation characterized by nonmetastatic tumors and which is associated with specific point mutations in the platelet-derived growth factor receptor B (PDGFRB) gene. Using primary tumor samples and the tumor-derived NSTS-47 cell line, Sramek et al.  demonstr+-dependent protein deacetylase, SIRT1. Addition of the SIRT1 activator, SRT1720, diminished p38 and JNK signaling and normalized function in psoriatic fibroblasts, suggesting therapeutic potential for this approach. Another chronic proliferative disease is psoriasis, a multisystemic disease characterized by abnormal keratinocyte proliferation resulting in erythematous lesions on the skin. Dermal fibroblasts can regulate the function of adjacent keratinocytes, and studies by Becatti et al.  identifiInflammatory bowel disease (IBD) is characterized by intestinal barrier dysfunction that is strongly associated with gastrointestinal infections caused by Gram-negative bacteria. Cecropin A is an antimicrobial peptide (AMP) that has been shown to exhibit antimicrobial activity, but its potential role in intestinal barrier function has remained unknown. In this Special Issue, Zhai et al. now show that cecropin A can increase the expression of tight junction proteins zonula occludens 1 (ZO-1), claudin 1 and occludin, thus increasing intestinal cell barrier function . CecropiRabdosia inflexa (RI) exhibits potent anti-inflammatory and gastro-protective effects by affecting proinflammatory cytokine release and MAPK signaling [MAPK signaling is also involved in other gastrointestinal inflammatory diseases such as gastric ulcer. In this issue, Akanda et al. show that an extract from the perennial shrub ignaling . RI was Atherosclerosis and aortic valve sclerosis are major cardiovascular diseases in Western societies. Reustle and Torzewski  discuss Bones are subject to continuous remodeling that involves osteoclasts that degrade the bone and the counteracting osteoblasts that reform the bone. Physiologically, it is important that the activity of these cells is kept in a delicate balance, and tipping of this balance in one or the other direction is involved in numerous diseases. In this issue, Lee et al. review the roles of MAPKs in osteoclast biology . Osteocl+ channel to inhibit the A-type current inactivation, leading to rapid repolarization towards resting potential, thus causing an increase in firing frequency, which may contribute to the neuronal differentiation process. ERK5, an atypical member of the ERK family, is thought to contribute to neuron differentiation and survival. In this context, Kashino et al.  used theParkinson’s disease (PD) is a neurodegenerative disorder caused by insufficient dopamine production due to the loss of dopaminergic neurons. Both oxidative stress and endoplasmic reticulum stress are directly implicated in the pathogenesis of PD. As reviewed by Bohush et al. , both ofN-heterocyclic carbene (NHC) moiety [Organometallic drugs such as naphthalimides and their derivatives have shown promise in the treatment of cancers, but their clinical use is limited by their toxic side effects . Thus, t) moiety . They shThere have been numerous setbacks in the clinical use of MAPK inhibitors, in particular due to acute liver toxicity with p38 inhibitors . However


The uterus is dynamically regulated in response to various signaling triggered by hormones during the estrous cycle. The Hippo signaling pathway is known as an important signaling for regulating cellular processes during development by balancing between cell growth and apoptosis. Serine/threonine protein kinase 3/4 (STK3/4) is a key component of the Hippo signaling network. However, the regulation of STK3/4-Hippo signaling in the uterus is little known. In this study, we investigated the regulation and expression of STK3/4 in the uterine endometrium during the estrous cycle. STK3/4 expression was dynamically regulated in the uterus during the estrous cycle. STK3/4 protein expression was gradually increased from the diestrus stage and reached the highest in the estrus stage. STK3/4 was exclusively localized in the luminal and glandular epithelial cells of the uterus, and phosphorylated STK3/4 was also increased at the estrus stage. Moreover, the increase of STK3/4 expression in uteri was induced by administration of estradiol, but not by progesterone injection in ovariectomized mice. Pretreatment with an estrogen receptor antagonist ICI 182,780 reduced estrogen-induced STK3/4 expression and its phosphorylation. The estrogen-induced STK3/4 expression was related to the increase in phosphorylation of downstream targets including LATS1/2 and YAP. These findings suggest that STK3/4-Hippo signaling acts a novel signaling pathway in the uterine epithelium and STK3/4-Hippo is one of key molecules for connecting between the estrogen downstream signaling pathway and the Hippo signaling pathway leading to regulate dynamic uterine epithelium during the estrous cycle. The uterus is one of most important reproductive organs for successful pregnancy on mammals ,2. UnlikThe Hippo signaling is known as a cellular signaling pathway that controls organ size to regulate cell death and proliferation . The sigMouse experiments were performed on 6- to 8-week-old ICR mice . Mice were maintained in optimal temperature and light controls and allowed freely to take food and water. Experimental and surgical procedures complied with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Agricultural Animal Care and Use Committee of CHA University .To determine the stages of the mouse estrus cycle, the vaginal smear assay was done as previously described . Vagina Uteri were fixed in 4% paraformaldehyde for histology and immunostaining. After embedded in paraffin block, uterine blocks were sectioned with 5 μm-thickness using a microtome and put on Superfrost Plus Stain slides . For immunofluorescent assay, the slides were deparaffinized and boiled in sodium citrate buffer  for 40 min using antigen retrieval steamer . The antigen-retrieved slides were washed in PBS-T (PBS containing 0.05% Tween-20) and blocked by incubating in blocking buffer (PBS-T with 4% BSA) for 4 h (h) at room temperature. Then, the slides were treated with the indicated primary antibodies for overnight at 4 °C. After washing with PBS-T, the slides were incubated in blocking buffer containing the secondary antibody conjugated with Alexa-Fluor antibodies 488 or 546  at room temperature for 1 h. The slides were washed with PBS and treated with DAPI  . After washing, the slides were mounted by treating with Mounting Medium  and covered with glass coverslip. The expression and localization of proteins were analyzed using confocal microscopy . Images were converted to JPEG format at the resolution of 1024 × 1024 pixels. The intensity of positive signals was calculated by the software LAS X  and Image J software.2O2 in distilled water to quench endogenous peroxidase activity before antigen retrieval. After antigen retrieval, the sections were incubated in a blocking buffer  and treated with the indicated antibody followed by HRP-conjugated secondary antibody . After washing, the signals from antibody were acquired by incubating with DAB solution . The slides were counterstained with hematoxylin. The slides were dehydrated and mounted with Permount Mounting Medium .To perform immunohistochemical staining, the deparaffinized slides were treated with 0.3% HThe primary antibodies used in this study are as follows; anti-STK4 , anti-STK4 phospho-Thr183 (FITC) , anti-STK4 phospho-Thr183/STK3 phospho-Thr180 , anti-LATS1 phospho-Thr1079 , anti-YAP , anti-YAP phospho-Ser127 , and anti-β-Actin 2, 200 ng/mouse, Sigma-Aldrich, St. Louis, MO, USA) or progesterone  was subcutaneously injected into mice. Mice were sacrificed and the uteri were collected at 0, 1, 3, 16, 12, and 24 h after hormone treatment. Five mice were used in each group. To test estrogen receptor-mediated regulation of gene expression, ovariectomized mice were either injected with E2 (200 ng/mouse) or pretreated with estrogen receptor antagonist, ICI 182,780  30 min before E2 injection. Sesame oil  was used as control.Mice were ovariectomized to investigate the effects of estrogen and progesterone on the expression of Hippo signaling-related genes in the uterus as previously described . After 1® III First-Strand Synthesis System . The reverse-transcribed cDNAs were used for RT-PCR and quantitative RT-PCR analyses. RT-PCR was performed using C1000 Touch™ Thermal Cycler . The annealing temperatures used in the thermal cycling were indicated in Total RNAs were isolated from uteri using RNeasy total RNA isolation kit  according to the manufacturer’s instruction. The total RNAs (2 μg) were reverse-transcribed using SuperScript® Green Supermix  and the CFX96 Touch™ Real-Time PCR Detection System . The results were analyzed with the iQ5™ Optical system software . The relative gene expression was calculated by the comparative CT (ΔΔCT) method [Rpl7) or Gapdh as reference genes.Semi-quantitative RT-PCR analysis was performed using The iQ™ SYBR) method . The relSTK4 knockdown on gene expression in human endometrial cells, Ishikawa cell line was used. Ishikawa cells were transfected with STK4 siRNAs  or universal scrambled negative control siRNA  using Lipofectamine 2000 .To examine the effect of Total proteins from cells or uteri were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking with ProNATM phospho-block solution , the membrane was incubated with the indicated primary antibody diluted in ProNATM phospho-block solution  at 4 °C for overnight. The membrane was treated with HRP-conjugated secondary antibody  in ProNATM phospho-block solution to detect protein expression. The immunoreactive bands were detected by chemiluminescence using ECL Western Blotting substrate kit . The relative expression was imaged by ChemiDoc XRS system  and analyzed by One-way ANOVA analysis.STK3 and STK4 in the uterus, we evaluated the relative level of STK3 and STK4 transcripts during the estrous cycle using RT-PCR and qRT-PCR. As shown in STK3 and STK4 expression was dynamically regulated during the estrous cycle divided into four stages; proestrus, estrus, metestrus, and diestrus. First, both STK3 and STK4 transcripts were highly increased at the estrus stage and the increase was significantly reduced at the stage of metestrus  in the uterus during the estrous cycle. STK3/4 is a cytoplasmic kinase which acts as the first factor in the core Hippo signaling pathway . If the STK4 transcripts was increased in time-dependent manner after administration of estrogen (Stk4 expression was highest at 6 h after administration of estrogen (Rasd1) and lactoferin (Lf) to confirm estrogen response in ovariectomized mice. Rasd1 and Lf were known as either early- or late-response genes, respectively [Stk4 in ovariectomized mice (Areg), and Hoxa10  30 min before estrogen injection. The expression of STK4 transcript and STK3/4 protein was examined 6 h after estrogen administration. As shown in STK3/4 transcript and protein was dramatically blocked by ICI pretreatment. Immunostaining showed that estrogen increased the STK3/4 expression in LE and GE and induced phosphorylation of STK3/4  kinase pathway . TherefoIn the following study, we investigated the effect of STK3/4 knockdown on the expression of Hippo signaling target genes in a human endometrial cell line, Ishikawa cells. Knockdown efficiency of STK3/4 using siRNA in Ishikawa cells was shown in VEGFA [KLF5) [BIRC5) [CTGF) [E2F [CTNNB1) [VEGFA and CTGF, whereas it reduced KLF5 expression  , baculov [BIRC5) , connect) [CTGF) , E2F [19pression C. Howevend BIRC5 C. These In this report, we investigated the regulation of Hippo signaling pathway in the uterus. We found that the expression of STK3/4 is dynamically changed in the uterine endometrium during the estrous cycle . In addiDrosophila and is evolutionarily conserved in mammals. The Hippo signaling pathway is one of well characterized cellular signaling pathways in various cells and tissues [The Hippo signaling pathway is originally found in  tissues . The pat tissues ,21. It c tissues . Zhan et tissues . YAP and tissues , implyin tissues . STK3/4  tissues . The phoDrosophila Happyhour) knockout reduced YAP phosphorylation more significantly than STK3/4 knockout in human cell line [Interestingly, we found that LATS1/2 phosphorylation in the uterine endometrium seems to be faster than phosphorylation of STK3/4 signaling . It mighell line . This isRasd1 was mediated via p38-mitogen-activated protein kinase (p38-MAPK) signaling in endometrium of mouse uterus [The uterine endometrium has cyclic changes during the estrous cycle for successful pregnancy. It is known that the dynamic change of the uterus is regulated by ovarian steroid hormones, estrogen and progesterone. However, it was not enough to explain the complicated regulation of uterine endometrium during processes. In addition to hormone regulation, other signals need to be associated. In the previous studies, we showed that estrogen could be associated with rapid expression of other signaling molecules such as dexamethasone-induced RAS-related protein 1 (RASD1) and aminoacyl-tRNA-synthetase-interacting multifunctional protein-1 (AIMP1) ,8. RASD1e uterus . And we e uterus . EstrogeThe uterus circulates repeatedly through the three processes of proliferation, differentiation, and degeneration by steroid hormones until menopause. The uterine cells in the endometrium are the most active undergoing the cellular proliferation, differentiation , and degeneration in response to the hormones . The uteIn conclusion, the present study demonstrated that a key Hippo signaling factor, STK4 is regulated by estrogen in the uterus and presents a novel potential signaling pathway for regulating uterine dynamics during the estrous cycle. The present report provides experimental evidence to support the further investigation of Hippo signaling factors including STK4 as a mediator in estrogen signaling. It will give us better understanding for complicated signaling networks in the uterine environment during the estrous cycle.


Adipocytes arising from mesenchymal stem cells (MSCs) requires MSC adipocyte commitment and differentiation of preadipocytes to mature adipocytes. Several signaling and some cytokines affect the adipogenesis of MSCs. This review focuses on the roles of TGF-β/SMAD signaling in adipocyte commitment of MSCs. BMP4 and BMP7 signaling are sufficient to induce adipocyte lineage determination of MSCs. The roles of BMP2, TGF-β, and myostatin signaling in this process are unclear. Other TGF-β/SMAD signaling such as BMP3 and BMP6 signaling have almost no effect on commitment because of limited research available, while GDF11 signaling inhibits adipocyte commitment in human MSCs. In this review, we summarize the available information on TGF-β/SMAD signaling regulation of MSCs in adipocyte commitment. Deeper study of this commitment mechanism will offer new approaches in treating obesity, diabetes mellitus, and obesity-related metabolism syndrome. Mesenchymal stem cells (MSCs) are a group of cells with multilineage differentiation potential and self-renewal capacity, which were discovered and isolated from the bone marrow in the 1960s . Many stTGF-β superfamily signaling regulates the progress of many cellular processes such as apoptosis, inflammation, fibrosis, and adipocyte differentiation . There aActivin, inhibin, BMPs, GDFs, TGF-β, Nodal, and other ligands involved in TGF-β/SMAD signaling bind to cell membrane receptors to activate the intracellular downstream SMAD family proteins. The SMAD family proteins constitute three functional classes: the receptor-regulated SMADs (R-SMADs), the common partner SMAD (Co-SMAD), and the inhibitory SMADs (I-SMADs) , 15. TheIn the resting state, R-SMADs mainly exist in the cytoplasm, Co-SMAD predominantly localizes to both the cytoplasm and nucleus, and I-SMADs mainly tend to be in the nucleus. Upon TGF-βs ligand binding, the receptors recruit and phosphorylate SMAD2/3, and SMAD1/5/8 by BMPs ligands. After C-terminal phosphorylation of the R-SMADs, these receptor-activated R-SMADs (SMAD2/3 or SMAD1/5/8) are released from the receptor complex to form a trimeric complex consisting of two R-SMADs and a SMAD4 molecule, which then translocates to the nucleus to regulate the expression of specific genes , 16, 17.− lineage mainly gives rise to white preadipocytes, and the Myf5+ lineage gives rise to brown preadipocytes, but may not be absolute. Some studies confirmed that brown adipocytes and a subset of white adipocytes originated from Myf5+ lineage [Adipose tissue dysfunction can lead to obesity, fatty liver, dyslipidemia, and type II diabetes , 21, 22. lineage . Most st lineage –55. The LOX (lysyl oxidase), TPT1 , and αB-crystallin which are target genes of BMP4 signaling in the adipocyte commitment of C3H10T1/2 cells [SMAD4 impeded adipocyte commitment by exogenous BMP4. Treatment with 40 ng/mL BMP4 triggered the adipocyte commitment of hASCs. The committed cells differentiation into adipocytes was associated with increased activation of PPARγ, APM1, AP2, and GLUT4 [The role of BMP4 signaling has been validated in the commitment process of MSCs. C3H10T1/2 cells were isolated from C3H mice in 1973 and can differentiate into various cell types such as adipocytes, osteocytes, chondrocytes, and myoblasts , 56. C3H/2 cells , 31. Tre/2 cells . The res/2 cells . A33 cel/2 cells . Exposur/2 cells . BMP4 al/2 cells . The hAS/2 cells . Inhibit/2 cells . SMAD1/5/2 cells . Also, knd GLUT4 . To datend GLUT4 . BMP4 alnd GLUT4 . The expnd GLUT4 . It alsond GLUT4 , 61.LOX, TPT1, and αB-crystallin in the adipocyte commitment of C3H10T1/2 cells [SMAD4 expression prevented the adipocyte commitment of C3H10T1/2 cells by BMP2 [Escherichia coli origin [BMP2 signaling  in the adipocyte commitment of MSCs remains unclear. Several studies found that BMP2 signaling can induce pluripotent C3H10T1/2 cells to commit to the adipocyte lineage –31, 33. /2 cells , 31. Kno by BMP2 . BMP2 in by BMP2 . SMAD6 b by BMP2 . The exp by BMP2 . The reli origin . Conversi origin , 62, 63.i origin . Thus, ii origin , 31. DesBMP7 knockout mice died shortly after birth, BAT mass was reduced and there was an almost complete absence of UCP1 in the knockout newborns [Many studies have demonstrated that BMP7 has an important role in brown adipocyte lineage determination , 44, 46.newborns . Mice innewborns . BMP7 alnewborns . Human anewborns .TGF-β is another important molecule that regulates the adipocyte commitment of MSCs. TGF-β signaling inhibited adipocyte commitment via SMAD3 signaling . TGF-β iMstn) knockout in mice (Mstn−/− mice) increased myogenesis and decreased adipogenesis [Mstn−/− mice had reduced adipogenesis and consequently decreased leptin secretion and C/EBPα and PPARγ levels [Mstn−/− mice also had suppressed obesity and glucose metabolism than control mice [Mstn−/− mice had reduced adipogenesis was that the absence of Mstn resulted in enhanced peripheral tissue fatty acid oxidation and brown adipose formation in white adipose tissue [Mstn−/− mouse embryonic fibroblasts (MEFs) had enhanced expression levels of brown adipocyte markers including UCP1, PRDM16, and PGC1 compared with the control group undergoing adipogenic differentiation [Myostatin, also called GDF8, is a well-known regulator of myogenesis inhibition. Some researchers found that myostatin can promote adipogenesis in MSCs. C3H10T1/2 cells were incubated in regular medium with graded concentrations of myostatin and found that there were many oil droplets present after a 2-week incubation, as visualized by Oil Red O staining . Myostatogenesis , 70. Mstγ levels . Mstn−/−e tissue . Some ste tissue . As preve tissue . Myostatntiation . From thSMAD4 in C3H10T1/2 cells. One study added BMP2 protein but the other did not, causing a different outcome because the function of SMAD4 was independent of the TGF-β signaling in some cases, and sometimes BMP2 had a role in adipocyte commitment independent of TGF-β/SMAD signaling. PPARγ is a master transcription factor in adipocyte commitment. BMP2/4 signaling induces adipocyte commitment of MSCs by activating PPARγ expression. A recent novel study reported the epigenetics of adipogenic commitment under TGF-β/SMAD signaling. miR-20a promoted C3H10T1/2 cells to commit, and this function may depend upon its direct inhibitory effect on lysine-specific demethylase 6B (KDM6B) and TGF-β signaling [TGF-β superfamily signaling has an important function in regulating the adipocyte commitment of MSCs. TGF-βs ligands such as TGF-β and myostatin use SMAD2/3 signaling to regulate adipocyte commitment. However, BMPs ligands such as BMP2/4/7 use SMAD1/5/8 signaling to regulate adipocyte commitment. BMP4 signaling is explicitly sufficient to induce adipocyte lineage determination and the formation of brown adipocytes in MSCs. BMP2 and myostatin signaling have an effect on the adipocyte commitment of MSCs. Some studies found that BMP2 or myostatin signaling can promote adipocyte commitment in MSCs, while animal experiments showed the same results. However, some studies found that BMP2 or myostatin signaling can inhibit this commitment. It is unclear whether they promote adipocyte commitment or inhibit it. It is well known that BMP2 and myostatin signaling were originally identified based on their ability to induce osteogenesis and myogenesis inhibition. BMP2 and myostatin signaling may play a small part in adipocyte commitment, perhaps due to different donor lines, the concentrations of an adipogenic induction cocktail, or serum types in culture medium. Previous articles have demonstrated that TGF-β signaling promoted the proliferation of MSCs and suppressed the adipocyte commitment of MSCs by inhibiting C/EBPs and PPARγ expression. Recent studies have showed that TGF-β promoted adipocyte commitment. The difference was that they studied the adipocyte commitment of BMSCs isolated from different species: mice and humans , 47. MSCignaling . Thus faThe TGF-β superfamily members have an effect on adipocytokines secreted from WAT and BAT as well as beige adipose tissue, influencing white-to-brown transition and disease. Future studies of TGF-β superfamily signaling will offer new approaches in treating obesity, diabetes mellitus, and obesity-related metabolism syndrome.


Synthetic biology uses living cells as the substrate for performing human-defined computations. Many current implementations of cellular computing are based on the “genetic circuit” metaphor, an approximation of the operation of silicon-based computers. Although this conceptual mapping has been relatively successful, we argue that it fundamentally limits the types of computation that may be engineered inside the cell, and fails to exploit the rich and diverse functionality available in natural living systems. We propose the notion of “cellular supremacy” to focus attention on domains in which biocomputing might offer superior performance over traditional computers. We consider potential pathways toward cellular supremacy, and suggest application areas in which it may be found. Synthetic biology uses cells as its computing substrate, often based on the genetic circuit concept. In this Perspective, the authors argue that existing synthetic biology approaches based on classical models of computation limit the potential of biocomputing, and propose that living organisms have under-exploited capabilities. The promise of synthetic biology lies in its potential to provide new substrates for computation3, production4, pollution control5 and medical diagnosis6 (among many areas), and to harness the “wetware”7 inside the living cell for human-defined purposes. Synthetic biology is set to become a significant component of the multi-billion dollar bio-economy8, but, in addition to tangible benefits such as cheaper drug production or more precise bio-sensing, many researchers in the field believe that the very process of re-engineering life will both require and inform a reexamination of our fundamental understanding of cellular processes9. This position underpins the current paper.Synthetic biology10 and the “repressilator”11).Much existing work in synthetic biology is concerned with the construction of “circuits” of biological components (such as genes and proteins) that receive some input , perform some transformation of that input, and produce a result determined by the “rules” encoded in the circuit. This input-transform-output pipeline is entirely consistent with the notion of a computation, as traditionally defined, and it is wholly unsurprising that synthetic biology has, so far, largely adhered to the genetic “logic circuit” model . Characterisations of the cell as a “factory”, a “little engine”, and a “chemical machine” dominated early discourse in cell biology, and the post-war development of cybernetics and computer science, combined with the emergence of molecular biology, provided significant support for this conceptual interpretation of living systemslac operon13 facilitates metabolic switching within E. coli in a manner that may be interpreted as a simple Boolean circuit. In his highly influential popular book, Chance and Necessity (a chapter of which is titled “Microscopic Cybernetics”), Monod made explicit the connection between biological processes and the basis of electronic circuits:The “machine model” of the cell was further cemented by the pioneering work of Jacob and Monod, which established that the 14“The logic of biological regulatory systems abides not by Hegelian laws but, like the workings of computers, by the propositional algebra of George Boole.”lac operon, the machine model has largely dominated molecular biology15, and the notion of the genome serving as a “genetic program” is still relatively common16. Of course, abstracting biological processes to the level of circuits (Boolean or otherwise) or even programs may be a necessary step in terms of making sense of their operation. Conversely, in the context of engineered biological systems, the availability of a set of well-characterised, modular and tunable components are perhaps necessary for creating controllable and predictable systems17. We emphasise here that we do not seek to overturn or abandon an extremely powerful and useful metaphor. However, it may give a misleading impression regarding the reliability and predictability of genetic circuits, compared to their electronic counterparts18. Moreover, as we argue in this paper, by restricting ourselves to the computational palette offered by (Boolean) combinatorial logic, we inherently (and seriously) limit the power and scope of synthetic biology.Since the discovery of the 19, which many see as a conceptual precursor to synthetic biology, if not a direct predecessor. Although the notion of computing with individual atoms and molecules was attributed to Richard Feynman as early as the 1950s20, the field was only reified in 1994, when Leonard Adleman published his groundbreaking paper on computing solutions to the Hamiltonian Circuit Problem using molecular operations with strands of DNA21. At the time, a commonly-held belief was that the future promise of DNA computation22 lay in solving instances of hard computational problems that were beyond the capabilities of traditional silicon-based computers23. Much early work on producing general DNA-based computational models focussed on molecular emulation of the Boolean circuit model25. Discussion of the field’s overall direction was, for a time, dominated by the quest for the “killer application”, the application of DNA computing that would establish its superiority over existing computational substrates27. In some ways, this may be viewed as a restricted form of the search for so-called “quantum supremacy” in the quantum computing domain28.It is perhaps instructive at this point to discuss the field of molecular computing22. For even small problem instances, the amount of DNA required to represent all possible solutions would be astronomical; Hartmanis calculated that if Adleman’s experiment were scaled up from 7 to 200 vertices, the amount of DNA required would weigh more than the Earth29. We note, however, that this inherent time-space tradeoff would appear to hold regardless of whether the underlying substrate is DNA, silicon, or a more exotic material (with the possible exception of substrates for quantum computers30).As the field of molecular computing evolved, it rapidly became clear that the killer application would not be found by solving large instances of computationally hard problems using any variant of Adleman’s original model. The elegance of the original solution derived from the encoding used to construct all possible paths through a given graph; vertices and edges were represented as strands of DNA that self-assembled into a collection of paths, which was then rapidly filtered in parallel using molecular manipulations to gradually extract valid solutions. Crucially, this reasonable run-time required the entire solution space for a given problem instance to be represented in an initial “test tube” at the start of the computation; the problem was to find the “needle” ) in a molecular “haystack”. Essentially, the entire space of solutions was tested in polynomial time using molecular operations, but this came at the cost of having to represent the whole solution set in a single volume of liquid31 and strand displacement architectures32 emulated traditional computational models such as cellular automata33 and artificial neurons34, but also took advantage of biochemical processes that provided power beyond mere miniaturisation. That is, rather than simply harnessing the potential for massively-parallel laboratory operations on large (but finite) numbers of molecules, these systems co-opted specific physical and chemical properties of the underlying substrate to generate the potential for autonomous operation and dynamic behaviour. In the context of synthetic biology, we suggest the killer application for engineered living systems will be similarly derived; one that will use features of the living system that are unavailable via traditional silicon-based substrates, for the purposes of applications that exploit autonomy and the ability to perform in uncertain environments.Importantly, this assessment emphasised the fact that the killer application would probably not be derived from single-use, finite DNA-based systems . Subsequent influential work on DNA self-assembly37). That is, we seek a set of problem domains in which cell-based systems will offer capabilities beyond the reach of existing computers, due to cost or technological constraints. Simply put, cellular supremacy will be determined by the set of problems that biocomputers can practically solve that traditional microprocessor-based devices cannot. To identify this set of problems, we naturally focus on the “added value” that living systems offer beyond silicon-based hardware. In the following sections, we discuss a number of features of living systems that we believe give synthetic biology-based solutions a distinct “edge” over traditional computers. Importantly, we focus on aspects of computation and information transformation, rather than simply on the ability of the cell to act as a micro-scale drug precursor “production plant” or miniaturised bio-sensor. While these applications are important and valuable, they are not the main focus of the current discussion, which centres on the general computational capabilities of living systems if we go beyond the limitations of Boolean logic-based systems. In what follows, we use the term “cellular computing”39 to emphasise this focus on computation.If we accept that a driving motivation for synthetic biology should be the search for a killer application, then this naturally leads to a consideration of the class of problems in which engineered living systems might offer what we call cellular supremacy  architectures, and on their inherent stochasticity.When assessing cellular computing against traditional computing, it may be useful to frame the comparison using a scheme that emerged from an exercise on roadmapping the so-called “unconventional computation” domain. The authors identified a number of criteria or benchmarks against which emerging computational models may be measured, each accompanied by its own motivating question41.This leads us to a consideration of the specific computationally expressive mechanisms that are available to us, and which act both inside and between living cells. In what follows we focus on five (non-exclusive) features of living cells that (we believe) offer the most potential in terms of the search for cellular supremacy; these are reconfigurability , noise , concurrency , representation , and evolution (the population-level ability to seek out novel solutions to problems over time). By harnessing these capabilities, we will obtain bio-computing systems that are self-organising, self-repairing, resilient, distributed, and adaptiveFirst, we consider aspects of computational theory that suggest how cellular computers may out-perform traditional machines. We then describe a number of features of biological systems that lend themselves to non-standard computation. Taken together, these two perspectives point towards a number of possible areas for investigation in which the killer application for cellular computing may reside.42, and the von Neumann architecture43 used to implement the types of computations performed by the Turing Machine. Although Turing’s model provides a framework for answering fundamental questions about computation, “…as soon as one leaves the comfort provided by the abundance of mathematical machinery used to describe digital computation, the world seems to be packed with paradoxes.”44.The current definition of digital computation is based on the abstract model defined by Turing in the 1930s45).The important thing to note here is that although genetic circuits may appear to behave digitally, it is only the collective behaviour of a large number of inherently analogue components that give rise to this property. The fact remains that we still currently lack any formal framework within which to argue that a cell computes, according to any understood model of computation , and this severely restricts the range of computations that are possible with a single circuit. Clearly, there are models of computation more powerful than combinatorial logic, that is, models which can describe all computations that are possible with combinatorial logic circuits and more. In this regard, two extremely powerful models of computation, the Lambda calculus42. From a practical perspective, these considerations of the limits of computability might be of less conceptual value than the strategy that TMs employ to decouple the complexity of the machine from the number of states on which they can operate.The output of stateful computations depends not only on the current input, but also on the current state, which encodes information about previous inputs that are no longer present. Models of computation such as finite state machines (FSMs)  its dynamics65. Therefore, cells may already be seen as “stochastic processors” that could provide a substrate for implementing probabilistic algorithms. Indeed, the utility of stochastic processors has already been recognised in the silicon world66.Combinatorial logic circuits, FSMs and TMs model deterministic computations, which are essentially step-wise descriptions of mathematical functions that map inputs to outputs, where each step follows in a predetermined way from the previous step. Deterministic computation can be generalised to include stochastic and non-deterministic computation. Stochastic  computing refers to algorithms which can make random choices during their execution. Specifically, a given input may generate a number of different computational paths, each with some probability, and the cumulative probability of all such paths is equal to one. Probabilistic algorithms are a cornerstone of computer science68 and at the scale of evolution69. Perhaps more speculatively, we might also consider the role of quantum effects in biology70, and whether or not these may be harnessed for the purposes of non-deterministic algorithms in biological systems.In non-deterministic models of computation, as in stochastic ones, there may be many computational paths from input to output, but crucially each of these computational paths is explored simultaneously by the algorithm, with a result analogous to parallel execution of multiple distinct deterministic algorithms. While non-determinism has considerable impact on theoretical computer science, practical implementations of non-deterministic computation remain elusive. Nevertheless, algorithmic solutions to some computational tasks can be significantly easier to express as non-deterministic, and biological systems extensively exploit non-determinism, both at the population levelFor some models of computation, the definition of an algorithm is “sequential”, and implies an ordering of the computational steps. Implementations of these models enforce these “sequential semantics”, because sequential execution may be a strict requirement to ensure the correctness of an algorithm. However, for certain computations, algorithms exist in which the ordering of computational steps may be relaxed, or abandoned entirely—in computer science terms, the algorithm displays a level of concurrency. In such situations, if multiple computing devices may interact and coordinate their efforts as a “distributed system”, concurrent parts of an algorithm may be executed in parallel, potentially speeding up computations significantly.71. For instance, at a fundamental level, and even within a single cell, natural cellular computation is concurrent and distributed. Computation is performed by stochastic biochemical processes—the probabilistic interactions of spatially separated molecular computing “agents”, many of which can happen in any order. Computer science has long used distributed and concurrent models of computation such as actors49, Petri nets51, process algebras72 and population protocols52 to describe computations in just these situations.The utility of such distributed computing is not limited to performance advantages. For some problems, sequentiality is desirable, but the nature of the computing substrate makes it impractical (or even impossible) to enforceThe nature of computation described so far has been digital; that is, information is represented using a set of discrete values. However, many physically relevant quantities vary continuously. Even the signals that encode binary values in electronic logic circuits are, in reality, stored as continuous-valued voltages. Such signals must be discretised in order to represent digital values. In contrast, analogue computation allows continuous signals to be used directly in the computational steps of an algorithm, and for representation of information as continuous values.73. Furthermore, the biochemical processes responsible for cellular computations involve discrete interactions of discrete molecules, but are also inherently stochastic. Cellular computing may, therefore, be viewed as both digital and stochastic, or as analogue computation with noise74, and the viability of the cell as a substrate for synthetic analogue computations has already been demonstrated75.Although many cellular computations involving binary “yes/no” decisions may be interpreted as digital computations, and digital logic computations are certainly suited to applications such as biosensors, cells often exhibit graded responses to stimuli that are more appropriately viewed as analogue computations53 or neural networks46, use arbitrary precision signals to perform super-Turing computation, it is not clear whether realisations of analogue computation will be able to provide more computational power than digital computation, since physical laws forbid the existence of such an idealised computer76. However, for digital models of computation, the abstraction of continuous signals into digital represents a semantic gap between the formal definition and implementation of a computation. Models of analogue computation admit continuous signals, which not only narrows this semantic gap, but also allows more powerful computational primitives to be used for defining algorithms77. Consequently, depending on the computation and the computing substrate, an analogue algorithm might have a more intuitive implementation, require fewer components to implement, and be considerably more energy-efficient than its digital counterpart75.Whilst some models of analogue computation such as the Blum-Shub-Smale machine78, but the fact remains that the cellular environment is a radically different computing substrate than silicon. Although this difference might make cells unsuitable for computational tasks traditionally dominated by conventional computers, it could also offer opportunities to explore more unconventional models of computation. Aside from gene regulation, which has been useful for engineering biological logic circuits, a number of processes and features exist in natural systems which may offer computational capabilities. Here, we identify four such resources as promising in terms of their information-processing capabilities  and constrains how algorithms run on the system. A program compiled for one type of computer architecture may not be executable by another due to these physical differences. The architecture of most electronic computers is fixed during a computation and has been honed to perform primitive digital operations in quick succession, guided by a set of instructions (the code). While this architecture is general enough for tackling many types of task, its static architecture makes it sub-optimal for most of them. In a cellular consortium, such a severe trade-off between flexibility and optimality (e.g. in regards to energy consumption or speed) need not exist, as the cellular community can dynamically change its physical composition and the interactions present to produce a “living architecture” that better matches the problem at hand. Furthermore, such reconfiguration is possible even during a computation itself, offering a level of control and flexibility that electronic computers are unable to match. Harnessing these unique capabilities will offer synthetic biologists new approaches to solving problems and require us to rethink what a computer architecture can be126.A feature of multicellular consortia that pushes us beyond the capabilities of classical electronic computers is their highly flexible and re-configurable nature127. Combinatorial logic is, therefore, a good example of how a given theoretical model of computation may be easily implemented with one physical toolkit (electronic), but not with another (molecular).Combinatorial logic, which relies on well separated on/off signals to encode information, constitutes a valuable framework with which to understand and build genetic circuits. Even if molecular signals  do not have absolute and constant values, the notion of a promoter being repressed or not (thus being binary) is intuitive. Nevertheless, the use of Boolean logic for engineering cellular circuits is often challenged by the fact that molecular signals are intrinsically noisy, and therefore stochastic. Each signal will have different on/off values Fig. , and not128, in producing the phenotypic variability that allows for bet-hedging strategies that promote survival in fluctuating environments129, and as a key component of evolution under tight selection criteria69. Therefore, models of computation incorporating stochasticity would seem to be necessary to best fit the intrinsic properties of living systems.The voltage that runs through electronic wires is also noisy, but computer engineers overcame this problem by arranging for thresholds at the input and output of logic gates, so that analogue values could be abstracted into two unique values: 0 and 1. In contrast to this, living systems have evolved to benefit from signal variability. Stochasticity has been shown to be useful in coordinating the expression of large sets of genes130 reveals information about the structural architecture of the cell. Also, noisy patterns in the cellular machinery of Staphylococcus aureus (that lead to different infectious cell types) originate in upstream molecular events131. These examples, among others75, highlight the fact that the shape of non-digital biological signals is used to effectively transmit information. Therefore, the challenge is to use noise for implementing computations — an area where cellular computers clearly outperform conventional ones. In the meantime, current efforts in the discretisation132, stabilisation134 and reusability135 of noisy signals may help to understand, and harness, the dynamic complexity of molecular interactions.Unlike silicon computing devices, where noise is a consequence of random signal fluctuations, cellular systems are capable of encoding important information into noise patterns. For example, the change in gene expression noise according to intracellular physical distances136. Different implementations of this idea exist, such as genetic algorithms, evolutionary strategies or genetic programming, but the underlying principles are similar: a set of candidate solutions to a problem  compete under some pre-defined fitness criteria, and the fittest individuals are selected to form the next generation (via crossover and/or mutation). An important advantage of these techniques is that they allow for the dynamic optimisation of solutions throughout potentially vast search spaces.Evolutionary computing is a field within computer science that develops and applies algorithms inspired by Darwinian evolution138, libraries of biological components139 and even to evolve non-functional genetic circuits into functional ones140. Despite this, evolutionary processes are often omitted when engineering computing circuits in living cells. Harnessing the power of such information-processing mechanisms for predefined functions seems to be difficult, and the rational engineering of autonomous evolutionary computing in living cells is still an overarching challenge.Both computer scientists and living systems use evolutionary algorithms to generate algorithmic solutions. Evolutionary processes have also been used to design biomolecules141, since, as evidenced above in “Towards whole-cell computation”, metabolism is a crucial omission from the point of view of information processing. A complete model of the flow of genetic information at a given point in time needs to include both genetic and metabolic processes, and dynamic models of computation inside living cells should include evolutionary processes that operate on genetic information.In order to start addressing this challenge, it will be important to formalise the effects of evolutionary dynamics on the flow of genetic information. A potential initial step would be to expand on the standard representation of the central dogma (CD) of molecular biology to include evolutionary dynamics, in an attempt to describe the model of computation of a living cell Fig. . Expansi142. Importantly, these studies linking environmental signals to intracellular responses in a predictable way, as well as directed evolution efforts143, are undertaken in tightly controlled systems. However, the mechanisms that allow a cell to thrive in challenging and dynamic environments are largely unpredictable. For problems like bio-remediation which intrinsically involve ever-changing environments, natural cellular computers clearly show superiority compared to conventional ones.Theoretical models of computation may find valuable and perhaps novel implementations within this dynamic representation of the CD. Recent developments in optogenetics are promising to this end, since these show how genetic elements dynamically shape their response in relation to external signals144, the benefits are largely limited to the exploitation of material factors such as miniaturisation and longevity of components.As already discussed, cellular computing is unlikely to compete with conventional computers in domains for which the latter are specifically engineered. Although future biological systems may offer competitive performance in related areas such as data storage145, bio-production146 and targeted therapeutics147 indicate that this is indeed a fruitful line of inquiry. Nevertheless, the implementation of conventional computations with unconventional computing substrates does not, in and of itself, constitute cellular supremacy. Rather, supremacy must be derived from the fact that the type of computation performed by a conventional computer is qualitatively different to that executed by living systems. Current implementations of computations within living cells are generally based on combinatorial or sequential logic circuits mapped onto equivalent genetic circuits; while these are useful metaphors for both understanding and engineering living systems, digital logic is not easily implemented using a cellular substrate. Furthermore, logic circuits offer a relatively bland computational palette compared to the richness of biology. For this reason, future developments in cellular computing should focus on models of computation that both accommodate and exploit the natural abilities of the cell, and avoid forcing biological systems into artificial (and often unsuitable) architectures148.The identification of promising applications for cellular computing has been largely guided by the observation that living systems operate best in application domains that are not easily reachable by conventional computers. Successes in bio-remediation151. Furthermore, the relationship between computer science and natural computing is often synergistic. As described earlier, models of computation that transcend logic circuits are available which have yet to be explored by cellular computing to any great extent. In return, biological systems offer computational features that are (and are expected to remain) unavailable to silicon implementations of existing computational models. It is at this intersection that exciting opportunities for cellular supremacy emerge. For example, in the past decade it has become increasingly clear that a number of biological processes show quantum mechanical properties153. In particular, there is strong experimental evidence that long-lived quantum coherence is involved in photosynthesis154, and that quantum tunnelling is active in enzyme catalysis155. Since most quantum computing devices are built and run under stringent environmental conditions (at temperatures approaching absolute zero), the opportunity to control quantum effects in a biological system that “runs” at room temperature, through the emergence of a “quantum synthetic biology”, could turn out to be a game changer in the quantum supremacy race. More to the point, realising models of quantum computation156 using quantum biology could yield a cellular computer capable of a radically different kind of computation than silicon153.This does not mean that cellular computing must necessarily “reinvent the wheel” when it comes to models of computation. Living systems share many characteristics with in vitro platforms for computation, and as well as the considerable body of theoretical work concerning molecular computing, cellular computing may draw on cell-free systems as a practical resource for faster prototyping with a greater degree of control157. Cultured neuronal cells have been used to control flight simulators158 and robots159, and slime mould has a rich repertoire of spatial computing capabilities160. Additionally, we do not dismiss the possibility of “hybrid” architectures, in which cellular systems are interfaced to more traditional, silicon-based machines. Such connections may allow for subtle and sensitive adjustments to cell state, for example161, but these hybrid systems will still fundamentally rely on the properties of the living material.Although we have, until now, focussed primarily on bacterial cells, there does, of course, exist a multiplicity of cell types that possess rich and complex computational capabilities. For example, plants can respond to environmental signals and stresses in a way that is reminiscent of signal processing in neural networks 162, especially at scales inaccessible to silicon. Robotics has also drawn inspiration from biological computation, particularly in relation to morphological computing, which takes advantage of the physical properties of computing agents in order to achieve more efficient computations163. By using intrinsic physical properties of the computational substrate to “outsource” parts of the computation, increasingly complex computations can be carried out whilst maintaining relatively simplistic control structures164. Living systems are an ideal implementation technology for morphological computation, since they not only compute solutions to individual instances of problems, but continuously compute and adapt in order to embody an efficient general solution. As we have previously argued, conventional silicon computers have inflexible architectures by comparison, which must sacrifice efficiency for generality. These qualitative differences between cellular and conventional computing suggest that applications such as terraforming and smart material production may remain beyond the reach of silicon computers, but in contrast, strategies for both applications based on living technologies have already been proposed166.As we have already argued, natural cellular computing operates at vast scales, in a distributed manner, and in the presence of considerable noise. Consequently, biological metaphors have served as inspirations for models of amorphous computation, for which cellular computing is a promising implementation technologyIn this perspective, we have championed the notion of cellular supremacy in an attempt to focus attention on the high-impact areas in which synthetic biology can have a truly transformational effect. We call on the community to consider cellular supremacy as a framing device for future work, and to explore in a systematic fashion how it may be established. By accepting the idea of cellular supremacy, we naturally acknowledge the richness and power of living systems. And by ceding a degree of control to biology, we may yet open up a much wider range of applications and perspectives on information processing in nature.


Citrus aurantium L.  has been used as a traditional medicine in China. Our previous study has shown that ZQ decoction may contribute to the antidepressant-like action of Chaihu-Shu-Gan-San. However, there are no reports on the chemical constituents of ZQ aqueous extract or its anti-depression effects. Firstly, this research reported the on-line identification of the chemical constituents in the aqueous extract of ZQ by coupling ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC-Q-TOF/MS). A total of 31 chemical constituents were identified in ZQ aqueous extract, including one tannic acid, five flavones, 13 flavanones, one limonoid, three coumarins, three cyclic peptides, and five polymethoxylated flavonoids. The antidepressant effect of ZQ aqueous extract was evaluated in vivo and the results indicated that the mice immobility time during the forced swimming test and the tail suspension test were significantly reduced with ZQ treatment. MTT assays showed both ZQ aqueous extract and its major constituents  had neuroprotective effect on corticosterone-induced neurotoxicity in PC12 cells. The in vivo and in vitro results suggest that ZQ has an antidepressant effect.The immature fruit of  Citrus aurantium L. (bitter orange) has been widely cultivated in tropics and subtropics. The extract of its immature fruit or peel is added to many weight loss formulas in dietary supplements. The immature fruit of C. aurantium  has also been used as a traditional medicine in China for treating the stagnation of dyspepsia, improving gastrointestinal function, and reducing chest pain + and/or [M+Na]+ adduct ions were observed. Due to the use of formic acid in mobile phase, the [M+46−H]− fragment ions corresponding to [M+HCOOH−H]− were also observed in negative ion mode. The results provided valuable information for confirming accurate molecular weights and the composition of the constituents. Based on the retention behaviors, accurate molecular weight and MSn fragment data, 31 compounds from the ZQ aqueous extract were tentatively identified  chromatograms of ZQ sample in positive and negative ESI modes are shown in entified  by compaentified . The ide− at m/z 609.1833, as well as its [M+H]+ at m/z 633.1792, is indicative of the molecular formula as C28H34O15. Additionally, the neutral loss of 308 Da (C12H20O9) from C28H33O15− in the MSE spectra was attributed to the characteristic ion [M−H−rutinose]− fragments. Then, the loss of 15 Da indicated the existence of “–CH3” group. The fragment ion at m/z 151.0063 (C7H3O4−) was produced from flavanone nucleus. Thus, this compound was tentatively identified as hesperidin, and further confirmed by comparing with reference compound 8 Da C12HO9 from C19, 26, and 29) were tentatively identified by analysis of their ESI(+)-MS spectra  in MS/MS spectrum. Thus, we tentatively identified compound 19 as cyclo, which has been previously reported from Citrus aurantium [26 and 29 were tentatively identified as citrusin I, and cyclo(-Gly-Gly-Leu-Leu-Leu-Pro-Pro-Phe-), respectively.Three cyclic peptides (compounds  spectra  and compurantium . Similiap < 0.05), similar to the positive control clomipramine hydrochloride. Furthermore, the significantly decrease on immobility time in the TST (p < 0.01) was also observed after treatment with the aqueous extract of ZQ. Both of the FST and TST experiments are suggestive of ZQ aqueous extract with significant antidepressant-like effect (The forced swimming test (FST) and tail suspension test (TST) are the most widely used animal models for antidepressant activity screening, partially because of their high predictive validity . In the e effect .in vitro experimental model of depression [The PC12 cell line, derived from rat pheochromocytoma tumors, possesses typical neuron features and expresses a high level of glucocorticoid receptors. The PC12 cells treated with high concentration of glucocorticoid to induce the neuronal damage have been widely used as an pression ,26. PC12The protection of four major components from ZQ aqueous extract against corticosterone-induced neurotoxicity in PC12 cells was also evaluated. When the cells were treated with naringin, hesperidin, neohesperidin, and nobiletin at 5, 10, 20 μM in the presence of 200 μM of corticosterone for 48 h, the cell viability was significantly increased as compared with the corticosterone treated group .In present study, we identified 31 constituents in the ZQ extracts using UPLC-Q-TOF/MS and the antidepressant effect of ZQ aqueous extract had antidepressive-like effect in both the FST and TST assays in mice. In order to explore its mechanism of action, the neuroprotective effect of both ZQ aqueous extract and its four major constituents was evaluated on corticosterone-induced neurotoxicity in PC12 cells. Naringin, hesperidin, neohesperidin, and nobiletin had protection against corticosterone-induced neurotoxicity in PC12 cells in a dose-dependent manner. ZQ aqueous extract also showed protection at concentrations of 5 and 10 mg/L, but had cytotoxicity at high concentrations . The results indicated that neuroprotective effect of ZQ may be involved in antidepressant mechanism. Its major active constituents should be naringin, hesperidin, neohesperidin, and nobiletin. Obviously, there exist constituents with cytotoxicity on PC12 cells in ZQ aqueous extract. l-arginine-NO-cGMP pathway [Flavonoids are a large group of phenolic compounds that are widely distributed in plants, and are known to antidepressant-like activity at least in animal models of depression . Many of pathway . Nobilet pathway . Our pre pathway . Thus, tReference substances of quinic acid, narirutin, naringin, hesperidin, neohesperidin, 3-methoxynobiletin, nobiletin, and tangeretin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products . All of the purities were above 98% by HPLC analysis. Clomipramine hydrochloride tablets were purchased from Jiangsu En Pharmaceutical Co. Ltd. . Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum, heat-inactivated horse serum, penicillin and streptomycin were purchased from Gibco . Corticosterone and MTT were purchased from Sigma-Aldrich . HPLC grade acetonitrile and methanol were purchased from Fisher . Formic acid (HPLC grade) was purchased from Tedia . Water was prepared using a Millipore Milli-Q purification system .Citrus aurantium L. by Yulin Lin of the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences and Peking Union Medical College. The voucher specimen is deposited in our laboratory of IMPLAD. The raw herb was purchased from Beijing Tongren Tang Pharmaceutical Co. Ltd.  and identified as the immature fruits of w/v) for 0.5 h at room temperature and thereafter boiled for 1 h. The filtrate was collected and the residue was then boiled again for 1 h. The filtrates were combined, concentrated under vacuum and lyophilized to give extract. The yield of ZQ extract was 20.28%. The accurately weighed ZQ extract (0.5 g) was dissolved in 25 mL of 50% methanol (v/v), and centrifuged at 13,000 rpm for 15 min at 4 °C. The 2 μL was injected for UPLC-Q-TOF/MS analysis after filtration through a 0.22 μm membrane filter. All samples were analyzed in triplicate.The raw herb was soaked in distilled water  equipped with an Acquity UPLC HSS T3 column  at a column temperature of 35 °C. The mobile phase was composed of water (A) and acetonitrile (B), each containing 0.1% formic acid. The line gradient program was carries out as follows: 10%–14% B at 0–2 min; 14%–20% B at 2–6 min; 20%–30% B at 6–9 min; 30%–40% B at 9–11 min; 40%–70% B at 11–14 min; 70%–99% B at 14–16 min; 99% B at 16–18 min; and 10% B at 18–20 min. The flow rate was 0.45 mL/min. The mass spectrometric data were collected using a Q-TOF analyzer in a SYNAPT HDMS system (Waters Corporation) in both positive and negative ion modes. The parameters were set as previously described [m/z 50 to 1200 with a scan time of 0.3 s and an interscan delay of 0.02 s over a 15 min analysis time. Leucine-enkephalin was used as the lock mass (m/z 556.2771 in positive mode and m/z 554.2615 in negative mode) at a concentration of 0.5 μg/mL with a flow rate of 80 μL/min. The lock spray frequency was set at 20 s.The sample was analyzed on a Waters Acquityescribed . The souThirty male ICR mice, weighing 18–22 g were purchased from the Institute of Laboratory Animal Science, CAMS and PUMC . The mice were housed individually in cages and maintained (23 ± 2 °C and 40%–60% humidity) under a standard 12-h light/dark cycle with free access to purified water and commercial diet. The mice were habituated for 7 days before the experiment. All experimental procedures were approved by the Ethics Committee of the Institute of Medicinal Plant Development, CAMS & PUMC. The mice were randomly divided into three groups. The mice in the vehicle, positive control and ZQ treated groups were administrated with normal saline (0.9% NaCl), clomipramine hydrochloride (40 mg/kg) and ZQ , for 14 consecutive days, respectively.The forced swimming test was performed according to the conventional method of Porsolt . BrieflyThe tail suspension test was performed as previously described . Briefly2.PC12 cells were obtained from the American Type Culture Collection . PC12 cells were maintained in DMEM medium supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), 5% fetal bovine serum and 10% horse serum at 37 °C in humidified atmosphere of 95% air and 5% COThe appropriate damaging concentration of corticosterone was selected based on the results of Gao . In brieTo research the neuroprotective effect of ZQ and its major components, the experimental design contained the treatment groups as follows: non-treated control, 200 μM of corticosterone, 200 μM of corticosterone plus ZQ , and 200 μM of corticosterone plus naringin, hesperidin, neohesperidin, and nobiletin . In the experiments, PC12 cells were seeded on a 96-well culture plates for 24 h, then corticosterone was added 48 h prior to treatment with ZQ or its major components, and then the cells were co-incubated with corticosterone and ZQ or its major components for another 24 h.5 cells/well. At the end of the treatment, the media was removed. Then the cells were washed with D-Hanks, and MTT solution  was added and further incubated for 4 h at 37 °C. Subsequently, the dark blue formazan crystals formed in intact cells were solubilized with DMSO. After shaking at room temperature for 10 min, absorbance at 570 nm was measured with a microplate reader . Cell viability was expressed as a percentage of the non-treated control.Cell survival was evaluated by 3--2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, PC12 cells were seeded on a 96-well culture plates at a density of 1 × 10The results were presented as mean ± standard deviation (SD). Data were analyzed using one-way analysis of variance (ANOVA) followed by post-hoc LSD test and differences were considered statistically significant at p < 0.05. All experiments were performed in triplicate.In the present study, an efficient and sensitive method employing ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was developed for qualitative analysis of chemical constituents of ZQ aqueous extract. A total of 31 compounds including one tannic acid, five flavones, 13 flavanones, one limonoid, three coumarins, three cyclic peptides, and five polymethoxylated flavonoids were identified. The results from FST and TST indicated that ZQ aqueous extract owned antidepressant effect. Our results also showed that the protections of ZQ aqueous extract against corticosterone-induced neurotoxicity in PC12 cells. Four major components  displayed the protection against corticosterone-induced cytotoxicity in PC12 cells in a dose-dependent manner, a further clue they are main chemical constituents of ZQ responsible for its antidepressant effect .