Strikingly, it predicts critical nodes following a shift from IGF to Wnt signaling. These findings indicate that this shift might cause age-related diseases.Aging is a complex biological process, which determines the life span of an organism. Insulin-like growth factor (IGF) and Wnt signaling pathways govern the process of aging. Both pathways share common downstream targets that allow competitive crosstalk between these branches. Of note, a shift from IGF to Wnt signaling has been observed during aging of satellite cells. Biological regulatory networks necessary to recreate aging have not yet been discovered. Here, we established a mathematical  Aging is a highly complex biological process, which impacts health-related quality of life and life expectancy. The underlying mechanisms of aging are still poorly understood. Several theories have been postulated concerning the cause of aging. On a cellular level aging is, for instance, provoked by DNA damage, protein aggregation or cellular dysdifferentiation –5. As a IGF and Wnt signaling pathways have been linked to aging –10 and bWnt molecules bind to seven-pass transmembrane Frizzled receptors and different co-receptors. Depending on the involved Wnt ligand, receptors and co-receptors, Wnt pathways are sub-divided into the canonical Wnt/β-catenin signaling cascade and the two non-canonical signaling branches Wnt/c-Jun N-terminal kinase (JNK) and Wnt/calcium pathway. Each Wnt pathway leads to distinct cellular responses. However, in satellite cells it could be shown that some Wnt ligands can regulate parts of canonical as well as non-canonical Wnt signaling ,15. CanoWnt signaling was shown to be deregulated during aging in diverse cell populations. For instance, in intestinal and hematopoetic stem cells a downregulation of canonical β-catenin signaling has been observed in aging ,23. In cIGF signaling regulates growth, differentiation, survival and the metabolism of carbohydrates, proteins and lipids . IGF iniThere is extensive crosstalk between both IGF and Wnt signaling. The canonical Wnt signaling mediator β-catenin not only interacts with TCF/LEF transcription factors but also with FoxO, which is regulated by IGF signaling ,25,35–37This raises the questions whether the known interactions between both pathways can be represented in a mathematical model and whether this model subsequently can represent changes during aging of satellite cells. Such a model of the dynamics and cross regulation of IGF and Wnt signaling might be beneficial for a better understanding of the mechanisms underlying aging.Boolean networks are a powerful tool to model dynamic cellular signaling pathways. They make use of the assumption that a gene is either expressed or not, resulting in two states: ON and OFF –51.All activating and repressing influences on a molecule are abstracted and summarized in its Boolean function. This Boolean function is then used to determine the subsequent state of all molecules of the model. To simulate a Boolean model usually an initial state is either given or randomly generated. Starting from this initial state, the states of the molecules are updated via the Boolean function creating a sequence of states until the model enters a circle of recurring states. Recurring states are called attractors and may represent known stable states. They are linked to biological phenotypes. Attractors may either consist of a series of states or just one single recurring state –47.The aim of this work was to create a Boolean network model, which accurately describes the crosstalk between IGF and Wnt signaling as it was observed in satellite cells. The knowledge incorporated into this model originates from extensive literature research. The model was then used to simulate the interaction of both IGF and Wnt signaling in the context of aging.t is specified by a binary string containing the value of each regulatory factor at that specific time point. In synchronous Boolean networks the state of each regulatory factor is updated at the same time. Consequently, a state transition is performed by synchronously applying all Boolean functions. A Boolean network with n regulatory factors has 2n possible states—one state for each possible combination of gene values. The state space can be depicted as a directed graph with one node for each state in the network. In this graph called state graph the transition from one state to another is represented by directed edges between the nodes.Boolean networks were introduced as dynamic mathematical models to simulate gene regulatory processes by Stuart Kauffman in 1969 . In BoolThe state space of a Boolean network is finite. Consequently, consecutive state transitions irrespective of the initial state eventually converge to a single state or a cycle of states called attractor. Attractors describe the long-term behavior of a Boolean network and can be associated with biological phenotypes . Each ofBiological systems are considered to be robust against perturbations . ComputeThe Boolean network model for the IGF/Wnt crosstalk was derived from collecting literature data. Modeling of the network functions was performed using ViSiBooL . The exh23 = 8,388,608 possible start states.To further analyze the effects of IGF/Wnt crosstalk in our network model, we divided the model in its IGF and Wnt sub-networks. Therefore, we removed the crosstalk elements from the Boolean functions. The attractors of the sub-networks were associated to those of the crosstalk model. This was done by comparing the overlapping regulatory factors of the complete network and the IGF and Wnt sub-network respectively in all the attractors. Two attractors are matched, if all overlapping factors are equal. The IGF/Wnt crosstalk model consists of 23 nodes. Therefore, there are 2To construct a regulatory network of the crosstalk between IGF and Wnt, we collected published data of both pathways and incorporated the core molecules into our model. An overview of this model and its internal interactions is given in Cellular signaling pathways are tightly controlled and depend on external stimuli. IGF and Wnt molecules can affect cells in a paracrine manner. Not necessarily being secreted by the Wnt or IGF affected cells themselves, they cannot be activated within the network. Therefore, in our model IGF and Wnt are considered inputs. Here, we briefly describe the rules implemented to model IGF and Wnt signaling and list the simplifications made within our approach.Canonical Wnt signaling is activated by Wnt binding to a Frizzled receptor (Fzd) and the co-receptor Lipoprotein receptor-related protein 5/6 (LRP5/6). This receptor complex in turn activates Dishevelled (Dvl) ,12,59. Iβ-catenin, the key effector of canonical Wnt signaling, can be phosphorylated and primed for proteasomal degradation by a destruction complex (DC) including the glykogen synthase kinase 3 beta (GSK3β) and axin 2, which triggers β-catenin inactivation ,12,59. DBinding of IGF to its receptor IGFR1 results in its activation and creates a Src homology 2 (SH2) binding site for activators of Ras ,30,72,73Caenorhabditis elegans gene age-1, being homologous to PI3K [In addition, activated IGFR1 (here via IGF) can also activate intracellular targets like Insulin receptor substrate 1 (IRS) ,71. Phos to PI3K ,81. PDK  to PI3K ,68,79. T to PI3K ,63,82–84 to PI3K . A downs to PI3K ,80,82,90Downstream of PI3K Akt inhibits Tuberous Sclerosis Factor 2 (TSC2) by phosphorylation ,80,91,92The conditions for mTORC2 activation are not yet completely understood. Association of the complex with ribosomes through PI3K seems to be necessary ,68,86,94In addition, GSK3β activates S6K ,91,97,98A very common interaction of the Wnt/calcium pathway and IGF signaling is the activation of Raf by PKC ,102,103 JNK is a potent activator of FoxO and thus contributes to the inhibition of TCF via FoxO. FoxO is a transcription factor for Rictor, which is a component of mTORC2. Since FoxO-dependent transcription of Rictor only boosts mTORC2 activity but cannot activate mTORC2 itself we did not include FoxO in the regulation of mTORC2 and further activation of Akt. However, Akt can phosphorylate FoxO and thus inhibit its function as a transcription factor. Thereby, it is acting as a negative feedback regulator ,99,107. The Boolean functions corresponding to the previously described regulatory interactions are depicted in Once the model of the IGF/Wnt crosstalk was established by the transformation of literature statements into Boolean functions we performed an exhaustive search to identify all attractors of the model. Five attractors named attractor 1 to attractor 5 were identified with different frequency . Four ofDepending on the initial state the nodes representing the Wnt and IGF inputs stay active or inactive over the entire time period. Thus, there are four possible combinations of external input factors for our model: I) both IGF and Wnt are active (attractor 3), II) only IGF or only Wnt is active , or III) both are inactive (attractor 1).Attractor 1 represents the state of an un-stimulated cell; neither IGF nor Wnt are active and thus no input is given. During aging IGF signaling slowly declines while both canonical and non-canonical Wnt signaling increases . Based oHowever, when starting a simulation from an initial state where all nodes except the input factors are off, every attractor could be reached, except for attractor 5. Since attractor 5 could not be reached starting from the chosen initial state it was excluded from further analysis. Attractors 3 and 4 represent the age-related shift from IGF to Wnt signaling. Due to the slow transformation process first both IGF and Wnt are active (attractor 3) and afterwards only Wnt as external input is active (attractor 4).in vivo situation as closely as possible. Therefore, the created model is supposed to follow the same temporal expression pattern as during biological IGF/Wnt crosstalk.Simulations of a biological network by computational Boolean models should resemble the IGF and Wnt are extracellular signaling molecules and thus cannot be activated from within the network. If the simulation of the signaling cascade starts with an initial state where all nodes are inactive , the netStarting from an initial state in which all nodes are inactive expect the external input IGF, the simulation of a signaling cascade reaches the three-states attractor 2 . Here, IAs this attractor consists of three states, it probably does not represent any fixed state of a cell but rather the transition from one cellular state to another. Here, IGF and Ras as well as the canonical Wnt antagonist axin 2 are the only nodes that are active in all three states. All other nodes switch between their active and inactive state during iterations. This represents crosstalk and internal regulations of the signaling pathways too avoid permanent activation of signaling cascades. For instance it was previously demonstrated that IRS is inhibited by S6K to avoid an excess of IGF signaling ,69,79,91As mentioned before, IGF signaling is more active at the beginning of a lifespan and declines with time . TherefoSimulation of a signaling cascade starting from an initial state in which only the two external input factors IGF and Wnt are active , the modOriginating from a cell with active Wnt as single active node , canonicIn addition, Ras-Raf-MAPK signaling is also activated by crosstalk, even if IGF is not present. This crosstalk is mediated by factors of non-canonical Wnt signaling such as PKC, Rac and JNK.The resulting attractor is a single state attractor with active canonical and non-canonical Wnt signaling as well as components of IGF signaling. Wnt signaling increases with age . TherefoDuring aging of muscle cells, IGF signaling slowly declines while Wnt signaling increases . Also, nAfterwards, the shift from IGF to Wnt signaling was simulated by adding Wnt as an additional input factor. However, a slow shift from IGF to Wnt is supposed to occur. Therefore, we first activated Wnt as external input of the model in addition to IGF and performed another simulation based on the states of the prior attractor 2. This time the model reached attractor 3 . It turnIn a last step we turned off IGF signaling and simulated a signaling cascade with only Wnt as external input, thereby reaching attractor 4 . The effDuring aging of muscle cells, IGF signaling slowly declines while Wnt signaling increases  and thusOf note, the aged phenotype might be prone to develop aging-related diseases. Here, TCF is currently active and can further induce the expression of the canonical Wnt target gene cyclin D ,115. TheTo investigate the relevance of the IGF/Wnt crosstalk in our model we have simulated both pathways separately. As seen in The simulation of the IGF sub-network leads to three attractors , called This leads to the conclusion that the modeling of the aging process of satellite cells with the IGF/Wnt crosstalk model is only possible through the interaction of both pathways while the simulation of only one of the two pathways could not realize aging.Biomolecular networks are considered to be robust. This means that a perturbation of single molecules in most cases does not influence the behavior of the system. We evaluated the robustness of both the IGF/Wnt crosstalk model as well as the sub-networks.Biological networks can adapt to environmental changes and their functions are resistant to damage . The net6) in comparison to the randomly generated networks  the network stabilize and the simulation ends in a single state attractor which favors the behavior of Wnt signaling. As mentioned before, IGF signaling shifts to Wnt signaling during aging of satellite cells. By use of the model we demonstrate that the imbalance between these two pathways potentially leads to aging-related diseases. In particular, the permanent activation of TCF and Akt can lead to the development of cancer due to cell cycle activation and inhibition of apoptosis. Another well-known age-related disease is diabetes mellitus type II. It is defined by the reduced response of insulin receptors regarding normal insulin concentrations. A common underlying mechanism for the development of diabetes is the dysregulation of IRS . In our The simulation of IGF and Wnt pathways separately did not yield the young and old phenotype attractors (attractor 2 and attractor 4), which could be found in the IGF/Wnt crosstalk model. This further supports our thesis that crosstalk between IGF and Wnt pathways exists. Furthermore, the crosstalk is necessary to represent the aging process whereas Wnt or IGF signaling on their own are not sufficient to describe the aging process.Beside IGF and Wnt, there is a multitude of other growth factors and external stimuli, which can also influence the behavior of a cell. It stands to reason that signaling molecules and pathways other than IGF and Wnt interact with each other and shape the behavior of these two signaling pathways. Especially the simulation of the IGF/Wnt shift supports this theory. As aging is a highly complex process, IGF and Wnt signaling might certainly not be the only players involved.Despite being a commonly examined pathway, Wnt signaling is still not fully understood. Examples for this are the activation of the mTOR complexes or the activation of canonical and non-canonical Wnt signaling pathways by different Wnt molecules. Especially the latter is dependent on the cell type. Our model was simplified to include the main signaling pathways.During aging, the functionality of stem cells decreases in adult tissues ,122, whiIt can be concluded that there are different mechanisms in Wnt signaling in different cell types. There are 19 Wnt proteins encoded in the human genome , which cAfter constructing and simulating our model, we evaluated its dynamic behavior by confirming the results with published laboratory experiments which were not included in the model construction. We could indeed confirm that IGF activates Akt, S6K and ERK  which ar6). In contrast, transition robustness of the sub-network models of Wnt (p = 0.051) and IGF (p = 0.2) does only borderline or not differ statistically from random networks. Comparison of the transition robustness of the IGF/Wnt crosstalk model Click here for additional data file.


The identification of components of the kallikrein–kinin system in the vitreous from patients with microvascular retinal diseases suggests that bradykinin (BK) signaling may contribute to pathogenesis of retinal vascular complications. BK receptor 2 (B2R) signaling has been implicated in both pro-inflammatory and pro-angiogenic effects promoted by BK. Here, we investigated the role of BK/B2R signaling in the retinal neovascularization in the oxygen-induced retinopathy (OIR) model. Blockade of B2R signaling by the antagonist fasitibant delayed retinal vascularization in mouse pups, indicating that the retinal endothelium is a target of the BK/B2R system. In the rabbit cornea assay, a model of pathological neoangiogenesis, the B2 agonist kallidin induced vessel sprouting and promoted cornea opacity, a sign of edema and tissue inflammation. In agreement with these results, in the OIR model, a blockade of B2R signaling significantly reduced retinal neovascularization, as determined by the area of retinal tufts, and, in the retinal vessel, it also reduced vascular endothelial growth factor and fibroblast growth factor-2 expression. All together, these findings show that B2R blockade reduces retinal neovascularization and inhibits the expression of proangiogenic and pro-inflammatory cytokines, suggesting that targeting B2R signaling may be an effective strategy for treating ischemic retinopathy. The kallikrein–kinin system (KKS) has long been recognized as a key player of inflammatory processes in various organs . In partBK exerts potent pro-inflammatory and pro-angiogenic effects through the activation of two G-protein-coupled receptors, BK receptor 1 and 2 , widely expressed in vascular tissues, including retinal vessels . PreviouIn endothelial cells, we demonstrated that BK/B2R signaling promotes cell proliferation and migration through activation of the pro-inflammatory nuclear factor kB and by the upregulation of cyclooxygenase-2, prostaglandin-E2 and vascular endothelial growth factor (VEGF) . A similOther studies suggested that the BK/B2R-dependent angiogenic response might be mediated by the upregulation of fibroblast growth factor-2 (FGF-2) and through transactivation of the VEGF-receptor 2 signaling ,9. VEGF Here, we investigate the role of BK/B2R signaling in two in vivo models of neovascularization, the rabbit cornea assay and the mouse retinal vascularization assay. We demonstrate that the B2R signaling is involved in physiological retinal vascularization. In fact, the B2R blockade by a selective antagonist significantly delayed the extension of retinal vessels in mouse pups. At high concentration, BK significantly induced pathological vessel sprouting in avascular rabbit cornea accompanied by an inflammatory response. Kallidin, a B2R agonist, mimicked the activity of BK in rabbit cornea, inducing a marked neoangiogenesis associated with a persistent corneal opacity indicating that B2R signaling plays a key role in inflammation-related angiogenesis. In the OIR model, characterized by hypoxia-induced pathological pre-retinal neovascularization, the B2R blockade reduced retinal neovessels, as determined by the area of retinal tufts , and the expression of VEGF and FGF-2 in pathological retinal vessels, suggesting that drugs targeting B2R signaling might have a role in proliferative retinal diseases.Mouse pups have an immature retinal vascularization at birth. The superficial vascular plexus forms during the first week after birth by radial outgrowth of vessels from the optic nerve into the periphery, reaching the retinal edges at approximately postnatal day (P) 8. Preliminary experiments were performed on mice (C57Bl/6) from P4 to P8 to investigate the B2R signaling activity on physiological radial outgrowth of retinal vessels and the maximum tolerate dose of the B2R antagonist fasitibant. Mice were treated daily with fasitibant  and then retinal vascularization was evaluated by immunohistochemistry. In all mice, no effects were observed after vehicle treatment  [In order to investigate whether B2R signaling was also involved in pathological neoangiogenesis in adult tissues, inflammatory concentrations of BK or kallidin were evaluated in the avascular rabbit cornea assay. This in vivo model was used to specifically investigate whether B2R signaling was associated to pathological neoangiogenesis, thus BK and the selective B2 receptor agonists, kallidin, were implanted in the corneal stroma as slow-release preparations. BK (1 µg/pellet) induced a marked pathological neoangiogenesis a, charac−12 M) , the datNext, based on above results, we assessed whether B2R signaling contributed to the pathogenesis of retinal microvascular complications by using the model of oxygen-induced retinopathy (OIR) in mice, which is well characterized by retinal vascular regression and hypoxia-induced pathological pre-retinal neovascularization. In OIR mice, a large avascular area was observed at the center of the retina, while the mid-peripheral area showed regrowth of superficial vessels leading to pre-retinal neovascular tufts, which were, mostly, at the border between the vascularized peripheral area and the avascular central area. Fasitibant did not affect the extension of the avascular central area a–d, whilFurthermore, in OIR mice, B2R blockade markedly reduced FGF-2 and VEGF protein expression in retinal microvessels a. It alsIn this study, we demonstrated that the BK/B2R signaling affects the pattern of physiological retinal vascularization in mouse pups, and induces pathological inflammation-related neoangiogenesis in the model of rabbit cornea. In mice from OIR model, characterized by hypoxia-induced pathological retinal neovascularization, the blockade of B2R by the antagonist fasitibant significantly reduced retinal neovessels and expression of the proangiogenic and pro-inflammatory growth factors FGF-2 and VEGF .The kallikrein–kinin system (KKS) has been implicated in the pathogenesis of retinal vascular inflammation and neoangiogenesis observed in several retinal diseases . BK and The half-life of BK in plasma is short , suggestIn light of the results, we propose a model for the angiogenic switch in retinal endothelial cells based on the BK/B2R signaling activation and, in turn, proangiogenic factor upregulation. We previously reported that FGFR-1 acts as a master switch in neovascularization mediated by inflammatory mediators by initiating a positive autocrine/paracrine cycle of FGF-2 synthesis and FGFR-1 activation . FurtherIn conclusion, the blockade of the B2R signaling by a selective antagonist might restrict the pathological angiogenesis in retinal diseases, reducing the acute inflammatory and angiogenic responses of the vascular endothelium, and the concomitant amplification and propagation through the FGF-2 and VEGF pathway .Mice were housed in a controlled environment and provided with standard rodent chow and water. All animals were subjected to a 12 h light–12 h dark schedule. The experimental procedures, according to Italian (DL 26/2014) and European (No. 63/2010/UE) regulations on the protection of animals used for experimental and other scientific purposes, were approved by the Italian Ministry of Health . To investigate the role of B2R signaling on retinal vascularization, neonatal mice with their nursing mothers (C57Bl/6) were treated from postnatal day P3 until P8  daily with 0.5, 1 or 2 mg/kg fasitibant or NaCl 0.9% (control condition), in 50 µL, i.p. Mice were sacrificed at day 8 by cervical dislocation, and the eyes were enucleated for retinal dissection and evaluation of retinal vascularization by immunofluorescence and Western blot analysis.Enucleated eyes were immersion-fixed for 2 h in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at room temperature. Retinal dissection was performed as reported . To visuThe experimental procedures, according to Italian (DL 26/2014) and European (No. 63/2010/UE) regulations on the protection of animals used for experimental and other scientific purposes, were approved by the Italian Ministry of Health .The angiogenic activity was assessed in vivo by using the avascular rabbit cornea assay ,31. New Oxygen-induced retinopathy, OIR, is a reliable model of vascular retinopathy. In brief, neonatal mice with their nursing mothers (C57Bl/6) were exposed to 75% oxygen from postnatal day P7 until P12 and then returned to room air (21% oxygen) . On retug for 30 min at 4 °C. The pellet was re-suspended in 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4 containing 150 mM NaCl, 5 mM EDTA (Ethylenediaminetetraacetic acid), 3 mM EGTA (Ethylene-bis(oxyethylenenitrilo)tetraacetic acid), 1 mM phenylmethylsulphonyl fluoride, 1 μM peptistatin, 10 μg/mL leupeptin and 2 μg/mL aprotinin, and centrifuged at 22,000× g for 30 min at 4 °C. The supernatant was used to detect FGF-2, VEGF, HIF-1alpha and B2R. Proteins from cell extracts were electrophoresed in SDS (sodium dodecyl sulfate)/4–12% polyacrylamide gels . Proteins were then blotted onto activated nitrocellulose membranes, incubated overnight with the indicated antibodies, and antigen–antibody complexes were detected with enhanced chemiluminescence kit . Band intensity was measured by scanning densitometry.Retinas  were homogenized and centrifuged at 22,000× Cell culture reagents, BK, kallidin, anti-B2R and anti-β-actin were purchased from Sigma Aldrich . Fasitibant was kindly provided by Menarini Ricerche, Florence, Italy. Anti-VEGF, anti-FGF-2 was from Merk Millipore . Anti-HIF-1α was from Bioss .p < 0.05 was considered statistically significant.Results are either representative or an average of at least three independent experiments done in triplicate. Statistical analysis was performed using a two-way ANOVA test followed by Bonferroni test .


Irrespective of various efforts, malaria persist the most debilitating effect in terms of morbidity and mortality. Moreover, the existing drugs are also vulnerable to the emergence of drug resistance. To explore the potential targets for designing the most effective antimalarial therapies, it is required to focus on the facts of biochemical mechanism underlying the process of parasite survival and disease pathogenesis. This review is intended to bring out the existing knowledge about the functions and components of the major signaling pathways such as kinase signaling, calcium signaling, and cyclic nucleotide-based signaling, serving the various aspects of the parasitic asexual stage and highlighted the Toll-like receptors, glycosylphosphatidylinositol-mediated signaling, and molecular events in cytoadhesion, which elicit the host immune response. This discussion will facilitate a look over essential components for parasite survival and disease progression to be implemented in discovery of novel antimalarial drugs and vaccines. Plasmodium falciparum is one of the major afflictions to the human health. The annual report of malaria speculated around 2.1 million cases of disease and more than 0.4 million mortality cases in 2015  and cyclic guanylyl monophosphate (cGMP). The homologous genes for enzymatic components involved in cyclic nucleotide-based signaling like adenylyl cyclase (AC), guanylyl cyclase (GC), cGMP-dependent protein kinase [protein kinase G (PKG)], and a regulatory and catalytic subunit of cAMP-dependent protein kinase, and nucleotide phosphodiesterase (PDE) have been identified , 19 in mPlasmodium culture was shown to positively affect the exflagellation or gametocyte formation during the ring stage of the parasite  pathway, which subsequently cause an increase in PfPKA coupled with elevation of Ca2+ level  or by developing transgenic parasite with higher expression of PfPKA-R (phosphokinase A-regulatory subunit), which binds to cAMP, thereby downregulating the process and directly affecting the parasitic growth  at late liver sporozoites (LS) stage depicted that parasite can infect hepatic HePG2 cell lines, but failed to get released from hepatocytes as merosome. Sporozoites at this stage elicited protective immune response in the host  and PfMSP-1 by P. falciparum subtilisin-like protease (PfSUB1) , resistant to compound 2 further demonstrated the role of PKG in the signaling event. Comparative analysis of the effect of treatment with compound 2 in wild-type and PfPKG mutant brought out various cellular targets of PfPKG involved in egress and invasion  Figure , 51. Them to PKG . The phoinvasion . The sigt manner , 53. Mort manner . The keyblocking .Plasmodium ineffective to infect the fresh erythrocytes . In this pathway, signals are transmitted successively to its respective components, MAPKKKs (MEKKs), MAPKKs [MEK/extracellular signal regulated kinase (ERK)], and finally to MAPK , 72. The Pfmap-1 , 74 and , similar to Ca2+/H+ exchanger , PfATP6 (PF3D7_0106300), and PfATP4  independent of extracellular Ca2+ ions.Live cell fluorescent video microscopy involving the use of fluo-4 Ca2+ ATPase from ER inhibitors and addition of Ca2+ ionophore enhanced the process of egress, while chelating agents like bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra acetic acid inhibited the process of egress (2+ is found to regulate the release of perforin-like protein 1 (PLP1) from microneme to the membrane where it begins its lytic activity. Likewise, the expression of PLP2 also occurs during the asexual blood stage, but its role is not clear. The reverse genetic studies would be helpful in disclosure of the details of PLP1 and PLP2 is another effector of calcium signaling, which is found to be required for the PfSERAs. The inhibition of PfSUB1 by the use of Ca2+ chelators prevented the rupture of the parasitophorous vacuole and consequently the egress of merozoites  and AMA-1 from microneme to merozoite surface , present in trace amounts in rhoptry neck, was found to trigger the release of calcium ions, which further initiates the cascade. The use of PfRH1 antibody inhibited the invasion process by blocking the calcium signaling and halting the interaction of EBA175 with host receptors. Therefore, it is assumed that an alternative pathway, namely, K+ ion-dependent pathway for the release of protein from apical organs, might exist. In spite of the above findings, the mechanism of triggering of signals to release calcium ions is yet to be probed , expressed during asexual and the gametocyte stage of parasite, was found to be colocalized with the centrosome. The role of centrin in cell division of Plasmodium yet concealed, while the knockout studies in Leishmania donovani revealed their involvement in the growth and cytokinesis  channels. It was demonstrated that exogenous addition of IP3 also causes the release of ions. Melatonin hormone was found to induce the production of IP3 through PLC, which further opens the Can the ER –93. An ie growth . Calciumlidation . Expressered yet . A diffeokinesis .There is an extensive involvement of calcium signaling in various important pathways of parasite. Any interruption would be deleterious for invasion, egress, and ultimately the growth of parasite. On these grounds, components of calcium signaling are considered for therapeutic interventions.via intracellular adaptors such as MyD88, TLR2, TLR4, TLR9, and TLR7 -12, IL-8, and interferon (IFN)-γ, has been noticed in infected individuals –103. Anyand TLR7 .P. yoelii infection, an unknown protein of parasite stimulates the TLR9 present on dendritic cells. These signals ultimately trigger off the Treg cells, which in turn suppress the effector T cells. Evidence suggests that mice deficient in TLR9 are more resistant to malaria infection due to the activation of the effective response of T cells  with TLR1 and TLR6 in a heterodimeric form  and induPlasmodium can differentially stimulate the MAPK pathway like ERK, P38, and c jun N-terminal kinase (JNK)  in macrophage. The induced cytokines lead to the development of symptoms like hypoglycemia, pyrexia, fever, illness, and lethal cachexia –129. Conse (JNK) , 134. Ofse (JNK) . Reflectnd IL-12 . Targetind IL-12 . GPIs exnd IL-12 , 137.P. falciparum, and this binding inhibits the invasion of parasite to fresh erythrocyte and also it obstructs the interaction of iRBC to CD36 and chondroitin sulfate A, thereby halting parasite sequestration and consequently suppress the production of the pro-inflammatory cytokine as well  with an anti-inflammatory effect, secreted by liver cell, was brought to attention . C1INH d as well . The efflciparum , 140 butPfEMP1 with endothelial cell surface receptors or receptors on immune cells is most widely explored  Crk-associated substrate (P130Cas) adaptor protein  activates protease activation receptor-1, which leads to induction of protective signals. APC on getting released binds to membrane phospholipids on platelets and causes the inactivation of the coagulation factor (PfEMP1 with EPCR inhibits the activation of protein C then pro-inflammatory cytokines from EC cause the shedding of EPCR (In childhood malaria, n factor . However of EPCR . But it  of EPCR .P. falciparum infection, interaction of iRBCs with ICAM-1 is one of the reasons for the development of cerebral pathologies (in silico approach, which can specifically block the binding of PfEMP1 to ICAM-1(Host immune response causes the subtle changes in the functioning of blood–brain barrier, which affects the cellular trafficking of lymphocytes . The infhologies , 162. Duhologies . Severithologies . The appto ICAM-1.PfPKG, PfPKA, and PfCDK gives an opportunity to exploit them as targets. The uniqueness of these molecules to the Apicomplexan provides an additional benefit. Despite this, several host receptors such as TLRs and immune cells are also awaiting (The impression of the involvement of signaling pathways during the asexual stage of parasite is clear. The different signaling strategies are involved to gain access or to modulate the host environment. Several techniques such as pharmacological inhibition and reverse genetic approach and techniques for single-cell live imaging revealed various indispensable components responsible for the invasion, egress, parasite growth, and survival. The non-homologous nature of most of the signaling molecules such as awaiting  for a liRS, DS, PR, and BS collected the data. RS wrote the manuscript. Overall monitoring was done by TB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vitamin A is an essential micronutrient throughout life. Its physiologically active metabolite retinoic acid (RA), acting through nuclear retinoic acid receptors (RARs), is a potent regulator of patterning during embryonic development, as well as being necessary for adult tissue homeostasis. Vitamin A deficiency during pregnancy increases risk of maternal night blindness and anemia and may be a cause of congenital malformations. Childhood Vitamin A deficiency can cause xerophthalmia, lower resistance to infection and increased risk of mortality. RA signaling appears to be essential for expression of genes involved in developmental hematopoiesis, regulating the endothelial/blood cells balance in the yolk sac, promoting the hemogenic program in the aorta-gonad-mesonephros area and stimulating eryrthropoiesis in fetal liver by activating the expression of erythropoietin. In adults, RA signaling regulates differentiation of granulocytes and enhances erythropoiesis. Vitamin A may facilitate iron absorption and metabolism to prevent anemia and plays a key role in mucosal immune responses, modulating the function of regulatory T cells. Furthermore, defective RA/RARα signaling is involved in the pathogenesis of acute promyelocytic leukemia due to a failure in differentiation of promyelocytes. This review focuses on the different roles played by vitamin A/RA signaling in physiological and pathological mouse hematopoiesis duddurring both, embryonic and adult life, and the consequences of vitamin A deficiency for the blood system. In mammals, this latter family of enzymes includes three cytosolic aldehyde dehydrogenases: RALDH1 (encoded by the ALDH1A1 gene) [ALDH1A2), which is the main RA-synthesizing enzyme in the mesoderm [ALDH1A3) [Vitamin A was discovered in the second decade of the twentieth century by Elmer McCollum and Marguerite Davis ; also knA1 gene) , RALDH2 mesoderm , and RALALDH1A3) ,14,15.RA signaling is mediated by two families of nuclear receptors: RARs and RXRs, including three members α, β and γ for each family . NuclearVitamin A deficiency is widespread in developing countries . BesidesRA signaling plays an essential role in vascular development and in the early embryonic hematopoiesis. In the mouse embryo, hematopoiesis starts around E7.0–E7.5, when a first generation of primitive erythroid cells and endothelial cells forms clusters or blood-islands in the splanchnic mesoderm of the yolk sac. Later, these clusters connect with the developing vessels of the embryo. These primitive erythroid cells are soon replaced by a second wave of hematopoiesis, when hematopoietic progenitors and the definitive hematopoietic stem cells (HSC) arise from hemogenic endothelial cells ,22,23. TRA signaling is involved in the differentiation of the endothelium, as well as in the emergence of the hematopoietic progenitors . The embRALDH2 is expressed in visceral endoderm and generates RA, which activates the RARs of the visceral mesoderm. The lack of RA in RALDH2 knockout mice leads to death at E10.5 due to multiple defects, including disorganization of yolk sac extraembryonic vascular network ,30,31,32Cre/RALDH2flox system) precludes the HSC formation in yolk sac and dorsal aorta, demonstrating that the endothelium itself is a main source of RA. These authors also demonstrated that the RA signal for the endothelial-hematopoietic transition is transduced through RARα, a receptor highly expressed in the endothelium [As well as in the yolk sac, RA signaling in AGM is essential for emergence of hematopoietic progenitors, including definitive HSC, from the hemogenic endothelium of the dorsal aorta. RALDH2 is expressed in coelomic mesothelium at E9.5 and later in mesenchymal cells of AGM region . Activatothelium . Howeverothelium ,35.RA signaling is also involved in the regulation of endothelial cell cycle and the specification of the hemogenic endothelium . The c-KFetal liver HSCs probably require RA signaling for induction of HOXA medial gene expression and control of HSC identity and function, as suggested by experiments with human embryonic stem cells and fetal liver HSC . Inciden-/- fetal liver than in controls at E10, but there are no differences at E12. Other elements of the retinoic signaling pathway, such as RALDH1/2, cellular retinol-binding protein-1 (CRBP1) and cellular retinoic acid-binding protein-1 and 2 (CRABP1/2) are highly expressed in the early fetal liver, when the hematopoiesis is taking place, and are downregulated by midgestation, suggesting that they may play a role in hematopoiesis [RA signaling has a role in the erythropoiesis from the fetal liver. Erythropoietin (EPO) is a glycoprotein hormone produced by the kidney and promotes the formation of red blood cells by the bone marrow. The early wave of erythropoiesis in the yolk sac is EPO independent; however, the early phase of fetal liver erythropoiesis requires EPO, retinoic acid signaling through RAR/RXR receptors and also stabilization of the hypoxia inducible factor (HIF1). Interestingly, RXRα requirement is only active during this early phase (E9.5–E11.5), as erythropoiesis can continue without RXRα signaling from E12 on. In fact, EPO expression is 10 folds lower in RXRαopoiesis . -/-/RXRβ-/- double mutant embryos die between E9.5 and E10.5 due to a lack of formation of the labyrinthine zone [Hematopoiesis in the placental labyrinthine zone and umbilical arteries has been well described throughout development ,44,45,46ine zone . Howeverine zone . Thus, mDanio rerio) is a powerful animal model to study the role of RA during development. Some recent reports have shown that RA signaling is critical in early steps of zebrafish hematopoiesis, including the formation of the HSCs. RA signaling is required for HSC marker expression even before the formation of the aorta, when the main RA synthesis enzyme, RALDH2, is expressed in the paraxial mesoderm. This requirement is related with the expression of junctional adhesion molecules JAM1a and JAM2a , the chemokine CXCL12 and its main receptor CXCR4 [The zebrafish  treatment leads to increased expression of GATA1-expressing erythroid cells [Later, RA signaling is involved in the generation of a first wave of erythrocytes and myeloid cells, but playing in this case an inhibitory role. This primitive wave of hematopoiesis is strongly reduced by treatment with exogenous RA at the 5 somite stage, while expression of the vascular marker FLI1 is increased. This effect can be rescued by injection of mRNA of the hematopoietic transcription factor SCL. Inhibition of the enzyme RALDH2 by 4id cells ,52. Primid cells .Vitamin A (Retinol) and its derivatives (retinoids) play a key role in development and are important in the homeostasis of many organs in adults . The receptors RARα and γ, but not the RARβ receptor, are highly expressed in the adult hematopoietic system. RARα is expressed in a variety of bone marrow cells, while RARγ is expressed in primitive hematopoietic progenitors, a deficiency of which causes a reduction in the number of HSC . Loss of-/- mice show no defects in the early steps of adult hematopoiesis and this receptor is not involved in the self-renewal potential of HSC. However, RA signaling transduced through RARα is critically required for granulocytic lineage modulation. The presence of free RARα receptor not bound to RA inhibits neutrophil differentiation while the binding of RA induces its differentiation [RARαntiation . Howeverntiation .-/- mice are anemic [RARγe anemic  and devee anemic . The erye anemic . NOTCH ae anemic ,59; while anemic . In contrast, it has been demonstrated that RARγ regulates B and T lymphopoiesis in the thymus and bone marrow. It was initially described that RARγ mutants only display defective primary and memory CD8+ T cell response, and the production of inflammatory cytokines by macrophages appears impaired . HoweverIn humans, the RA/RAR signaling system also plays a key role in the balance between self-renewal and differentiation in the bone marrow niche. Bone marrow human primitive CD34+/CD38− HSC express RALDH1 and RARα. The inhibition of the RA signaling on cultured HSC induces HOXB4 upregulation, which prevents their differentiation and enhances their proliferation and expansion in an undifferentiated stage . RA degrRA/RARα signaling is also essential for lymphocyte function and plays a key role in the regulation of the immune homeostasis, the differentiation of T cell subsets, the migration of T cells into tissues, and the development of T cell-dependent antibody responses ,66,67. IRA also modulates proliferation and differentiation of B cells ,73. RA sThe role played by RARα in modulation of the granulocyte differentiation above described is related with the pathogeny of acute promyelocytic leukemia (APL). This disease is produced by a chromosomal rearrangement, where the RARα gene fuses with other genes and generate a dominant defective form of RARα. Lack of RA signaling through the RARα receptor results in blockage of promyelocytic progenitors’ differentiation. The most frequent translocation is the fusing of the PML gene, which codes for the promyelocytic leukemia protein, a tumor suppressor transcription factor, with the RARα gene, although up to eight genes more have been identified in these translocations .2O3, ATO) has improved dramatically the prognostic of this disease, lethal some decades ago, and currently cured in about 95% of the cases [The treatment of APL with all trans-retinoic acid (ATRA) combined with arsenic trioxide  is a disease characterized by accumulation of myeloid blasts in the bone marrow, blood and other tissues, which lack the ability to differentiate, consequently causing a decreased production of normal blood cells . A numbeDespite these excellent therapeutic outcomes, treatment of leukemia with ATRA can produce inflammatory reactions in about 25% of patients. These adverse reactions are known as differentiation syndrome, previously known as ATRA syndrome, and it is characterized by fever, peripheral edema, pleuropericardial effusion, respiratory distress, hypotension and renal/hepatic dysfunction . The difThe molecular pathways leading to differentiation of APL cells induced by ATRA are being unveiled by studies in vitro. RA induces a neutrophil-like phenotype in HL-60 leukemia cells  and incrOther applications of RA have been reported in immunotherapy of T cell leukemia. ATRA, combined with interferon-α, upregulate CD38 in malignant cells, rendering them sensitive to a chimeric anti-CD38 antibody . As described above, vitamin A/RA signaling exerts their regulation on hematopoietic processes through the transcriptional activity of the RAR and RXR receptors. However, epigenetic mechanisms also regulate hematopoiesis  and areCRABPI locus (encoding cellular retinoic acid binding protein I) by an epigenetic mechanism involving chromatin condensation [CDKN1A promoter [One percent of APL cases are due to a translocation fusing the promyelocytic leukemia zinc finger (PLZF) gene with the RARα gene. These APL are refractory to ATRA treatment. PLZF represses the ensation . As wellensation ,106. IncMyelofibrosis with myeloid metaplasia (MMM) is another hematopoietic malignancy related with RA signaling and its epigenetic regulation. Expression of RARβ2 is downregulated in CD34+ cells from MMM patients, but only in 25% allelic loss has occurred due to a 3p24 chromosomal deletion. Instead, hypermethylation of RARβ2 locus was found in most MMM patients but not in normal individuals . This RAFinally, miRNAs post-transcriptional regulation in hematopoietic cells can also play a role in retinoid-based treatment of leukemia. For example, regulation by miR-223 is essential for ATRA-induced differentiation of acute promyelocytic leukemia (APL) blasts to granulocytes .As stated in the introduction, RXRs can form heterodimers with the vitamin D receptor (VDR). In this way, physiological functions mediated by vitamin D, via the binding of its active metabolite 1α,25-dihydroxyvitamin D3, are closely related to retinoid signaling and this modulation is relevant for hematopoiesis and also for AML treatment, as described below. Vitamin D enhances binding of RXR/VDR heterodimer to vitamin D response elements and it restricts the ability of 9-cis-RA to signal through the RXRα receptor, modulating the retinoid signaling pathway ,110. Vitamin D signaling seems to be dispensable for hematopoiesis, since vitamin D signaling-deficient mice show no phenotype related with blood cells production ,112,113.ATRA treatment is ineffective for AML, as stated above. Since vitamin D3 induces monocytic differentiation of some lines of leukemic cells, vitamin D analogs could represent a therapeutic option ,119,121.Given the role played by vitamin A/RA signaling in hematopoiesis, it seems clear that vitamin A deficiency (VAD) must constitute a serious problem for the functions of the blood system, and this has been demonstrated in a number of animal models. In mice fed with a vitamin A deficient diet or treated with RAR antagonists, an increase of myeloid cells is observed in bone marrow, spleen and peripheral blood ,125. ThiIn humans, particularly in children and pregnant/lactating women, VAD has been recognized as a public health issue in developing countries . The estSevere VAD leads to xerophthalmia, the most common cause of preventable blindness among children. According to WHO data, 250,000 to 500,000 vitamin A-deficient children become blind every year, half of them dying within 12 months of losing their sight . VAD is The beneficial effects of vitamin A or β-carotene supplements to prevent conditions derived from VAD have been described in a number of papers. Just to mention some instances, biscuits fortified with vitamin A reduced VAD and anemia in Chinese pre-school children . SupplemA number of strategies can be followed for prevention of VAD, i.e., dietary diversification, high-dose supplementation, conventional food fortification , biofortification  and genetic modification of crops . PromotiSupplementation with high doses of vitamin A periodically dispensed to children between six months and five years of age have demonstrated to be associated with large reductions in mortality, morbidity, and vision problems in an extensive meta-analysis . This coFortification of sugar, vegetable oils and cereal-based products by addition of vitamin A has been extensively explored for VAD control in developing countries, having already provided good results in Central and South America .Biofortification of maize by conventional breeding (the “orange” maize) has been developed in Zambia. Orange maize contains enhanced β-carotene concentrations and it appears to be as efficacious as vitamin A supplementation in Zambian children ,152. BioFinally, a strategy for fighting against VAD was the introduction of the “golden rice”, a transgenic variety of rice that accumulates β-carotene in its endosperm . Golden In recent years, researchers have become increasingly interested in the multiple functions played by vitamin A/RA signaling in developmental and adult hematopoiesis. This basic knowledge of the molecular mechanisms modulated by RA signaling has led to impressive advances in the therapy of some forms of leukemia, such as the APL. The induction of differentiation of malignant cells has been possible by a better understanding of the cellular and molecular control by RA signaling of myeloid differentiation. A major challenge for the near future will be to overcome the mechanisms of RA resistance in the treatment of other types of leukemia. It will also be very important to advance in the knowledge of the complex interrelationships between vitamin A/RA and vitamin D signaling, particularly in processes related with the modulation of the immune response. Given the critical importance of vitamin A/RA signaling in the immune defense and other essential processes for human health, the fight against vitamin A deficiency is a priority in developing countries. More efforts will be necessary to assess the current practices and to develop new, more effective and efficient strategies to reduce the prevalence of this serious worldwide problem.


KISS1) and neurokinin B (NKB) and their receptors  result in an abnormal timing of puberty or the absence of puberty. To understand the neuroendocrine mechanism of puberty, we investigated the contribution of kisspeptin and NKB signaling to the pubertal increase in GnRH release using rhesus monkeys as a model. Direct measurements of GnRH and kisspeptin in the median eminence of the hypothalamus with infusion of agonists and antagonists for kisspeptin and NKB reveal that kisspeptin and NKB signaling stimulate GnRH release independently or collaboratively by forming kisspeptin and NKB neuronal networks depending on the developmental age. For example, while in prepubertal females, kisspeptin and NKB signaling independently stimulate GnRH release, in pubertal females, the formation of a collaborative kisspeptin and NKB network further accelerates the pubertal increase in GnRH release. It is speculated that the collaborative mechanism between kisspeptin and NKB signaling to GnRH neurons is necessary for the complex reproductive function in females.In human patients, loss-of-function mutations in the genes encoding kisspeptin ( KISS1) and its receptor (KISS1R) in human patients result in delayed puberty or no puberty (NK3R) in humans also result in delayed puberty or no puberty  are 100% co-localized in the ARC and express estrogen receptor alpha in sheep. Based on the anatomical characteristics along with the self-regulating stimulatory and inhibitory circuitry between NKB, kisspeptin, and dynorphin, Goodman and co-workers , 49 haveAs the first step to test this hypothesis, we measured kisspeptin in the same samples collected from the median eminence, in which the effects of senktide on GnRH release were examined. The effects of senktide on kisspeptin release in females are strikingly parallel to its effects on GnRH . KisspepThe important question here is why a larger release of kisspeptin induced by senktide in pubertal females than prepubertal females is not directly transduced to a larger GnRH release? We speculate that this is due to involvement of opioid input, as opioid tone increases after puberty onset. In fact, it has been shown that opioid tone increases along with the pubertal increase in estradiol/testosterone. For example, while administration of antagonists for opioid peptides, such as naloxone and naltrexone, in prepubertal children, chimpanzees, and rhesus monkeys failed to stimulate LH/GnRH release –64, thesAs described above, both hKP10 and senktide stimulate GnRH release in a dose-responsive manner in prepubertal as well as pubertal female monkeys , 35. We Our findings reported in this manuscript are obtained from dialysates collected from the median eminence, where agonists and antagonists for kisspeptin and NKB are directly infused. Because of our technical precision , interacGenetic findings in humans indicate that both kisspeptin and NKB signaling is critical for the mechanism governing puberty onset –4. Similmakorin RING finger protein 3 gene (MKRN3) result in precocious puberty in humans  in prepubertal monkeys is halted by the cessation of the infusion , starts to decrease at P15, and becomes the lowest by P30, just prior to vaginal opening , and ove monkeys . CollectWe have shown that both kisspeptin signaling and NKB signaling appear to contribute to the pubertal increase in GnRH release independently or in concert in females. That is, while there is no interaction between kisspeptin and NKB signaling in sexually immature females, increases in kisspeptin signaling through NKB neurons and NKB signaling through kisspeptin neurons both augment the pubertal increase in GnRH release during the progress of puberty. The contribution of direct NKB signaling to GnRH release, however, may be secondary, as NKB signaling to GnRH release does not change across puberty, whereas NKB signaling to kisspeptin release greatly increases Figure . Thus, iWe speculate that, in females, reciprocal signaling pathways between kisspeptin and NKB neurons would provide efficiency and flexibility for the stimulation of GnRH release, which ensures complex reproductive functions, such as cyclic ovulations and pregnancy. In summary, kisspeptin signaling and NKB signaling are both indispensable to facilitate the pubertal increase in GnRH after removal or diminution of “Central Inhibition.” Further studies, such as measurements of NKB release in the hypothalamus and examination of the role of dynorphin would strengthen our views.ET and JPG designed experiments. JPG and KLK conducted experiments. JPG analyzed the data, and ET, JPG, and SBS wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer VN declared a shared affiliation, with no collaboration, with one of the authors, SBS, to the handling Editor.


Cultural evolutionary theory and human behavioural ecology offer different, but compatible approaches to understanding human demographic behaviour. For much of their 30 history, these approaches have been deployed in parallel, with few explicit attempts to integrate them empirically. In this paper, we test hypotheses drawn from both approaches to explore how reproductive behaviour responds to cultural changes among Mosuo agriculturalists of China. Specifically, we focus on how age at last birth (ALB) varies in association with temporal shifts in fertility policies, spatial variation and kinship ecologies. We interpret temporal declines in ALB as plausibly consistent with demographic front-loading of reproduction in light of fertility constraints and later ages at last birth in matrilineal populations relative to patrilineal ones as consistent with greater household cooperation for reproductive purposes in the former. We find little evidence suggesting specific transmission pathways for the spread of norms regulating ALB, but emphasize that the rapid pace of change strongly suggests that learning processes were involved in the general decline in ALB over time. The different predictions of models we employ belie their considerable overlap and the potential for a synthetic approach to generate more refined tests of evolutionary hypotheses of demographic behaviour.This article is part of the theme issue ‘Bridging cultural gaps: interdisciplinary studies in human cultural evolution’. One indJust as there are no singular evolutionary predictions, there are no singular behavioural ecological or cultural evolutionary predictions for a given phenomenon, including age at last birth (ALB). Rather, several behavioural ecological and cultural evolutionary models with diverse assumptions suggest different predictions. Human behavioural ecologists emphasize the concept of trade-offs  and straThe compatibilities and disjunctures between evolutionary frameworks, including HBE and CET, have been debated –10, but This type of integration falls short of truly synthetic approaches envisioned by recent proponents of a ‘new’, or extended, evolutionary synthesis. One of these proponents' main goals is to investigate the ways that other inheritance systems, such as cultural ones, affect evolutionary dynamics and adaptation ,9. Such dynamics of evolutionary processes and less clearly affects inferences drawn about the current adaptive value of a trait as it is measured at one place and time . For thsee also ; at the timing decisions in a context where they are unlikely to reflect downstream effects of decisions made with respect to total fertility. Furthermore, the context affords the opportunity to study the impacts of (i) top–down institutional policy changes such as the ‘one child policy’ on temporal variation, (ii) matri- versus patri-focal kinship institutions on spatial variation and (iii) educational variation on both spatial and temporal variation in individuals' strategies towards the end of their reproductive careers.While we tried to devise comprehensive hypotheses sets before seeing the data, our analysis is largely exploratory. We review several hypotheses drawn from HBE and CET to posit explanations for the pathways influencing spatial and temporal variation in the timing of ALB. We focus on a population of ethnic Mosuo in Southwest China where fertility has been officially restricted for nearly 40 years. This allows us to examine reproductive 2.later ALB could also be construed as an investment in offspring quality if this were associated with increased time-sensitive parental inputs (e.g. breastfeeding) into offspring who are more widely spaced, or higher-quality parenting after accumulation of greater social or economic capital. Particularly in the former case, we would anticipate a corresponding shift to lower lifetime reproductive output and improved indicators of child quality . Mattison et al. [later ALB, reflecting a longer reproductive span with no trade-off in child quality.Several evolutionary models used by human behavioural ecologists suggest possible adaptive motivations for earlier reproductive stopping. Such models have close ties to related phenomena, including fertility decline and the evolution of menopause and a long post-reproductive lifespan. First, earlier reproductive stopping may be used to manage quality/quantity trade-offs when intermediate levels of reproductive output maximize fitness ,23. The n et al.  have argIn the absence of strong quality–quantity trade-offs, a more potent consideration in evolutionary models concerns the effects of demography on reproductive timing. Earlier reproduction is favoured in stationary and growing populations. This idea is encapsulated by Fisher's  concept A third class of evolutionary game theoretic models focuses on reproductive negotiations between members of a household. Sometimes termed ‘cooperative breeding models’, various accounts posit that the cost of reproduction may be alleviated by the availability of allocarers  who may promote continued reproduction –33. Theret al. [However, within cooperative systems, there is often conflict and competition over household resources and the recipients of allocare ,36, whicet al. . In the et al. [The above models are agnostic as to the ways that people achieve these equilibria or fitness-enhancing strategies. While individual learning, evolved psychological mechanisms and physiological responses may play roles, it is likely that cultural learning contributes to the spread of strategies about timing of ALB. This is because (i) learning what is optimal reproductive timing in a vast range of socio-ecological conditions is a difficult task that does not lend itself to trial-and-error learning and (ii) the moralization of reproductive behaviours is cross-culturally pervasive ,39 and ret al.  found thet al.  find thaet al.  also shoet al.  and indiet al.  educatio3.wan-xi-shao (late–long–few) messaging of the earlier part of the decade, by which the government encouraged voluntary later births, longer birth intervals and fewer children, resulted in lower fertility in China [Before we test more specific hypotheses derived from HBE and CET frameworks, we examine whether there is spatial (across villages) and temporal (across cohorts) variation in ALB. There are many reasons why ALB might be structured at the village level. This could be due to village-level norms surrounding reproductive strategies specifically (e.g. regarding the timing of births or the length of reproductive careers), or other norms that have downstream effects on reproduction . Villages could also differ in their ecologies (e.g. terrain ruggedness) in ways that would affect reproductive timing . The most obvious reason for temporal variation in ALB in this context is the top–down implementation of the Chinese fertility policy in the late 1970s that restricted fertility to a maximum of three children for ethnic minorities such as the Mosuo living in rural areas . The wanin China ,43 and min China , suggestin China .All of these cultural and institutional shifts changed the fitness landscapes for different reproductive strategies among the Mosuo. Possible implications of different findings are outlined below in H1–H5.(a) Top–down policies to reduce fertility may result in earlier ALB if earlier reproduction is favoured  as paren(b) If fertility policies served as a cue that population sizes were likely to start decreasing, people may have pursued later fertility schedules, as later ALB is hypothesized to be advantageous in shrinking populations .(c) Bottom–up shifts in fertility may have preceded policy implementation ,46; if sH1. Temporal variation in ALB across the twentieth century will be partly explained by the late–long–few messages of the 1970s, and the so-called one-child policy, which began being implemented in 1979. This can produce various patterns.H2. Spatial variation in village-level ALB will be partly explained by kinship systems. If reproductive conflict between women in stable reproductive unions is a determinant of female ALB, we expect earlier ALB in patrilocal communities.(a) If the higher embodied capital and norms associated with education mean pushing reproductive careers later, we would expect a positive association between education and ALB, controlling for fertility.(b) Insofar as education accounts for the historical shifts associated with demographic transitions, we expect cohort effects to drop out once we account for education.H3. Education both changes life-history trade-offs and introduces new norms.(a) If ALB changes as a consequence of total fertility, we expect the effects of cohort, education and village-level variance to attenuate once we account for fertility.(b) If ALB changes as a consequence of a later shift in reproductive career, we expect AFB to show similar cohort effects to ALB.H4. Differences seen in ALB might be by-products of other changes in reproductive decisions that shift in response to policies or local socio-ecology.Finally, patterns of spatial and temporal heterogeneity can also speak to how ALB and related norms are culturally transmitted and spread. Villages in the matrilineal region are in less rugged and more accessible terrain than those in the patrilineal region. Therefore we might expect that:(a) greater homogeneity in ALB in matrilineal regions or(b) faster changes in ALB in matrilineal regions and in villages nearer to the market town.H5. Information spreads more quickly in matrilineal than patrilineal regions and in villages close to the major market town. This may be manifested as:4.The data for this study were collected over nine months in 2008 in both patrilineal and matrilineal communities of the ethnic Mosuo of Southwest China. The Mosuo are one of 55 officially recognized ethnic minorities in China  now numbca. 1979, the Mosuo have been subject to the Chinese fertility policy. As a minority population, they were allowed a maximum of three children at the time of our surveys. Although the implementation of the fertility policy was variable across China [By contrast, the Chinese fertility policy provides a relatively clean test of how a pinpointed change in the cultural regulation of reproduction may have affected ALB. Neither subpopulation of the Mosuo could be considered to display natural fertility . Barrierss China , it leftss China ,57. The ss China  through N = 5 villages) and matrilineal (N = 7 villages) subpopulations of the ethnic Mosuo. These records were obtained via direct interviewing of a single respondent in each of 228 households. Interviews were administered in Mandarin Chinese or in the local dialect by a member of the research team, or translated into Naru, the Mosuo language, by a local assistant [To do so, we examine the demographic records of women drawn from 12 villages in both patrilineal (ssistant . Each re5.n = 320 women). This sample includes all resident women who were at least 30 years old, who had had one or more children and who had complete data on the modelled variables. Variables include ALB—our dependent variable—and the following covariates: age; cohort; education; normative lineality of community of residence  and village of residence; distance to market town; fertility (number of surviving children); and age at first birth (AFB); see To study ALB, we examine a sample of reproductive women from the 2008 household surveys  than a model without cohort, despite the jump in the number of estimated parameters  over a model with cohort only (H2. We do not find evidence of structured village-level variation when village is entered as a random effect in models of ALB . That isw = 0.98) than the cohort-only model, with women with the highest levels of education being more likely to have a later ALB , but does not completely attenuate the cohort effects  when compared with the lineality (w = 0.64) and temporal models (w = 0.01). That said, the distance to market is confounded by the patrilineal and matrilineal regions, because the patrilineal villages are all farther from town. Moreover, the matrilineal villages show less change over time in ALB, counter to the predicted direction if norms were flowing from the market town to the nearer smaller villages. Finally, the matrilineal villages are not more homogeneous than the patrilineal ones, counter to the prediction of an easier flow of information in the less rugged matrilineal areas .H5. There is little evidence that ALB variation is structured in ways that reflect recent cultural transmission events in the region. We find that a model with distance to market town does receive some support , ,64, 24,6In particular, we show that ALB varies across time among the Mosuo, with women from earlier cohorts reproducing until later ages. The drops in ALB are fairly consistent through time, casting doubt on a primary causal role of 1970s Chinese fertility policies in motivating different timing decisions. Such temporal shifts in the timing of reproduction plausibly reflect increasing orientation towards front-loading of reproduction in response to third-party regulation of total fertility behaviour in growing populations. Second, we show that almost all spatial variation in ALB is accounted for by villages being in the matrilineal versus patrilineal areas. Specifically, ALB is later among women residing in matrilineal areas. Third, we show that variation in education does not account for cohort effects, while shifts in fertility and AFB only partially account for the temporal shifts in ALB. This suggests that shifts in embodied capital investments for mothers or investments in child quality are unlikely explanations for ALB variation. Finally, we find no evidence that ALB-relevant norms spread geographically—neither proximity to cities nor terrain ruggedness associated with the patrilineal areas was associated with ALB homogeneity or the rate of ALB change.The timescale at which we see changes to the ALB suggests that cultural  shifts have influenced reproductive decision-making among the Mosuo. Mean ALB decreases 10 or more years within two decades . Furtherper se, could be targeted as a strategy for investing in children while one is a younger and healthier parent, or as a mechanism of reducing quantity of offspring to invest in their quality . As argOne of our more intriguing results provides evidence that is consistent with models of ALB emphasizing the potential for household cooperation and conflict to affect the timing of reproduction. In particular, our models show relatively early ALB among Mosuo residing in patrilineal areas compared with Mosuo residing in matrilineal areas. At face value, this may seem to support the idea that daughters-in-law win reproductive competition with their mothers-in-law in patrilocal households  and higher fertility among the matrilineal villages than the patrilineal ones . There ithe nest , our ownthe nest . Better We have limited ability to speak to how mothers use ALB strategically to allocate resources between reproduction and other goals, or as a way to manage the quality versus quantity of her children. However, we do find that higher fertility is associated with a slower progression to last birth. Relatively early stopping may therefore be part of a fertility reduction strategy. Similarly, women may be shifting their reproductive careers in response to educational investments that they make in themselves. Women with middle school or higher educational levels have the slowest progressions to last births. This interpretation must remain fairly guarded given that women with intermediate educational levels in fact show the fastest progression to last births, meaning that any effects of education on ALB are not linear. Furthermore, the highest levels of education observed in these communities at the time data were collected (i.e. high school) are unlikely to directly interfere with earlier reproductive careers . This meIt is worth reiterating that HBE and CET perspectives often do not make divergent predictions insofar as CET proposes mechanisms of transmission whereby equilibria predicted by HBE models are reached. However, in contexts of change, CET perspectives have the potential to offer additional insights regarding the dynamics of change. For example, during these periods of change, it may be possible to examine humans’ reproductive decision-making rules and the extent to which they may deviate from fitness maximization because of changing norms or institutions. This helps us make sense of the otherwise puzzling evidence that early adopters of low fertility norms suffer long-term fitness losses even as their descendants benefit in social status  are systematically under-represented in the younger age cohorts, possibly masking effects of delayed childbearing on ALB and other reproductive behaviours. Similarly, women who have not given birth in at least 6 years were classified as post-reproductive, which may also underestimate ALB if some of these women plan to continue reproducing. However, the reverse would also be true (some women with completed fertility would be censored). Furthermore, it should be noted that no women reproducing prior to 1979 were censored by these criteria. However, for earlier cohorts, the data may include survivorship biases, which could be problematic if, for example, women who lived longer also had later ALBs. This may be important when evaluating the impacts of the Chinese fertility policy on reproductive behaviours. Given that our results suggest that ALB began declining prior to implementation of the fertility policy, artefacts arising from this censorship or selection biases are likely to be small.Another challenge for analysis is that cross-sectional demographic data are limited in the degree to which they can characterize cultural transmission of reproductive behaviours. While cultural evolution studies typically emphasize equilibria resulting from dynamic models of social learning processes, we are limited to the use of crude proxies for transmission dynamics such as geographic proximity to market towns and kinship norms . Without being able to link transmission mechanisms directly with outcomes, we are unable to interpret patterns in terms of specific social learning mechanisms or their predicted effects on the adoption and spread of reproductive norms. Social network studies might be a slight improvement, because they can identify assortative clustering and frequency-dependent behaviours (e.g. ), but mo8.The timing of reproductive events in human life history can have important consequences for individual fitness, a fact that may be increasingly salient as variation in fertility continues to decline worldwide. Given that ALB is often a stronger predictor of reproductive success than more commonly used proxies such as onset of menarche or AFB ,70, we a


We found that Cx40 expression is decreased in carotid regions of oscillatory shear stress but conserved in high and low laminar shear stress regions. These results were confirmed in vitro. Using phage display, we retrieved a binding motif for the intracellular regulatory Cx40 C-terminus (Cx40CT), i.e. HS. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5-to-16 of IκBα, a member of the protein complex inhibiting NFκB activation. Binding of IκBα (peptide) and Cx40 was confirmed by crosslinking and en face proximity ligation assay on carotid arteries. TNFα-induced nuclear translocation of NFκB in ECs was enhanced after reducing Cx40 with siRNA. Transfection of HeLa cells with either full-length Cx40 or Cx40CT demonstrated that Cx40CT was sufficient for inhibition of TNFα-induced NFκB phosphorylation. Finally, TgCx40fl/flApoe-/-Tie2Cre mice showed exaggerated shear stress-induced atherosclerosis and enhanced NFκB nuclear translocation. Our data show a novel functional IκBα-Cx40 interaction that may be relevant for the control of NFκB activation by shear stress in atherogenesis.Connexins are proteins forming gap junction channels for intercellular communication. Connexin40 (Cx40) is highly expressed by endothelial cells (ECs) of healthy arteries but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is atherogenic partly through activation of the pro-inflammatory NFκB pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NFκB. Shear stress-modifying casts were placed around carotid arteries of mice expressing eGFP under the Cx40 promoter ( These probabilities were then compared to the obtained data.3 at pH 7.45. The reaction was subsequently blocked with 100 mM ethanolamine for 10 min. The samples were separated by SDS-PAGE (4-20%) and stained with Coomassie-Blue.For cross-linking, recombinant Cx40CT (0.25 mM) and a peptide (0.5 mM) were incubated for 1 hour at room temperature (RT) with 1 mM of the cross-linker reagent BS4Cl in PBS for 15 min and blocked in 2% bovine serum albumin (BSA) for 30 min. Subsequently, primary antibodies against Cx40  and IκBα  in blocking solution were applied overnight at 4°C. Next, secondary PLA probes (PLUS and MINUS) in blocking solution were applied and proximity ligation was performed using the Duolink detection reagent RED kit according to the manufacturer's protocol. Cell-cell junctions were counterstained with Cx37  diluted in 2% BSA and nuclei were stained with DAPI. Finally, the sections were mounted using Vectashield (Vector Laboratories) and analysed using a LSM510-Meta confocal microscope (Zeiss).Proximity ligation assays (PLA) were performed using the DUOLink™ kit (Olink) according to the manufacturer's protocol. In brief, rat carotid arteries were opened longitudinally, pinned out on silicone dishes, fixed with 100% methanol for 5 min at -20°C, permeabilized with 0.2% Triton X-100 in PBS for 1 hour, charges neutralized with 0.5 M NH0.4 (Ibidi). Immunosignals in LLSS, HLSS and OSS were normalized to the signal in static conditions.Different cell lines were used: a) a mouse endothelial cell line (bEnd.3) which endogenously expresses all three endothelial connexins , b) a coSilencing Cx40 in bEnd.3 cells using short interfering RNA (siRNA) was performed using SMARTpool ON-TARGETplus Gja5 siRNA (Dharmacon) specific for mouse Cx40 (mCx40). The ON-TARGETplus Non-targetting Pool (Dharmacon), which does not target known mouse sequences, was used as negative control. Transfections were performed under serum-free conditions, with 25 nM siRNA and 2 mg/l DharmaFECT4. Efficient Cx40 silencing was determined by qPCR and immunofluorescence.6 communication-incompetent HeLa cells were grown for 24 hours in 6-well plates. For transfection, 2.5 μg of plasmid, 2.5 μl of PLUS Reagent and 12.5 μl of Lipofectamine LTX were mixed in 300 μl Opti-MEM Medium (Gibco) and incubated at RT for 5 min. Then, the plasmid-Lipofectamine LTX complexes were added to each well supplemented with 1.7 ml DMEM and incubated for 24 hours at 37°C. Transfected cells were selected with 500 μg/ml G418 (Gibco) added to the complete medium (changed every 2 days). Purity of the transfectants was followed every passage by FACS (BD Accuri C6). Experiments were performed when 70-80% of the cells were eGFP positive.Transient transfections with pIRES2-eGFP containing cDNA coding for murine Cx40 or murine Cx40CT  were perTotal RNA from bEnd.3 cells was obtained using the NucleoSpin kit (Macherey-Naegel). Reverse transcription was performed using the Quantitect Reverse Transcription kit (Qiagen) and quantitative PCR was performed with the ABI Prism StepOnePlus Sequence Detection System (Applied Biosystems) using the TaqMan Fast Universal master mix (Applied Biosystem). Mouse Cx40  and GAPDH  were used as Taqman probes. Cx40 expression was normalized to GAPDH expression.4Cl in PBS for 15 min and blocked in 2% BSA. Subsequently, primary antibodies against Cx40 , Cx37 , NFκB (NFκB p65 (A) sc-109; Santa Cruz; 1/100) or Phospho-NFκB (Phospho-NFκB (S536)(93H1); Cell Signaling; 1/100) in blocking solution were applied overnight at 4°C. An Alexa Fluor 488 fluorochrome-conjugated goat anti-rabbit antibody  was used for signal detection. Cytoplasm and nucleic acids were counterstained with 0.003% Evans Blue (Sigma) and 1/20000 DAPI (Invitrogen), respectively. For eGFP signal detection, arteries were fixed with 4% PFA for 20 min at RT and processed for microscopy. Samples were mounted with Vectashield (Vector Laboratories) and analyzed with a confocal microscope  using LSM AIM software, or an epifluorescent microscope  equipped with an AxioCam color CCD camera using Zeiss Axiovision 4.6 software. Quantification of Cx40 immunosignal was performed using ImageJ software.Cx40 and NFκB immunofluorescent staining on carotid arteries, HeLa cells and bEnd.3 cells was performed as previously described . In brieCell cultures were rinsed with PBS, pH=7.4, and lysed in RIPA buffer as previously described . After pDye transfer experiments on HeLa cells were performed as previously described . In shorP < 0.05 were considered statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.Results are presented as mean ± SEM. Unpaired Student's t-tests or one-way ANOVAs were used to compare differences between groups. Differences with a


Tetrapyrroles make critical contributions to a number of important processes in diverse organisms. In plants, tetrapyrroles are essential for light signaling, the detoxification of reactive oxygen species, the assimilation of nitrate and sulfate, respiration, photosynthesis, and programed cell death. The misregulation of tetrapyrrole metabolism can produce toxic reactive oxygen species. Thus, it is not surprising that tetrapyrrole metabolism is strictly regulated and that tetrapyrrole metabolism affects signaling mechanisms that regulate gene expression. In plants and algae, tetrapyrroles are synthesized in plastids and were some of the first plastid signals demonstrated to regulate nuclear gene expression. In plants, the mechanism of tetrapyrrole-dependent plastid-to-nucleus signaling remains poorly understood. Additionally, some of experiments that tested ideas for possible signaling mechanisms appeared to produce conflicting data. In some instances, these conflicts are potentially explained by different experimental conditions. Although the biological function of tetrapyrrole signaling is poorly understood, there is compelling evidence that this signaling is significant. Specifically, this signaling appears to affect the accumulation of starch and may promote abiotic stress tolerance. Tetrapyrrole-dependent plastid-to-nucleus signaling interacts with a distinct plastid-to-nucleus signaling mechanism that depends on GENOMES UNCUOPLED1 (GUN1). GUN1 contributes to a variety of processes, such as chloroplast biogenesis, the circadian rhythm, abiotic stress tolerance, and development. Thus, the contribution of tetrapyrrole signaling to plant function is potentially broader than we currently appreciate. In this review, I discuss these aspects of tetrapyrrole signaling. Figure 1).Tetrapyrroles contribute a number of functions to processes that are critical for the viability of diverse organisms including essential functions to the light reactions photosynthesis, which underpins life on earth. Tetrapyrroles contain four pyrroles— aromatic rings containing four carbon atoms and one nitrogen atom—often linked together with methine groups, in linear  or cyclic arrangements . Porphyrins chelate metal ions, such as cobalt, iron, or magnesium ions. These structural features along with a variety of ring substituents contribute to the diverse functions of tetrapyrroles . These tetrapyrroles provide critical functions to important processes, such as light signaling, the detoxification of reactive oxygen species, the assimilation of nitrate and sulfate, respiration, programmed cell death, and the light-harvesting reactions of photosynthesis. In vivo, many tetrapyrroles, such as porphyrins, are sequestered to prevent them from generating singlet oxygen (1O2), a toxic reactive oxygen species that causes stress and cell death. Electronically excited versions of porphyrins produce 1O2 when they transfer energy to molecular oxygen  to plants. ALA is the 5-carbon universal precursor of tetrapyrroles. ALA is hydrophilic and is readily taken up by plants. Exogenous ALA was demonstrated to flood the tetrapyrrole biosynthetic pathway with intermediates, induce increases in the levels of chlorophyll precursors , and dowthe dark . These dthe dark . More rethe dark genomes uncoupled (gun) mutant screen . GUN2 encodes heme oxygenase. GUN3 encodes phytochromobilin synthase , which synthesizes heme from protoporphyrin IX  to test for gun phenotypes. However, norflurazon appeared to block chloroplast biogenesis when Arabidopsis seedlings were grown in 10 μmol m-2 s-1 white light   mutants  . Additio mutants . Thus, tgun phenotypes  Attempts to show that norflurazon-treated gun4-1 and gun5 accumulate less reactive oxygen species (ROS) than norflurazon-treated wild type by quantifying ROS-responsive gene expression, staining with ROS responsive dyes, and by treating plants with ROS scavengers were not successful   reduced chlorophyll levels to 20% of the chlorophyll found in wild type. Thus, in contrast to norflurazon treatments, sig2 attenuates but does not block chloroplast biogenesis. sig2 mutants expressed lower levels of PhANGs than wild type. Etiolated gun5 sig2 expressed higher levels Lhcb2.2 and other PhANGs than etiolated sig2 (Arabidopsis seedlings that were not treated with norflurazon. (7) Tetrapyrrole metabolism regulated starch-related nuclear gene expression in Bright Yellow-2 (BY2) cells derived from tobacco, which lack photosynthetic plastids  No mutants) . (3) gun mutants . (4) Bloncreased . (5) Higinifolia . (6) Nulted sig2 . Thus, pplastids . (8) In flurazon . (9) Phy mutants .gun phenotypes  chloroplast biogenesis in Arabidopsis seedlings would need to exhibit such extreme sensitivity to this photooxidative stress that the perturbations in the tetrapyrrole biosynthetic pathway exhibited by gun2, gun3, gun4-1, gun5, and gun6-1D would not affect the photobleaching activity of norflurazon and (2) the perturbations in tetrapyrrole metabolism induced by gun5 would need to affect a distinct mechanism to affect the expression of PhANGs in the dark.The idea that no evidence of partial resistance to norflurazon was obtained in so many independent experiments because during chloroplast biogenesis, the norflurazon-induced increase in ROS is transient or localized  is diffienotypes , etiolatted sig2 , and nei mutants . If norfGUN2/HY1, GUN3/HY2, GUN4, and GUN5 that were isolated from gun mutant screens are loss-of-function alleles , aba-insentive4 (abi4), and phytochrome-associated protein phosphatase 5 (papp5)  . These fin vitro , 2011. Fin vitro . In appaArabidopsis when chloroplast biogenesis was blocked with norflurazon treatments  to eliminate the possibility of the circadian regulation of PhANG expression obscuring the plastid regulation of PhANG expression , Mg-Proto methyltransferase (ChlM), and Mg-ProtoME cyclase (Chl27) in 10-day-old Arabidopsis seedlings with an inducible RNAi system. Within 24 h, Mg-ProtoME accumulated—to higher levels than observed by Chl27 RNAi lines. It is not clear whether the approach of Chl27 RNAi lines—24 h after suppression was induced—there were no macroscopic changes in the plants and no significant differences in the expression of PhANGs or ROS-regulated genes. Four days of downregulated Chl27expression induced chlorophyll deficiencies, necrosis, and downregulated PhANG expression. Plastid-to-nucleus signaling was proposed to not cause the changes in gene expression that were observed 24 h after the RNAi-induced suppression of ChlH, ChlM, and Chl27  and 60-fold (tobacco). Mg-ProtoME accumulated 100 (tobacco) and 200-fold (barley). This accumulation occurred within 30 min (tobacco) and 60 min (barley) and declined within another hour . An indeic light . In tobater dawn , severalter dawn . Thus, iter dawn  and the hcb mRNA .An attempt to identify the cytosolic receptor for Mg-Proto in plants revealed that a large number of different cytosolic proteins bound to an affinity column constructed by linking Mg-Proto to Affi-Gel 102 . SeveralLhcb expression, Lhcb genes. RNAi lines that reduced the expression of all HSP90.1–HSP90.4 genes exhibited lower levels of PhANG expression relative to wild type, but only norflurazon- or methyl viologen-treated lines were tested , which binds to promoters of genes encoding HAP and stimulates their transcription. HAP and CBP induced transcription in general and thus, were proposed to promote the transient increases in the levels of cellular RNA that porphyrin signaling is proposed to induce  that overexpressing catalytically active FC1 within the physiologically relevant range and feeding ALA to norflurazon-treated seedlings induced an increase in the expression of PhANGs  and (2) flurazon . SIG2 isplastids . The synynthesis . The levignaling . Consist nucleus .Arabidopsis. The expression of FC1 is observed in non-photosynthetic tissues and is induced by stress. FC2 is coexpressed with PhANGs . The current data are also consistent with changes in the flux through the tetrapyrrole biosynthetic pathway activating a plastid-to-nucleus signaling mechanism  induces increases in the levels of Mg-Proto and Mg-ProtoME and (2) is correlated with downregulated expression of PhANGs was observed under distinct developmental and environmental conditions relative to the conditions used to screen for gun mutants. The evidence consistent with oxidative stress inducing an increase in the levels of Mg-Proto or Mg-ProtoME that affects gene expression in the nucleus is also consistent with oxidative stress attenuating heme signaling or at least in some instances, ROS signalingThe plastid-to-nucleus signaling mechanism that is specifically interrogated by the At this point, it is perhaps most important to understand the mechanism of tetrapyrrole signaling in plants. Understanding this mechanism may help us to understand why perturbations in tetrapyrrole metabolism sometimes upregulate, downregulate, or have no effect on the expression of nuclear genes. Understanding this mechanism may help us to understand the interactions between tetrapyrrole signaling and GUN1-dependent plastid-to-nucleus signaling, interactions between tetrapyrrole signaling and light-regulated development, and the biological function of tetrapyrrole signaling. Identifying a protein that specifically contributes to tetrapyrrole signaling  is perhaps the highest priority. Mutants with defects in such a gene are essential for learning the biological function of tetrapyrrole signaling in plants. The evidence for tetrapyrrole signaling contributing to abiotic stress tolerance and for the interactions between tetrapyrrole metabolism and GUN1-dependent plastid-to-to-nucleus signaling are consistent with an important role for tetrapyrrole signaling in plants. Thus, future research in this area is expected to advance our understanding of plant biology and possibly contribute to the development of new germplasm that will help to meet the needs of agriculture in our changing environment.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.