Embryonic stem cells (ESCs) have been used extensively as in vitro models of neural development and disease, with special efforts towards their conversion into forebrain progenitors and neurons. The forebrain is the most complex brain region, giving rise to several fundamental structures, such as the cerebral cortex, the hypothalamus, and the retina. Due to the multiplicity of signaling pathways playing different roles at distinct times of embryonic development, the specification and patterning of forebrain has been difficult to study in vivo. Research performed on ESCs in vitro has provided a large body of evidence to complement work in model organisms, but these studies have often been focused more on cell type production than on cell fate regulation. In this review, we systematically reassess the current literature in the field of forebrain development in mouse and human ESCs with a focus on the molecular mechanisms of early cell fate decisions, taking into consideration the specific culture conditions, exogenous and endogenous molecular cues as described in the original studies. The resulting model of early forebrain induction and patterning provides a useful framework for further studies aimed at reconstructing forebrain development in vitro for basic research or therapy. The forebrain is the most complex region of the vertebrate central nervous system (CNS), containing several elaborate neural centers, such as the cerebral cortex, the basal ganglia, the thalamus, the hypothalamus, and the retina.This anatomical complexity is achieved through sequential inductive interactions, progressively restricting the fate of pluripotent embryonic cells towards specific neural identities. The first step in forebrain development is neural induction, causing neuroectoderm formation in the dorsal region of the ectoderm germ layer –3. This The molecular mechanisms controlling these events have been studied extensively in vertebrate model organisms and especially fish, frog, chick, and mouse embryos. While studies in these systems have identified the main genetic pathways controlling early neural development, they have not entirely reconstructed the molecular events underlying forebrain regionalization. The reason for this is that, in whole organisms, it is difficult to separate the roles of multiple molecular pathways acting simultaneously and to manipulate specific signaling events without interfering with earlier or later roles for the same pathways.ESCs are pluripotent cells derived from pre-implantation mammalian embryos, which share many similarities with progenitors found in early embryos before gastrulation –11. MousHere, we review the journey of ESCs from pluripotency to specialized forebrain cells in a culture dish. We discuss how the specification of different forebrain fates in ESCs recapitulates the sequence of events known to take place in vivo, but we also highlight new insights obtained thanks to the use of ESCs. Furthermore, we discuss some of the challenges lying ahead in this field and especially the importance of fully characterizing the influence of the chosen culture conditions on neural specification and patterning of ESCs.Vertebrate neural induction was first discovered in amphibians, where the dorsal mesendoderm of gastrulating embryos induces neuroectoderm formation in the dorsal ectoderm, diverting it from epidermal differentiation . SimilarFrog embryos have been pivotal in elucidating the molecular signals produced by the organizer. Studies performed in this system have recently been reviewed elsewhere e.g., , 16). Es. Es16]).Genetic manipulation of TGFβ signaling in mice has shown that Activin/Nodal and BMP pathways are essential for early developmental steps preceding neural induction . For thamESCs and hESCs are pluripotent cells derived from the inner cell mass (ICM) of blastocyst stage embryos –12. In bUnexpectedly, mESCs and hESCs show different reliance on TGFβ signaling for the maintenance of pluripotency. The molecular mechanisms of pluripotency have been reviewed elsewhere e.g., –11, 24)., 24.11, Id genes that are necessary, and sufficient together with LIF, to allow self-renewal [Nanog gene [Nodal genes showed that TGFβ signaling prevents premature neural induction in the developing epiblast in vivo [BMP and Activin/Nodal signaling play corresponding roles in supporting pluripotency and repressing neural specification in mESCs and hESCs, respectively. In mESCs, LIF is insufficient to block neural differentiation in serum free cultures . By acti-renewal . Activat-renewal . Thus, tnog gene , 31. Nan in vivo , 33. In  in vivo –36, whil in vivo , 38, pri in vivo , mesendo in vivo , or non- in vivo , 42. ThuWhile high levels of TGFβ signaling are incompatible with neural induction in ESCs, a question of interest is whether culture conditions based on TGFβ-free media are sufficient for neuralization or whether exogenous TGFβ antagonists are needed. In other words, the contribution of ESCs to the levels of TGFβ signals in the culture and the effects of these endogenous TGFβ levels on ESC neuralization have to be carefully evaluated.mESCs cultured in serum-free media in either monolayer or floating aggregate conditions readily undergo neuralization, which is suppressed by exogenous BMP or Nodal factors –45. TreaThe situation in hESCs is more complex. When hESCs are cultured as floating aggregates at least for an initial period of differentiation, neuroectoderm generally forms in serum-free media even without exogenous BMP antagonists , 46, 47.Several studies have suggested that neural induction is not simply a default pathway of pluripotent cells shielded from TGFβ signaling and that a permissive influence of FGF signaling is implicated , 3. EvidResearch with pluripotent stem cells has shown that neural induction is a multistep process that is differently influenced by FGF signaling at different steps, leading to a unifying view of the role of FGF signaling in neuroectoderm specification. mESCs represent an ICM-like basal pluripotency state (or ground state) that is intrinsically self-maintaining if shielded from inductive differentiation stimuli exerted by autocrine FGF4 , 11. In The subsequent roles of FGF signaling in neural induction can be best modeled in EpiSCs or hESCs, representing a primed pluripotency state. In hESCs, Activin/Nodal and FGF signaling cooperate in promoting Nanog expression, maintaining pluripotency and restraining differentiation , 66. FolInhibition of TGFβ and FGF signaling, however, appears to drive hESCs to an early neural state that is not yet committed to evolve into definitive neuroectoderm. FGF inhibition beyond the first 4 days of hESC neural differentiation hampers neuroectoderm formation , 68, 69 Further issues remain to be addressed to clarify the roles of FGF signaling in neural induction. In particular, different FGFs may trigger distinct intracellular pathways , which mA tentative model of neural induction in ESCs based on the work described in this section is shown in Fig. In different experimental settings, once neural progenitors emerge from pluripotent cells, they almost invariably display AP positional identities, suggesting that allocation of AP fates is an integral part of neural induction or it is a very early process occurring in newly neuralized cells. Positional identities, however, are often initially plastic and can be modified by further exposure to patterning signals.Embryological experiments in amphibians led to a two-step model of neural induction and AP patterning, known as the activation–transformation model . In the The activation–transformation model has been supported by studies performed in model organisms. In many assays of neural induction, whenever neural tissue is induced, it initially expresses forebrain-specific genes, suggesting a link between induction of neural identity and acquisition of an anterior character. This activation step has been commonly associated with the downregulation of TGFβ signaling in competent ectoderm. For example, impaired TGFβ signaling in frog ectoderm  or mouseWork performed in ESCs has provided substantial confirmation for the activation–transformation model. In both mESCs and hESCs, protocols that allow spontaneous neural induction in the absence of exogenous signals lead to the generation of neural progenitors expressing markers of forebrain identity , 75–77. Pax6 gene and promote its expression in hESCs differentiating to neuroectoderm [Among the earliest genes induced during neural conversion of hESCs are those coding for the Otx2 and Lhx2 transcription factors, which are selectively expressed in the early rostral neuroectoderm in vivo . Both faectoderm , 79. Paxectoderm . These sRecent work in mESCs has revealed a novel function of BMP signaling in AP neural patterning distinct from its well-known role in neural induction . This stWork carried out in hESCs has shown that Activin/Nodal signaling too can affect both the induction of neuroectoderm and its AP patterning, since inhibition of Activin/Nodal signaling was found to impose a caudal positional identity on the induced neuroectoderm . This isAs predicted by the activation–transformation model, forebrain neuroectoderm generated from ESCs by spontaneous neuralization or by means of exogenous TGFβ inhibitors can be caudalized to midbrain, hindbrain, and/or spinal cord fates by exposure to signaling molecules promoting posterior specification in vivo, such as Wnts, FGFs, or RA. For example, exogenous RA promotes spinal cord fates in neuroectoderm derived from mESCs or hESCs , 81. DosStudies in ESCs are starting to provide interesting insights on how different caudalizing pathways interact during the specification of posterior neural fates. For example, in hESC-derived neuroectoderm, exogenous Wnt antagonists were shown to reduce the posteriorizing effects of FGF2 treatments , indicatThe crucial influence of the Wnt/β-catenin pathway in the AP patterning of ESC-derived neuroectoderm has been confirmed by enhanced forebrain specification following abrogation of endogenous Wnt/β-catenin during ESC neuralization. Recent reports have described activation of endogenous Wnt/β-catenin signaling during neural conversion of hESCs by TGFβ inhibition , 88. As Otx2, Lhx2 and Foxg1, at early stages of neural conversion [Consistently with the posteriorizing effects of exogenous FGF during ESC neuralization, inhibition of both FGF and TGFβ signaling in hESCs can facilitate the upregulation of early forebrain markers, such as nversion . While tA model of AP neural patterning in pluripotent stem cells is schematized in Fig. According to the prosomeric model , the forSpecification of caudal forebrain fates represents a mild form of posteriorization of the rostral neuroectoderm induced with the activation step. For example, moderate levels of BMP and/or Wnt signaling can cause specification of caudal forebrain and/or midbrain fates in neuralized ESCs at the expense of rostral forebrain fates , 82, 88.Chemically defined protocols of ESC neuralization represent an excellent system to investigate the intrinsic and extrinsic mechanisms governing the specification of different rostral forebrain fates. When mESCs were differentiated as floating aggregates in medium devoid of any undefined supplement and exogenous signaling factors, including insulin, they acquired rostral hypothalamic fates . In thesIn support of this idea, when mESCs were cultured in adherent conditions using a minimal medium devoid of exogenous morphogens and a low cell density to minimize endogenous extracellular signaling, they were efficiently specified to telencephalic progenitors . TelenceSeveral studies have investigated whether ESC-derived telencephalic progenitors acquire dorsal  or ventral  character in culture. mESCs or hESCs differentiated to telencephalon in conditions allowing expression of the endogenous Sonic hedgehog (Shh) signaling molecule, or including exogenous agonists of the Shh pathway, acquired ventral identities , 101–103Studies performed in hESCs have shown that endogenous Wnt/β-catenin signaling can play an instructive role in the specification of dorsal telencephalic fates in vitro , 101, whIn conclusion, it appears that, in ESC-derived telencephalic progenitors, active Shh signaling is necessary for ventral patterning, while active Wnt/β-catenin signaling can enhance, but it is not absolutely required for, dorsal specification. The model in Fig. During embryonic development, the region of the anterior neural plate comprised between the telencephalon and the diencephalon becomes specified as the eye field, which contains the progenitors fated to give rise to the retina and the adjoining optic stalk , 71, 108hESCs can acquire telencephalic fates when cultured in minimal media containing inhibitors of TGFβ and Wnt/β-catenin pathways , 103. ThOther studies have shown that hESCs can be converted to retinal progenitors by means of protocols employing floating aggregate culture conditions, which facilitate cell–cell interactions, and avoiding exogenous morphogens or inhibitors, thus leaving endogenous extracellular signaling intact , 109. InA few exogenous molecules or supplements increasing retinal conversion of ESCs have been described . DeliverUnexpectedly, the most important influence on retinal conversion of mouse or human ESCs has been shown to be exerted by Matrigel , a reconstituted basement membrane preparation. Culture of mESCs as floating aggregates in the presence of low KSR and Matrigel improved the yield of eye field progenitors from 15 % to up to 70 % compared to previous studies, with up to 80 % of the aggregates positives for retinal markers , 115. ImIn summary, studies performed in ESCs allow to draw a model of early forebrain induction and patterning in pluripotent progenitors, which provides a useful framework for future investigations on forebrain development both with ESCs in vitro and with model organisms in vivo.According to this model, downregulation of TGFβ and FGF signaling initiates neural lineage specification in primed epiblast-like progenitors (such as EpiSCs or hESCs), while stabilization of this primitive neuroectodermal state into definitive neuroectoderm requires FGF signaling and is hampered by increased BMP signaling Fig. .Inhibition of TGFβ signaling in primed pluripotent cells (EpiSCs or hESCs) induces a loosely defined anterior neuroectoderm population . On this ground, inhibition of Wnt/β-catenin signaling allows efficient specification of rostral forebrain fates. Moderate levels of BMP, Wnt/β-catenin, and FGF signaling during specific time windows promote caudal forebrain and/or midbrain fates at the expense of rostral forebrain fates. High levels of Wnt/β-catenin, FGF2, and/or RA signaling cause strong neuroectoderm posteriorization to hindbrain/spinal cord fates  fates and Wnt/β-catenin signaling promoting dorsal telencephalic identities Fig. . SpecifiIn this review, we have attempted a systematic scrutiny of the existing literature in the field of anterior neural development in ESCs, with a focus on the molecular mechanisms of early cell fate decisions. For this reason, our discussion has been limited to the early steps of forebrain development and it has not included, or only briefly mentioned, later steps of neuronal cell differentiation and maturation. A detailed description of the mechanism of forebrain development in vivo was beyond the scope of this review and it has been extensively covered elsewhere , 73, 74.Pioneering studies performed in ESCs have also greatly advanced our understanding of how complex anatomical structures are formed during development and opened new avenues of investigation in this field. It will be of great interest to work out whether forebrain morphogenesis from ESCs in vitro recapitulates the same mechanisms used by embryonic cells to form forebrain structures in vivo, or whether similar morphological structures can be formed using alternative, context-dependent routes. Last but not least, the tremendous progress of recent ESC research in neural development has represented a significant step forward in the in vitro generation of neural tissue for clinical applications and has paved the way to the next generation of studies for stem cell-based therapies.


G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that participate in many aspects of the endocrine function and are important targets for drug development. They transduce signals mainly, but not exclusively, via hetero-trimeric G proteins, leading to a diversity of intracellular signaling cascades. Ligands binding at the hormone orthosteric sites of receptors have been classified as agonists, antagonists, and/or inverse agonists based on their ability to mainly modulate G protein signaling. Accumulating evidence also indicates that such ligands, alone or in combination with other ones such as those acting outside the orthosteric hormone binding sites , have the ability to selectively engage subsets of signaling responses as compared to the natural endogenous ligands. Such modes of functioning have been variously referred to as “functional selectivity” or “ligand-biased signaling.” In this review, we provide an overview of the current knowledge regarding GPCR-biased signaling and their functional regulation with a focus on the evolving concept that receptor domains can also be targeted to allosterically bias signaling, and discuss the usefulness of such modes of regulation for the design of more efficient therapeutics. G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface receptors and are involved in almost all physiological and hormonal responses. Therefore, it is not surprising that they are the most targeted in drug discovery programs . Their aFunctional selectivity is not only limited to OLs, but is also a property that has been described for other allosteric ligands (AL)/effectors. These, which are also known as “allosteric modulators,” include ions, ligands, small and large molecules  and/or protein complexes  that modulate hormone binding and/or the intracellular coupling of receptors to their effectors, and affect responses in different ways: they can have differential cooperative effects – negative or positive ones – on the binding of the OL, the “conduit” of the information of the ligand to the effector through the receptor, and the biasing of receptor signaling. Moreover, an evolving concept also suggests that receptor domains that participate in ligand and/or effector binding, and in the “signaling conformations” of GPCRs, can also be targeted to bias signaling. Here, we will expand on this concept of targeting receptors’ domains to allosterically regulate their signaling for mainly class A and C GPCR, and review some of the potential clinical and pre-clinical uses of biased AL.2+, Na+, and Ca2+ are other examples of endogenous molecules that have been shown to allosterically modulate GPCRs (2 and the D4 dopamine receptors. PLG increases the affinity of dopamine for its receptors and the agonist-mediated inhibition of adenylyl cyclase (AC)  . Similarase (AC) . They caase (AC) , 9. Firsase (AC) . Second,ase (AC) .3+), a known allosteric modulator of the mGluR1α, was shown to act as a PAM on the agonist-mediated Gαq and a NAM on the Gαs signaling pathway  receptor. This compound was shown to act as a PAM on the agonist-mediated Gαq signaling pathway, while at the same time acting as a NAM on the Gα12 signaling cascade , which used a peptide consisting of residues 276–296 of the TM6 of the receptor (GIIMGTFTLCWLPFFIVNIVH), to prevent homodimerization, showed that the agonist-mediated cAMP production could be inhibited; thus suggesting that domains of GPCRs could be targeted to regulate receptors signaling in an allosteric fashion  and extracellular loops (ECL) and transmembrane domains (TM)] are known to participate in ligand and/or effector recognition and receptor dimerization, recent attention has also been drawn to understand the role of these domains in receptor conformation and signaling. It is well-recognized that GPCRs can form oligomers within which conformational rearrangements of the receptors can impact signaling Figure  18, 19), 1918, 1 fashion . In addi fashion . Despite1B and α2A (α1BAR and α2AAR), as well as the muscarinic acetylcholine receptors M1 and M2 (M1AChR and M2AChR), have revealed the importance of this domain in G protein coupling/activation (2R)  . Moreoveion (2R) . For exaion (2R) . Not onlion (2R) . Consistaffected . Targeti3AChR)  , the C–C3AChR) , the dop3AChR) , and the3AChR) . Other s3AChR) .The ECL domains of GPCR can also be targeted to allosterically modulate signaling. For instance, a recent study screened potential small molecule for their action on the relaxin/insulin-like family peptide receptor 1 (RXFP1), with the goal of finding agonists. They identified compound 8, which displayed agonistic effects on cellular responses such as cAMP production and cellular impedance. Interestingly, using different RXFP1 constructs and mutagenesis, they showed that the ECL3 of the receptor was required for this effect, suggesting that the compound interacts with an allosteric site within this ECL . Conversd-Ala-OH), a peptide antagonist of the AT1R-dependent Gαq pathway was recently shown to selectively induce β-arrestin signaling. TRV120027 increases cardiomyocyte contractility in vitro, and cardiac performance both in rats and dogs  involved in the underlying pathophysiology, hence limiting side effects that may be engendered from the non-selective engagement of other GPCR subtypes and/or from unwanted downstream signaling effectors/responses. However, much more is needed to be understood about directed signaling in order to develop better therapeutics; especially about GPCR interaction with biased ligands  and their ensuing effects on receptor conformation, and how such receptor–ligand complex can convey signaling information in a selective manner. Challenges also include a better understanding of the role played by GPCR domains such as the TMs, ECLs, and ICLs in the conformation of receptors and how different OL and AL ligands affect these domains to stabilize subsets of conformations for selective signaling. In particular, because allosteric compounds may represent a promising new class of drugs, predicting their sites of binding on GPCRs and modes of action – on the hormone binding, conformation, and “conduit” of signaling  – becomes fundamental. Moreover, because most GPCR can engage many downstream signaling pathways, which in many cases are cell-, tissue-, and/or context-specific, it is essential to better define the entire “signaling repertoire” for the hormones/drugs of reference and the putative-biased ligands to be studied, particularly, in normal vs. pathological conditions. Establishing such “signaling repertoire” becomes therefore essential in programs that aim at improving drug efficacy or at repurposing drugs; hence, future research efforts should be oriented toward the development of approaches for assessing the full spectrum of signaling of the ligands of interest, in different cell models, including those physiological and pathophysiological relevant ones, and integrative analytical methods for linking these “signaling signatures” to pre-clinical and/or existing clinical data.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.


While numerous articles have demonstrated the impacts of ROS on various signaling pathways and clarify the mechanism of action of cell-signaling proteins, their influence on the level of intracellular ROS, and their complex interactions among multiple ROS associated signaling pathways, the systemic summary is necessary. In this review paper, we particularly focus on the pattern of the generation and homeostasis of intracellular ROS, the mechanisms and targets of ROS impacting on cell-signaling proteins (NF- Reactive oxygen species (ROS), generated through a variety of extracellular and intracellular actions, have drawn attention as novel signal mediators which are involved in growth, differentiation, progression, and death of the cell , 2. As aIt has been demonstrated that ROS have impacts on several signaling pathways and the mechanisms of how ROS act on cell-signaling proteins, how the cell-signaling proteins influence the level of intracellular ROS in turn, and if there are complex interactions between different ROS associated signaling pathways have been clarified, but the systemic summary is necessary. In this review, we focus on the pattern of the generation and homeostasis of intracellular ROS, the mechanisms and targets of ROS impacting on cell-signaling proteins, ion channels and transporters, and modifying kinases and Ubiquitination/Proteasome System.Under a physiological state, the level of cellular ROS is stable in a dynamic equilibrium, and this balance is modulated by cellular processes that produce ROS and eliminate them .2−∙ mainly generated by complexes I and III and could be quickly transformed into H2O2 by the enzyme superoxide dismutase (SOD) and then could be reduced to water by catalase or glutathione peroxidase c) is determined by a dynamic balance between the mechanisms that pour Ca2+ into the cytoplasm, including Ca2+ influx from the extracellular medium and intracellular stores such as endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR), and those processes that remove it out, involving Ca2+ efflux across the plasma membrane and sequestration into mitochondria m): ROS promotes Ca2+ efflux from ER/SR to cytoplasm and from cytoplasm to mitochondria. The increase of Ca2+ concentration in turn favors ATP production and ROS generation during oxidative phosphorylation and promotes the opening of mPTP [The mechanism of ROS mediating the mPTP formation involves several pathways. Firstly, ROS directly modulate mPTP opening by oxidizing four different sites: Cysmobimane ; Cys56 omobimane ; externaocks MPT . Besides of mPTP , 132. In of mPTP –135.Recently, it is becoming increasingly apparent that, like physiological second messengers in signal transduction, ROS function in various cellular processes via oxidating sulfhydryl (SH) groups of cysteine residues in protein kinases including protein kinase A (PKA) , protein2+ channel and phospholamban (PLN) [PKA, also called cAMP-dependent protein kinase A, is organized as tetramers comprising two catalytic and two regulatory subunits. The activation of PKA can occur by binding of two molecules of cAMP to each regulatory subunit and then this activated PKA phosphorylates its targeting proteins, including RyR and L-type Caan (PLN) , 142. Rean (PLN) . Meanwhian (PLN) , 145.2O2 stimulated the activation of tyrosine kinases and was able to indirectly regulate the tyrosine phosphorylation of PKC-δ at residues 512 and 523 [Protein kinase C (PKC), containing four homologous domains termed C1, C2, C3, and C4, is a superfamily of structurally correlated serine-threonine kinases that catalyze numerous critical biochemical reactions, like cellular responses, gene expression, cell proliferation, survival, and migration . In an i and 523 .β and PLCγ [β and PLCγ. Then PLCβ cleave PI  P2 that generates DAG and IP3. Subsequently, membranal DAG binds to and activates PKC and recruits PKD, which then is phosphorylated and activated by PKC on Ser744 and Ser748 residues [δ at the outer mitochondrial membrane [93 residue by Src that creates a binding site for the PKCδ C2 domain which facilitates the binding between PKCδ with PKD and the activation of PKD [463 residue by the tyrosine kinase Abl. Additionally, expression of mitochondrial Mn-SOD induced by PKD1-NF-κB signaling removes toxic ROS [PKD isoforms , the effectors of diacylglycerol (DAG), and protein kinase c (PKC) effectors have been described as vital regulators of diverse cellular pathways and mediate the actions of growth factors, neurotransmitters, hormones, and other stimuli that activate PLCand PLCγ –153. Theresidues , 155. ROmembrane ; ROS pron of PKD ; ROS alsoxic ROS .281 and Met282 residues in the regulatory domain results in the activation of CaMKII [Moreover, the activation of RTK and CaMKII could be affected by the level of intracellular ROS. The oxidation on Metf CaMKII . And RTKUbiquitination/Proteasome System (UPS) includes four components: proteasome, ubiquitin, the ubiquitination machinery, and the deubiquitinases (DUBs) . UPS plaβ-TrCP complex renders it unable to carry out ubiquitination and is thus making it inactive [β, and Bcl-2/Bcl-xL, also being disturbed by ROS via modifying the reactive cysteines  and then inducing a conformational change that leads to the release of Nrf2, IKKβ, and Bcl-2/Bcl-xL from Keap1 and the suspending of their ubiquitination and degradation [Recently, an increasing number of studies have documented the interactions between ROS and UPS –168. Theinactive . Additioradation –174. Mearadation .κB and both could mediate the level of ROS by their downstream antioxidative proteins [In turn, UPS regulates cellular redox status via the degradation of Nrf2 and the activation of NF-proteins . In addiproteins –179.It has been clearly demonstrated that redox equilibrium plays pivotal roles in cells' physiological and pathological events due to ROS's ability to activate or deactivate a variety of receptors, proteins, ions, and other signaling molecules. When the redox equilibrium is disturbed due to the excessive accumulation or depletion of ROS, many cellular signaling pathways are influenced which confers to the cellular dysfunction and subsequently the development of various pathologies. Therefore, unveiling the mechanisms of ROS regulating redox-associated signaling pathways is essential in providing relevant targets in order to develop innovative and effective therapeutic strategies. However, due to numerous signaling pathways which are sensitive to ROS and the high degree of complexity in simultaneous actions of ROS, even though we have learnt much about the mechanisms by which ROS influences signaling, in particular, the interactions between different ROS associated signaling pathways are yet to be elucidated.


HTLV-1 Tax oncoprotein is considered a key factor in HTLV-1 pathogenicity. BRCA1 gene dysfunction can lead to breast cancer development. In contrast to the tumor suppressor nature of BRCA1, Tax is a potent oncoprotein, most of its activities are strictly opposing those of BRCA1. Therefore, we hypothesize that HTLV-1 Tax expression in breast epithelial cells can antagonize BRCA1 expression and functionality, thereby sensitizing these cells to malignant transformation by environmental carcinogens. Our objective was to provide molecular and cellular indications to validate this hypothesis. Based on earlier findings that the milk of HTLV-1 infected women is rich in HTLV-1 infected lymphocytes that can transfer the virus into breast epithelial cells, the outcomes of this project may point that HTLV-1 can be a risk factor for the development of breast cancer, with a substantially higher risk to women who practice long-term breastfeeding. Our results showed that Tax strongly inhibited estrogen induced activation of BRCA-1 expression in breast cells by sequestering CBP/p300 co-activators. Trying to explore the CBP/p300 associated mechanism of Tax effect on BRCA1 expression, our results suggest that Tax does not prevent the binding of CBP/p300 to ERα but rather physically associates with the ERα-CBP/p300 to form a tertiary reporter. Since CBP/p300 complex has several binding domains, we believe that Tax associates with ERα-CBP/p300 complex through binding to CBP/p300 rather than to the ERα protein. We have also found that Tax inhibits BRCA1-mediated activation of p53-target promoters. These results support the possibility of HTLV-1 involvement in breast cancer development.


The role of platelets in hemostasis and thrombosis is dependent on a complex balance of activatory and inhibitory signaling pathways. Inhibitory signals released from the healthy vasculature suppress platelet activation in the absence of platelet receptor agonists. Activatory signals present at a site of injury initiate platelet activation and thrombus formation; subsequently, endogenous negative signaling regulators dampen activatory signals to control thrombus growth. Understanding the complex interplay between activatory and inhibitory signaling networks is an emerging challenge in the study of platelet biology, and necessitates a systematic approach to utilize experimental data effectively. In this review, we will explore the key points of platelet regulation and signaling that maintain platelets in a resting state, mediate activation to elicit thrombus formation, or provide negative feedback. Platelet signaling will be described in terms of key signaling molecules that are common to the pathways activated by platelet agonists and can be described as regulatory nodes for both positive and negative regulators. The primary role of platelets lies in their ability to form aggregates when they encounter areas of blood vessel damage, allowing them to limit blood loss by forming a hemostatic clot. The capacity of platelets to aggregate also forms the basis of their role in disease, whereby platelet thrombus formation is triggered by an inappropriate stimulus such as the rupture of an atherosclerotic plaque. If the thrombus causes vessel occlusion in the heart or brain, this can result in a heart attack or stroke. The way in which platelets respond to their environment, often referred to as ‘platelet function’, defines their ability to fulfill their role in hemostasis and also their propensity to contribute towards disease. The environmental stimuli encountered by platelets are complex, as are the intracellular signaling pathways that regulate their responses to these stimuli.To understand how platelets function within the vasculature, the three types of signals encountered by platelets and the signaling responses that they trigger must be considered simultaneously. Platelets encounter inhibitory signals that keep them in a quiescent state in the healthy vasculature, activatory signals at sites of vascular damage that rapidly trigger adhesion and aggregation, and negative regulatory signals that provide negative feedback once platelet activation is initiated and serve to limit thrombus formation Fig. . There iIIbβ3 activation, we can understand the factors that define platelet function. In this review of platelet signaling pathways, we will refrain from describing signaling pathways as such, and instead focus on the impact of molecules that activate or inhibit platelet function on the core signaling processes that control platelet function, to begin to appreciate the interconnectedness of these systems.Many of the complex positive and negative platelet signaling pathways converge on key regulators of platelet signaling. By understanding how platelets regulate important signaling molecules such as phospholipase C (PLC), protein kinase C (PKC), and phosphatidylinositide‐3‐kinase (PI3K), which underpin critical events in platelet activation such as granule secretion and integrin αWithin the vasculature, platelets exist in a complex, flowing environment where both rheologic and hemodynamic factors force platelets into close proximity to the vessel wall. The signals generated by healthy, undamaged blood vessels promote quiescence, and allow platelets to circulate in a resting state 2 [PGI2]), which regulate levels of intracellular cyclic nucleotides that suppress platelet activity by activating protein kinase G (PKG) and protein kinase A (PKA). The inhibitory signaling pathways are relatively few in number, but suppress several key nodes in the platelet signaling network that support activation. The primary platelet‐inhibiting signals generated by healthy, intact vascular endothelial cells are nitric oxide (NO) and prostacyclin (prostaglandin I2 binds to the prostaglandin receptor (IP receptor) on the platelet surface NO suppresses platelet activation by regulating intracellular levels of cGMP IIbβ3 affinity, and is phosphorylated at Ser179 by PKA and PKG, causing it to be relocated away from the cell membrane, and downstream effectors of platelet activation 2+ into the cytosol, which underpins many events in platelet activation, including regulation of the Ca2+‐dependent Rap1 guanine nucleotide exchange factor (GEF), CALDAG‐GEFI 3) receptor by PKA and PKG inhibits its function as a Ca2+ channel, and prevents Ca2+ release into the cytosol from the dense tubular system (DTS) Many substrates of PKA and PKG are yet to be thoroughly characterized, but there is significant overlap in the well‐established targets. These targets include regulators of platelet activation such as Rap1b, which controls integrin α2+ responses to agonists, indicating a role for cGMP‐mediated inhibition of Ca2+ release pathways Platelets from humans deficient in PKG have enhanced CaThe endothelium and red blood cells can release several molecules, such as ADP and ATP, that are capable of stimulating platelets, and removal of these molecules is essential for the prevention of unwanted activation. CD39, an ectonucleotidase found at the platelet membrane and on endothelial cells, prevents platelet activation by hydrolyzing secreted ADP and ATP to AMP and adenosine IIbβ3. In resting platelets, JAM‐A is phosphorylated and associates with integrin αIIbβ3 to allow the recruitment of C‐terminal src kinase (Csk). Csk regulates autoinhibition of c‐Src through phosphorylation of the inhibitory Tyr529 site of c‐Src, thus preventing c‐Src‐dependent phosphorylation and activation of integrin αIIbβ3IIbβ3 activation. As a consequence of this, mice deficient in JAM‐A show a hyper‐reactive platelet phenotype JAM‐A, a member of the immunoglobulin superfamily of surface membrane proteins, has been identified in human platelets, and is thought to prevent platelet aggregation and thrombus formation in resting platelets through inactivation of integrin α2β1 and GPVI enables stable adhesion and initiates platelet signaling and activation 2 (TxA2) amplifies the platelet response and subsequent activation of the coagulation pathway at the platelet surface. Exposure of phosphatidylserine (PS) on the surface of highly activated platelets enables assembly of the prothrombinase complex and the generation of thrombin. Thrombin is a potent platelet activator that can stimulate and enhance platelet aggregation and thrombus formation through the protease‐activated receptors (PARs) Following damage to the vascular endothelium, endogenous inhibitory signals are overcome, enabling platelets to react instantly to limit blood loss at sites of vascular injury. Platelets located close to the vessel wall within the blood flow come into contact with exposed extracellular matrix proteins such as collagen, and this, in combination with vascular shear forces, enables them to adhere to the site of injury and aggregate to form a thrombus IIbβ3. These are by no means the only critical regulators of platelet function, but they represent key nodes in the complex network of platelet signaling that regulate many other important signaling elements  to generate diacylglycerol (DAG), which activates PKC, and IP3, which binds to IP3 receptors on the DTS of platelets to activate Ca2+ flux into the cytosol. Patients with low platelet PLC activity fail to respond normally to platelet agonists and even to Ca2+ ionophore, only achieving normal aggregation following addition of the direct PKC activator DiC82+ release and PKC activation are critical for integrin αIIbβ3 activation and secretion. How and when the two main PLC isoforms in platelets, PLCγ2 and PLCβ, are activated therefore determines platelet activation.Activation of PLC is a critical event in platelet activation, because it gives rise to two of the most important activatory signaling molecules in platelets. PLC family members catalyze the cleavage of phosphatidylinositol 4,5‐bisphosphate (PIP6–12Yxx[L/I]) adhesion receptors in platelets, i.e. GPVI‐FcRγ/FcγRIIA and C‐type lectin receptor 2 (CLEC‐2)  2β1 and αIIbβ3, respectively, also stimulates PLCγ2 signaling, although for this LAT is not required PLCγ2 is activated downstream of immunoreceptor tyrosine‐based activation motif (ITAM)‐bearing (consensus sequence Yxx[L/I]x1 (ADP receptor), TP (TxA2 receptor), and PAR1 and PAR4 (activated by thrombin). PLCβ is an important mediator of signaling evoked by the secreted, soluble secondary mediators ADP and TxA2, and therefore mediates positive feedback signaling and recruitment of platelets to growing thrombi. The consequences of preventing PLCβ activation via gene deletion of Gq in mice is profound, because it is the major route to Ca2+ release and PKC activation in platelets that are not in direct contact with matrix proteins PLCβ is activated by Gq‐coupled GPCRs such as P2Y2+ for full activation; the novel PKCs, i.e. PKCδ, PKCθ, PKCη, and PKCε (only in mice), which require DAG binding but are insensitive to Ca2+; and the atypical PKCs, i.e. PKCζ and PKCι/λ, which are insensitive to both DAG and Ca2+. Expression of some isoforms in platelets is controversial 2 synthesis, integrin activation, aggregation, and thrombus formation 2+ release, although some of the isoform‐specific roles remain controversial PKC constitutes a family of several different isoforms that play key regulatory roles in platelet intracellular signaling. Several isotypes/isoforms of PKC have been identified in platelets, and are divided into three different subtypes according to their structures and mechanisms of regulation: the conventional (classic) PKCs, i.e. PKCα, PKCβI/II, and PKCγ, which require both DAG and Ca2 to generate phosphatidylinositol 3,4,5‐trisphosphate (PIP3), enabling signaling to or recruitment of proteins containing PH domains to the plasma membrane. This is believed to localize signaling kinases within close proximity of their downstream effectors. One such protein is the Ser/Thr kinase protein kinase B (Akt), which has numerous targets but is believed to exert some of its effects via inhibition of glycogen synthase kinase 3 (GSK3). Inhibition of GSK3 appears to potentiate responses to thrombin but not to GPVI agonists such as collagen Appreciation of the role of class I PI3K in platelets as a promoter of sustained activation and stable thrombus formation has developed as its different functions have become understood. All class I PI3K isoforms phosphorylate PIP12, but plays a semi‐redundant role with PI3Kβ downstream of GPCRs. PI3Kβ is the dominant form downstream of GPVI, where it has a role in PLCγ2 activation and Ca2+ elevation Mice deficient in each type I PI3K expressed in platelets have been generated and characterized 2+ elevation mediated by Gq or activation of G13, which couples to the small GTPase Rho. Rho mediates cytoskeletal reorganization through activation of myosin light chain kinase. Cytoskeletal reorganization enables relocalization of platelet granules and organelles to the center of the platelet, the transient formation of filopodia, and the sustained formation of lamellipodia, which enables secretion and spreading over the area of vessel damage 2+, the generation of phosphoinositides and activation of integrin αIIbβ3 are required Platelet activation is associated with major changes in the actin cytoskeleton that initially support shape change and later allow platelets to spread once they come into contact with adhesive surfaces such as exposed collagen or other platelets. Shape change occurs rapidly when platelets are stimulated; it results in the formation of pseudopodia that increase platelet surface area, and is dependent on Ca2. Like integrin αIIbβ3 activation, the secretion pathways are dependent on several convergent signaling pathways activated by multiple receptors.Secretion or release of secondary mediators is critical for the positive feedback that supports clot consolidation, but can also serve as the primary activating signal for platelets encountering an established and growing thrombus. The most prominent secondary mediators are ADP and TxA2+ has a critical role in granule secretion in all cell types, but the precise mechanism of Ca2+‐mediated granule secretion in platelets is unclear. It is believed that the Ca2+‐sensing protein calmodulin mediates phosphorylation of myosin light chain 2+ in the process is unclear Platelets contain different types of granule, including the dense granules, which contain high concentrations of the secondary mediators ADP and 5‐hydroxytryptamine, and the α‐granules, which contain many coagulation and adhesive proteins. The mechanisms governing the secretion of both types of platelet granule appears to be largely shared, and involve assembly of a secretion complex containing soluble NSF‐associated attachment protein receptor (SNARE) proteins such as the vesicle‐associated membrane proteins (VAMPs) 2+ elevation The DAG mimetic and PKC activator phorbol 12‐myristate 13‐acetate can induce granule secretion in the absence of intracellular Ca12 responds to secreted ADP by potentiating granule secretion via activation of Rac1, leading to an increase in ADP secretion and further activation of P2Y12 and P2Y1As a consequence of the complex crosstalk between pathways, the secreted secondary mediators have a role in potentiating secretion, creating an unintuitive, cyclical relationship. For example, following GPVI activation, P2Y2 is synthesized de novo upon platelet activation. Synthesis is mediated by a cascade of enzymes, including cyclooxygenase‐1, the target of aspirin, but it is regulated at the level of liberation of arachidonic acid from membranes by phospholipase A2. This enzyme is activated by elevated Ca2+, which induces translocation to the plasma membrane and phosphorylation by the stress kinase P38 12 has a regulatory role in TxA2 synthesis, whereby stimulation of platelets with ADP causes PI3K‐dependent TxA2 generation, which potentiates Ca2+ signaling TxA1, the only ligand‐gated Ca2+ channel in platelets, causing influx of Ca2+. This enhances activation stimulated by low concentrations of GPVI agonists A novel platelet secretion mechanism has recently been reported whereby ATP is secreted from pannexin hemichannels to activate P2XIIbβ3 activation represents the central point of platelet signaling pathways, as the majority of inhibitory and activatory signaling pathways contribute towards its regulation in some way. The activation of integrin αIIbβ3 is the culmination of a chain of signaling events that induce a conformational change enabling high‐affinity binding of fibrinogen and VWF. Without the capacity to bind fibrinogen, platelets are unable to adhere to each other and form aggregates. The binding partners that are assembled around integrin αIIbβ3 and support activation and association with the actin cytoskeleton include talin IIbβ3 activation 2+ modulates Rap1 activation via the highly expressed platelet GEF CalDAG‐GEFI 2+ via EF‐hand domains to become activated 12, are unable to elicit integrin αIIbβ3 activation unless positive regulation via Ca2+ elevation or PKC occurs simultaneously. A study using genetic reconstitution of the integrin αIIbβ3 activation pathway in cell lines, RIAM, indicated that the Rap1b effector was a critical mediator of integrin activation in platelets, linking the GTPase to integrin αIIbβ3In many ways, integrin αIIbβ3 is only part of the story. Coordination of the cytoskeleton with clustering of adhesive receptors and mediators of activation underlies the ability of platelets to form stable aggregates under shear stress Advanced microscopy techniques are now revealing the spatial and temporal regulation of integrin activation, and have demonstrated that knowledge of the signaling cascades that support activation of key platelet receptors such as integrin αFollowing initiation of platelet activation and thrombus formation, negative signaling mechanisms restrain activation to ensure that platelet aggregation does not progress out of control, thus preventing excessive thrombus formation.IIbβ3IIbβ3 activity, phosphatases that limit phosphorylation‐dependent mechanisms, receptor desensitization, which limits the response to secondary mediator signaling, and intracellular nuclear receptors with different mechanisms of action The pathways that contribute to negative regulation have been studied less extensively than those that mediate platelet activation, and consequently have not been fully characterized. Negative regulators, such as immunoreceptor tyrosine‐based inhibition motif (ITIM)‐containing receptors, are thought to reduce the activation of critical players such as PLC, PI3K, and integrin α3 leads to inactivation of PI3K and Akt, and inhibition of downstream signaling pathways ITIM‐containing proteins are predominantly associated with negative regulation of platelet signaling and activation. The ITIM consensus sequence (L/I/V/S‐x‐Y‐x‐x‐L/V) in the cytoplasmic tail has been identified in several proteins that are expressed in platelets, including platelet endothelial cell adhesion molecule‐1 (PECAM‐1) IIbβ3 function, suggesting that their role is not solely limited to inhibition of ITAM signaling. In support of an inhibitory role in the regulation and limitation of platelet activation, studies of thrombus formation in PECAM‐1 ITIM‐containing proteins were generally considered to constitute the off ‘switch’ that counteracts the positive signaling initiated by ITAM‐bearing receptors, such as GPVI‐FcRγ/FcγRIIA, and CLEC‐2 (which contains a hemITAM). However, studies on PECAM‐1 −/− or CEACAM‐1−/− mouse platelets show impaired outside‐in signaling, as spreading and adhesion on fibrinogen, clot retraction and phosphorylation of focal adhesion kinase are reduced in comparison with wild‐type platelets IIbβ3 signaling following fibrinogen binding.Interestingly, despite the negative roles of PECAM‐1 and CEACAM‐1 in the regulation of platelet activity, studies using either PECAM‐1Human studies have revealed that the expression levels of PECAM‐1 on the surface of human platelets correlates negatively with platelet reactivity to CRP‐XL and ADP. The contribution of PECAM‐1 expression levels as a determinant of platelet reactivity is around half that of GPVI levels, demonstrating the importance of both activatory and inhibitory pathways in controlling responsiveness. IIbβ3 outside‐in signaling, as ESAM‐deficient mouse platelets show increased aggregation in response to GPCR agonists, inhibition of clot retraction, increased thrombus formation in vivo, and reduced tail bleeding ESAM, which is usually contained in the α‐granules, is translocated to the cell surface following platelet activation IIbβ3. Integrin activation, aggregation, adhesion and spreading are all impaired following Sema3A binding. The exact mechanisms by which Sema3A binding inhibits platelet function have yet to be fully elucidated; however, inhibition of the GTPase Rac1 could play a role The neurophillin‐1–plexin A receptor complex, which has been identified in platelets IIbβ3 activation, and aggregation, possibly through regulation of the activity of small GTPases, including Rap1, Cdc42, Rac1, and RhoA Wnt3a is a GP that has been found to be released from TRAP‐stimulated platelets, enabling platelets to self‐regulate and limit activation Intracellular nuclear receptors have recently been identified in platelets and found to have a role in platelet inhibition. Usually associated with genomic regulation of transcription, these receptors have also been shown to have non‐genomic regulatory roles. Several intracellular nuclear receptors have been identified and characterized in human platelets, including peroxisome proliferator‐activating receptor (PPAR)α, PPARβ/δ, PPARγ, retinoid X receptor (RXR), liver X receptor (LXR), and glucocorticoid receptor 2+, granule secretion, and integrin activation. SHP2 has well‐established roles in the negative regulation of platelet signaling downstream of both GPCRs and GPVI, and is a key mediator of ITIM receptor signaling downstream of PECAM‐1 and CEACAM‐1, negatively regulating proximal signaling events, leading to reduced PLC activation and therefore reduced mobilization of Ca2+IIbβ3 function and signaling has been attributed to the dephosphorylation of PKCζ, and protein‐tyrosine phosphatase 1B, which subsequently reduces Src phosphorylation and activation Protein modification by phosphorylation of tyrosine or serine and threonine residues is a key reversible mechanism of signal transduction. Dephosphorylation by phosphatases enables bidirectional regulation of signal transduction and platelet function. Phosphatases, including SHP1/SHP2, SHIP1/SHIP2, protein phosphatase (PP)2, phosphatase and tensin homolog (PTEN), and T‐cell ubiquitin ligand‐2 (TULA2), are integral mediators of several negative signaling mechanisms in platelets preventing key platelet processes such as mobilization of intracellular Ca1 and P2Y12 receptors via agonist‐mediated internalization through two different protein kinase‐mediated pathways, with P2Y1 desensitization being mediated by both classic and novel isoforms of PKC, and P2Y12 desensitization being mediated by GPCR kinase and the novel PKC isoforms Receptor desensitization enables platelets to regulate their responsiveness to platelet agonists. The process of receptor desensitization includes inactivation via phosphorylation events, and internalization, which removes receptors from the plasma membrane. As previously described, ADP is a critical secondary mediator of platelet activation, but the receptors for ADP are rapidly desensitized following exposure to ADP, resulting in a subsequently reduced agonist response The understanding of platelet signaling has increased exponentially in recent decades, and it is now clear that the signaling that underpins platelet function is best described as a heavily interlinked network rather than as linear pathways Fig. . The vas2+ signaling, as it is easy to measure with high temporal resolution and to quantify, and is a point of signal integration downstream of many platelet pathways. Ca2+ models have proved to be useful in understanding how platelets organize important functional events, such as PS exposure, in subpopulations 2+ levels has provided insights into the mechanism of store‐operated Ca2+ entry and release from the DTS via IP3 receptors Systems approaches that utilize mathematical modeling are increasingly being applied to help in our understanding of platelet biology. These can take two distinct forms: those using a top‐down approach, whereby the properties of relatively intact and complex systems are modeled; and those using a reductionist approach, whereby relatively few aspects of platelet signaling or function are modeled, but in greater detail. For example, the top‐down approach has revealed important details of how platelet thrombi are organized into a highly activated, dense ‘core’ of PS‐exposing platelets surrounded by a loosely packed and partially activated ‘shell’ Future innovations will be required to integrate pathways into networks and to develop top‐down and reductionist models until they are able to meet in the middle. Such complex models will need to be validated by the use of data from many different platelet donors, in order to reflect the variability in platelet responsiveness that is present within the human population. Such modeling of platelet signaling networks will prove to be an important future direction for the field, as it will be a requirement for understanding how combinations of regulatory factors contribute collectively to platelet function and predicting how changes within specific parts of the network alter platelet reactivity or levels of activation. This knowledge will aid in the selection of proteins  representing promising new antithrombotic targets, and will help in understanding the defects associated with an increased risk of thrombosis.The authors state that they have no conflict of interest.


The region known as pX in the 3’ end of the human T-cell leukemia/lymphoma virus type-1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. The expression of ORF-I produces the protein p12 which can be cleavage resulting in the p8 protein. These proteins interfere with the ability of a biologically active of HTLV-1, influencing at the virus infectivity and persistence. Here, we evaluated whether natural mutations in HTLV-1 ORF-I can influence the proviral load and clinical manifestation of HAM/TSP. For that, we analyzed 1530 HTLV-1 ORF-I sequences from different clones of 156 patients with negative or positive diagnosis for HAM/TSP and demonstrated that some mutations may be associated with the outcome of HAM/TSP  or with proviral load (P34L and F61L). We further examined the presence of mutations in motifs of HBZ and observed that P45L mutation is located within the HBZ nuclear localization signal and was found more frequently between patients with HAM/TSP and high proviral load. This results suggest that some HTLV-1 ORF-I mutations may be associated with the development of HAM/TSP and the proviral load levels.


ZYZ-488, which exhibited significant cardioprotective property in significantly increasing the viability of hypoxia-induced H9c2 cardiomyocytes and reducing CK and LDH leakage. Further study suggested the protective activity of ZYZ-488 dependent on its anti-apoptosis effect. This anti-apoptotic effect is most probably related to its disturbing the interaction between Apaf-1 and procaspase-9 as the target fishing and molecular docking indicated. The suppression on the activation of procaspase-9 and procaspase-3 with ZYZ-488 strongly suggested that compound ZYZ-488 could be a novel inhibitor of Apaf-1. In conclusion, ZYZ-488 as a novel small molecule competitive inhibitor of Apaf-1, with the great potential for treating cardiac ischemia.Apaf-1 is a central component in the apoptosis regulatory network for the treatment of apoptosis related diseases. Excessive Apaf-1 activity induced by myocardial ischemia causes cell injury. No drug targeted to Apaf-1 for treating myocardial ischemia has been reported to the best of our knowledge. In the present work, we synthesized a novel compound,  LEO, also named as SCM-198) is a natural alkaloid chemically synthesized by our laboratory from Herba leonuri which has long been used in chinese traditional medicine. The studies on LEO show that it has cardio1in vitro and in vivo. The evidence in animal studies of LEO exhibits cardioprotective effects on myocardial ischemia disease in rats after oral administration. Therefore, LEO has been considered as a potential drug candidate in our laboratory for further studies. However, pharmacokinetic studies showed that the major of LEO was quickly metabolized into leonurine-10-O-ß-D-glucuronide  than its parent drug in the pooled plasma samples after oral administration at 30 mg/kgZYZ-488 has been further performed.Leonurine , a central nucleotide-binding and oligomerization domain (NOD), and a C-terminal WD40 repeats domainZYZ-488 most probably binding to Apaf-1 in procaspase-9 binding site. As expected, western blot confirmed ZYZ-488 inhibited the activation of binding protein procaspase-9, while not tampering the expression of Apaf-1, and studies on target specificity by siRNA based approaches further proved ZYZ-488 is a novel inhibitor of Apaf-1. These studies provide us a fresh insight for agent design and modification in treating ischemia.The previous studies of our group show that ZYZ-488 and key intermediate 5 is outlined in 2). After treatment of NaOH and methanol, the resulting methyl ester 3 was acetylated by Ac2O and HClO4 to afford the intermediate 4. Subsequently, 4 was treated with HBr in acetic acid to give desired intermediate 5.The synthesis of compound ZYZ-488, intermediate 5 condensed with another key intermediate 6 which was prepared as previously described7. Finally, the Boc groups in compound 7 were removed by trifluoroacetic acid (TFA) followed by hydrolyzation by guadine to give target compound ZYZ-488.To prepare target compound ZYZ-488 and LEO were evaluated on their protective effects against hypoxia in H9c2 rat ventricular cells. The effects of compound ZYZ-488, and LEO were investigated by cell counting kit-8 (CCK8) assay which demonstrates cell viability. It showed that the hypoxic condition had an effect on H9c2 rat ventricular viability compared with the normoxic control group (P < 0.001). Cells were treated with 0.1, 1, and 10 μM of ZYZ-488 and 10 μM of LEO during hypoxia for 12 hours. Treatment of ZYZ-488 led to increase in the number of surviving cells at a concentration-dependent manner  compared to vehicle group (41.76 ± 1.90). Most interestingly, 10 μM of compound ZYZ-488 showed stronger activity on promoting the cell vitality compared with the group treated by LEO in same concentration (P < 0.01) (ZYZ-488 and 10 μM of LEO remarkably inhibited LDH leakage into medium which is the marker of cell membrane integrityP < 0.05) (ZYZ-488 at 1 μM [(44.49 ± 3.92)%] and 10 μM [(7.848 ± 7.39)%] and LEO at 10 μM [(19.75 ± 9.93%)](ZYZ-488 was more potent than LEO in increasing cell viability against hypoxia (P < 0.01). Thus, the results above confirmed that ZYZ-488 exhibited significant, even more potent cardioprotective activity than LEO in hypoxia-induced H9c2 cells injury.The synthetic  < 0.01) . Lactic  < 0.05) . Elevate± 9.93%)], suggestZYZ-488 and LEO decreased the number of apoptotic cells (ZYZ-488 at 1 μM [(14.00 ± 0.59)%], 10 μM [(13.1 ± 0.26)%] and LEO at 10 μM [(15.28 ± 0.92)%] compared with vehicle group [(16.38 ± 0.13)%] %] . Imaging 0.13)%] . As demoZYZ-488, in-silico targets screening was conducted. PharmMapper server, a reverse pharmacophore mapping approach was performed using an in-house pharmacophore database (PharmTargetDB)ZYZ-488, we speculated that compound ZYZ-488 might interact with Apaf-1 to suppress the apoptosis, then elicit the protective effect on H9c2 cells. It has been established that binding of caspase recruitment domain (CARD) of Apaf-1 to procaspase-9 leads to apoptotic cell deathet al.ZYZ-488 may be capable of binding to the acidic residues of Apaf-1 CARD as mimics of Arg 13/Arg 52/Arg 56 in procaspase-9 prodomain. Interestingly, this presumption was perfectly supported by molecular docking studies on leonurine or ZYZ-488/Apaf-1 CARD complex using Schrodinger software suite 2015. As depicted in ZYZ-488/Apaf-1 CARD (ZYZ-488 and the two acidic residues of Apaf-1 CARD (Asp 27 and Glu 40) could be observed. Apart from that, ZYZ-488 anchors the glucuronic acid moiety in a positive center formed by Lys 21, Arg 44, and Arg 52 of Apaf-1 CARD. Hence, the binding of ZYZ-488 are more stable. These results indicate that leonurine and ZYZ-488 could occupy the caspase recruitment site of Apaf-1, thus block its interaction with procaspase-9, further resulting in the observed anti-apoptotic effect in vitro. Moreover, the more significant anti-apoptotic effect of ZYZ-488 as compared with leonurine may be attributed to the additional interactions of the glucuronic acid moiety binding to the positive center in Apaf-1 CARD.Cell apoptosis involves complex signaling network. To identify potential targeting candidates of compound f-1 CARD , more faZYZ-488 and Apaf-1 described as above, we infer that ZYZ-488 could bind to Apaf-1 at the Apaf-1/procaspase-9 binding site, antagonizing the activation of procaspase-9. To validate this deduction, the effect of compound ZYZ-488 on the Apaf-1’s activation of procaspase-9 and downstream signaling pathways were determined in H9c2 cells by western blot analysis. The results showed that hypoxia promoted activation of procaspase-9 compared with control group, this activation were remarkably inhibited in a dose-dependent manner after the treatment of compound ZYZ-488 %] display no significant change with vehicle group [(17.70 ± 2.56)%]. Results present the inhibition of apoptosis is lost when Apaf-1 is silenced. These results correlated with the western blot of caspase-9  that carries multiple functional domains, namely an N-terminal caspase activation recruitment domain (CARD), the CED4 homology domain (which include the dATP/ATP-binding motif), as well as 13 C-terminal WD-40 repeats, which allow for its interaction with Cyt c and for its oligomerization. Apaf-1 is the molecular core of the apoptosome, a multiproteic complex mediating the so-called mitochondrial pathway of cell deathLEO, compound ZYZ-488. Pharmacological evaluation showed ZYZ-488 possessed potent cardioprotective effects, which was elicited through its suppression of hypoxia-induced apoptosis. It is important to note that ZYZ-488 has stronger anti-apoptotic effects than its parent drug LEO. Different from the inhibitors mentioned above, molecular docking results suggest the similarity between ZYZ-488 and the arginine of procaspase-9 prodomain makes ZYZ-488 capable of inhibiting Apaf-1 recruitment and activation of procaspase-9 as a mimic of procaspase-9 which could competitively bind to Apaf-1 against procaspase-9, and thus tampering the activation of proprocaspase-9 and procaspase-3. Most importantly, compound ZYZ-488 did not show any noticeable effect in H9c2 cells where Apaf-1 was knocked down, which demonstrate the anti-apoptotic effect of ZYZ-488 indeed dependent on Apaf-1 inhibition. In summary, we propose compound ZYZ-488 as a novel competitive, synthetic small molecule inhibitor of Apaf-1 acting through interaction with Apaf-1 in procaspase-9 binding site.Here, we have successfully synthesized an active metabolite of All chemical reagents and solvents were purchased from commercial sources and used without further purification. Thin-layer chromatography (TLC) was performed on silica gel plates. Column chromatography were performed using silica gel , 200–300 mesh and MCI gel CHP20P . NMR spectra were recorded employing a 400 or 600 MHz Bruker spectrometer. Mass spectral data was collected on a HP5973 N analytical mass spectrometer. HRMS data were determined on an IonSpec 4.7 T FTMS instrument. All reagents of cell culture were purchased from Sigma  unless otherwise stated. The following substances Dulbecco’s Modified Eagle medium (DMEM) and calf serum were purchased from Invitrogen Inc. . All antibodies used for western blotting in this study were from Cell Signaling Technology, Inc. . The kits for, Lactic Dehydrogenase (LDH), Creatine Kinase (CK), were obtained from Jiancheng Bioengineering Institute . The kit for cell counting kit-8, Hoechst staining was obtained from Beyotime Bioengineering Institute . AnnexinV-FITC/PI kit were obtained from Becton, Dickinson and Company (USA).The purities of all newly synthesized compounds were analyzed by HPLC, with over 95% of purity. Analytical HPLC was performed on a Waters 600E system chromatograph equipped with photodiode array detector using a MG-C18 5 μm 250 mm 4.6 mm column (reverse phase) to detect the purity of the products. The mobile phase was a gradient of 30% methanol flow rate of 1.0 mL/min.5  and 6  was dissolved in CHCl3 (25 mL). To the above stirred solution Tetrabutyl ammonium bromide  and K2CO3  were added. The reaction mixture was heated at 45 °C for 12 h. After completion of the reaction, CHCl3 (25 mL) was added, and then washed by 1N HCl, saturated NaHCO3 (2 × 25 mL) and brine (2 × 25 mL). The organic layer was separated, dried over Na2SO4, the solvent was evaporated. The residue was purified by column chromatography on silica gel to afford compound 7 11.2 g (40% yield). 1H NMR : δ 6.93 , 5.60 , 5.47–5.21 , 4.34 , 4.12 , 3.88 , 3.68 , 3.52 , 2.05 , 1.83 , 1.74 , 1.49 , 1.48 . 13C NMR : δ 170.24, 169.40, 169.19, 167.08, 165.93, 155.99, 153.25, 152.73, 152.73, 137.84, 126.67, 106.93, 100.33, 83.47, 79.88, 77.39, 77.07, 76.75, 72.75, 72.40, 71.86, 56.43, 52.69, 40.65, 28.21, 26.15, 25.82, 20.65. HRMS (ESI): calculated for C37H53N3O18 [M + H]+ 828.3397, found 828.3404.Bromo-sugar 7  was dissolved in 10 mL CH2Cl2:TFA  solution and stirred for 12 h. Saturated NaHCO3 solution (100 mL) was added to the reaction mixture and stirred for 30 min. Then extracted with CH2Cl2, The organic layer was separated, dried over Na2SO4, the solvent was evaporated to provide yellow solid compound 8 (yield 92%) 1H NMR : δ 7.20 , 5.47-5.21 , 5.04 , 4.34 , 3.71 , 3.68 , 3.25–.01 , 2.75 , 2.05 ,1.61 ,13C NMR : δ 173.75, 172.83, 168.44, 168.37, 159.64, 149.93, 141.04, 128.57, 109.40, 105.29, 103.73, 78.4, 78.11, 76.31, 74.06, 66.03, 58.06, 58.00, 57.84, 43.04, 27.96, 27.56 24.48, 22.03. HRMS (ESI): calculated for C27H37N3O14 [M + H]+ 628.2348, found 628.2357.Compound 8  was dissolved in 50 mL CH2Cl2:MeOH solution and cooled to 0 °C, and then fresh guadine in ethanol was added and stirred for 2 h, immediately there was yellow solid deposited. The solid was filtrated and then purified by column chromatography on MCI CHP20 gel to provide ZYZ-488 . 1H NMR : δ 6.93 , 5.04 , 4.04 , 3.71 , 3.59-3.47 , 3.09 , 1.61 . 13C NMR : δ 175.24, 167.11, 156.65, 151.93, 137.51, 125.69, 106.66, 102.29, 77.12, 75.47, 73.56, 71.80, 65.43, 56.05, 40.67, 25.01, 24.66. HRMS (ESI) calculated for C20H29N3O11 [M + H]+ 488.1875, found 488.187Compund 2. The cells were fed every 2–3 days and subcultured once they reached 70%–80% confluence. Hypoxia was induced based on the technique described by Rakhit et al.2 1.8; MgCl2·6H2O 2; NaHCO3 26; NaH2PO4·2H2O 1) in an anaerobic chamber  maintained at 37 °C with a humidified atmosphere of 5% CO2, 10% H2 and 85% N2. Chambers were sealed before incubation at 37 °C for 5 h. Normoxic incubation of myocytes in serum-free DMEM was conducted in a water-jacked incubator gassed with 95% air and 5% CO2 at 37 °C for the same length of time. LipofectamineTM 2000 (Invitrogen) was used according to the manufacturer’s instructions to transfect H9c2 cells and Apaf-1 siRNA .H9c2 rat ventricular cardiomyocytes  were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum, in tissue culture flasks at 37 °C in a humidified atmosphere of 5% COZYZ-488 and LEO on cardiomyocyte viability were obtained using cell counting kit-8 (CCK8) assay. Briefly, H9c2 cells were seeded on 96-well plates (approximately 8000 cells/well) in Dulbecco’s modified Eagle medium culture medium and maintained in regular growth medium for 2 days. The culture medium was then changed to ischemia solution with or without drugs, placed in an anaerobic chamber for hypoxia induction, then incubated at 37 °C for 12 h. After adding 10 μL of the CCK-8 reagent to each well, the wells were incubated for 1 h at 37 °C and 5% CO2. The absorbance of each well were measured at 450 nm in a microtiter plate reader.The effects of Creatine Kinase leakage into the culture medium was analyzed using Creatine Kinase Kit . CK release was expressed as a fold relative to the activity in normoxic cells.5 cells per sample) were loaded with 5 μL PI and 10 μL Annexin V-FITC  at room temperature for 15 minutes in the dark. Flow cytometric analysis was performed using a flow cytometer , after of Annexin-PI labeling.Apoptosis and necrosis were identified by means of double fluorescence staining with annexin V-propidium iodide. Cardiomyocytes (1 × 10H9c2 nuclei were stained with chromatin dye (Hoechst 33258). Briefly, cells were fixed with 3.7% paraformaldehyde for 10 minutes at room temperature, washed twice with phosphate buffered solution (PBS), and incubated with 10 μM hoechst 33258 in PBS at room temperature for 30 min. After three washes, cells were observed under a confocal microscope .ZYZ-488 were prepared with the default pKa range of 7.0 ± 2.0 in the ligand preparation program LigPrepDocking studies were carried out using Schrodinger modeling suiteCultured H9c2 cells were harvested by scraping and centrifugation, washed with PBS, and re-suspended in RIPA buffer. Soluble proteins were collected by centrifugation at 12,000 g. Protein lysates were subjected to 10% and 12% SDS-PAGE and transferred onto a NC membrane (Millipore Corporation). After blocking with 5% skim milk, the membranes were incubated with the respective primary antibodies  in Tris-buffered saline (TBS) containing 0.1% Tween-20 overnight at 4 °C. The membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated IgG antibodies at a 1:5000 dilution . Immunoreactive proteins were then visualized using ECL. The signals were quantified by densitometry using a Western blotting detection system . Actin served as the loading control.Values are shown as means ± S.E.M of at least 3 independent preparations. The significance of differences between groups was evaluated with t tests or one-way ANOVA followed by the Dunnett’s multiple comparison tests. Values of P < 0.05 were considered significant. All statistics were carried out using PRISM 6.0 for MAC.How to cite this article: Wang, Y. et al. The discovery of a novel inhibitor of apoptotic protease activating factor-1 (Apaf-1) for ischemic heart: synthesis, activity and target identification. Sci. Rep.6, 29820; doi: 10.1038/srep29820 (2016).


Earlier work has shown that the association between the fertility of parents and the fertility of children has become stronger over time in some societies. This article updates and broadens the geographic coverage to assess the magnitude of intergenerational continuities in childbearing in developed and middle-income societies using data for 46 populations from 28 developed countries drawn from a number of recent large-scale survey programs. Robust positive intergenerational fertility correlations are found across these countries into the most recent period, and although there is no indication that the strength of the relationship is declining, the increasing trend does not appear to be continuing. Fertility patterns of parents and their children tend to be positively correlated. Results comparing the completed fertility of successive generations for both historical and more recent populations in a number of now-developed countries up to the later part of the twentieth century are presented in The association between parents' fertility and their children's fertility has been a subject of scientific interest since the late nineteenth century. p < 10−6) for completed fertility between couples and their sons and of 0.23 (p < .001) between couples and their daughters were found for a set of 161 genealogies in the Hutterite archetypal natural fertility population  or parous individuals. The results are usually not representative of the overall population concerned, since, for example, they exclude immigrants who arrive during the analysis period. While findings from these pretransitional populations need to be interpreted carefully because of the selected nature of samples used, such populations tend to show positive but modest and statistically nonsignificant intergenerational fertility correlations that are usually only slightly larger than the early values of For a slightly later period of childbearing of the second generation, around the 1920s and 1930s, studies in Britain by The overall conclusion is that the relationship between the fertility of successive generations in developed countries tended to become stronger during the demographic transition until the latest period analyzed in the 1970s when the younger generation was bearing children.Human Nature and a recent volume of History of the Family dedicated to the topic of intergenerational transmission  are required. Studies by A key issue is the extent to which simple intergenerational correlations are attenuated or even eliminated by controlling for socioeconomic factors. Some studies have attempted to control for heterogeneity—for example, by dividing populations into more homogenous units or by restricting analysis to particular subpopulations. However, the availability of micro-level data means that multivariate analysis can be used to control for population heterogeneity more efficiently. Analyses over the past 100 years on the association between fertility of parents and fertility of children have highlighted a number of issues that will be considered here. One is whether the increase in intergenerational fertility correlations has continued for the most recent cohorts when fertility has been relatively stable. There are two reasons why an increase is particularly likely to be observed when fertility changes rapidly. First, there could be greater heterogeneity between groups in the adoption of fertility control, which would be likely to lead to intergenerational correlations if fertility differed, for example, between urban and rural areas or socioeconomic groups. Second, at such periods, individuals may have greater choice regarding their family-building decisions, whereas earlier and later periods are characterized by greater homogeneity . On the The data sources used are as follows:http://www.unece.org/fileadmin/DAM/pau/_docs/ffs/FFS_2000_Prog_SurveyDesign.pdf.During the 1990s, 24 countries participated in the UNECE FFS project using a standardized questionnaire. Countries complied with this standard questionnaire to different degrees in their national instruments. There was a minimal core questionnaire and optional modules, so information on siblings is available for only 11 countries: Austria, Canada, Finland, Hungary, Italy, Latvia, Lithuania, Norway, Poland, Slovenia, and Switzerland. The question used was: “Including yourself, how many children has your mother had in all, who were born alive?” Further information is available at The 2001 round of the ISSP collected information on social relations and social networks, replicating the 1986 round that included a much smaller number of countries. Topics included number of adult brothers and sisters, as well as frequency of contact with parents, brothers and sisters, and own children. It also collected a range of other information, including respondents' number of children younger than 18 years, number of children 18 or older, age, education, religious denomination, and church service attendance. An example of question wording was as follows: “How many adult brothers and/or sisters—we mean brothers or sisters who are age 18 and older—do you have? .”http://www.issp.org/.However, a number of countries did not include the question on number of younger children. Some countries did not collect information about number of siblings, and in some cases, the proportion of missing cases was substantial, so I excluded countries with more than 15 percent of cases missing. Twenty ISSP surveys that included questions on siblings and all children are analyzed here: those for Austria, Canada, Chile, Cyprus, Czech Republic, Denmark, France, Germany , Hungary, Italy, Japan, Latvia, Northern Ireland (the question on younger children was not included in the rest of the United Kingdom), Poland, the Russian Federation, Slovenia, Spain, Switzerlandm and the United States. For more information, see http://www.unece.org/pau/ggp/welcome.html.This set of panel surveys of representative samples of people aged 18 to 79 is conducted by the UNECE. The surveys collect information on a range of socioeconomic topics, including social networks and education. Information was collected on total and living siblings in a subset of countries and was made available in early 2012 for 13 countries: Austria, Belgium, Bulgaria, Estonia, France, Georgia, Germany, Italy, Lithuania, the Netherlands, Norway, Romania, and the Russian Federation; some results are also available for the special German Turkish sample. Questions include both how many brothers and sisters the respondent ever had and how many are currently alive, including full, half, adopted, and foster siblings. For further details, see http://www.understandingsociety.org.uk/.Understanding Society is a panel study of the socioeconomic circumstances and attitudes of 100,000 individuals in 40,000 British households. The survey collects information on the total number of siblings and children living both in the household and elsewhere. The sample size and detail of information available is more substantial than that for the previously mentioned surveys conducted by international programs, which in any case do not include Great Britain. For more information, see The main indicator of socioeconomic status available in all surveys is the education level of the respondent from the younger generation , with the exception of Northern Ireland (in the ISSP) and France (in the GGP). The three international data sources code education as consistently as possible within each set, but they are not consistently coded across sets, so we use them principally as control variables to assess how far their inclusion alters the magnitude of sibling coefficients, rather than the magnitude of the specific education level per se. The FFS and GGP data include a relatively homogenous set of countries, so the highest recorded level is based on the UNESCO International Standard Classification of Education (ISCED), although the classification was updated between the two surveys. UNSOC uses a classification based on the highest examination level achieved that does not map directly into ISCED but also produces an ordinal scale. However, since the ISSP includes wider geographical and developmental ranges, the education variable is based on tertiles of years of education within each country, age, and sex group, that is, it indicates the level relative to similar individuals.Although the effect of religious denomination on fertility is weakening, religious participation remains a strong predictor . The ISSThe FFS is largely confined to respondents younger than age 50 , and the GGP to those younger than age 80. The other sources have no upper age limit, although relatively few respondents are older than age 75 . Basic descriptive data for the surveys included are given in Appendix A.•Life expectancy at birth•Real gross national income per head (GNI)•Average number of siblings among respondents aged 40 and over•Average number of children among respondents aged 40 and overI also present national-level comparisons, since it is possible that international patterns may be very different from intranational ones, as is the case for mortality and income , rather than those from around the time of childbearing, but they indicate the general relative positions of the countries. The last two values are indicators of fertility of the older and younger generations, computed as national average values from the surveys analyzed here and shown in Appendix A.The first two indicators are key components of the UN Human Development Index (HDI) and are the latest values in the 2011 HDI Report , which are needed for comparisons with earlier studies, so I also included analyses based on these measures.1. ijChildren) = μ + α jCountry + β ijAge + γ ijSex + ϵijln(2. ijChildren) = μ + α jCountry + β ijAge + γ ijSex + δ ijSibs + ϵijln(3. ijChildren) = μ + α jCountry + β ijAge + γ ijSex + δ ijSibs + θ ijEducation + ϵijln(4. ijChildren) = μ + α jCountry + β ijAge + γ ijSex + δ ijSibs + θ ijEducation + λ ijSibs* ijSex + ϵijln of respondent, ijSex is sex of respondent, ijEducation is highest education level of respondent, ijSibs is number of siblings of respondent, jCountry is the indicator for country j, and ϵij is an error term. Note that ISSP models also include the religious commitment variable in addition to education and that UNSOC does not include country as a covariate.Where for respondent 1. iChildren) = μ + β iAge + γ iSex +δ iSibs + ϵiln(2. iChildren) = μ + β iAge + γ iSex +δ iSibs +θ iEducation + ϵiln to each separate population, with notation as previously outlined, apart from the superfluous country index:The datasets used collected information on respondents' total number of children and number of siblings, although the precise form of the questions varied slightly. Results for overall correlation coefficients of the number of children of respondents aged 40 and over  and their number of siblings are shown in Only one value per country is shown in Only one value is negative, Chile (in ISSP), and that value is close to zero. ISSP sample sizes are smaller for each country than those used by the other surveys, and its results are based on living rather than ever-born siblings, but the number and geographical coverage of countries is wider than that of the other sets, including a number of middle-income countries that have not been analyzed in other studies so far. All the coefficients for the FFS, GGP, and UNSOC datasets are statistically significantly greater than zero at the 5 percent level, as are the great majority of ISSP values. The ISSP collects information on living siblings only, whereas the other surveys include all siblings. However, the GGP includes information on both living and total siblings, permitting comparison of the alternative indicators. The correlation between number of children and living siblings is 0.15 but reaches 0.18 for ever-born siblings, about one-fifth higher. The ISSP figures are therefore likely to produce slightly lower values than the other sources.These results confirm that intergenerational fertility continuities remain positive for the most up-to-date data available for these mainly developed countries. However, there do not appear to be any obvious national-level factors that distinguish countries with particularly high or low values. The second issue that we consider is the role of individual-level covariates in relation to the effect of parental characteristics, including fertility. Fertility is influenced by a number of factors, such as religion and education, in addition to the size of family of orientation. There is a clear monotonic relationship between completed fertility and simple overall measures of both sibship size and highest education level .Table 2The availability of individual-level, nationally representative studies permits a comprehensive assessment of the relationship of sibship size and completed fertility. In order to compare the magnitude of such effects and to investigate how far intergenerational patterns may be attenuated by the inclusion of socioeconomic variables, regression models described in the Methods section were fitted to those individuals aged 40 and over. The main variable used was education level, together with religious participation, as recorded in the ISSP data analyses.N” columns in Appendix A. Inclusion of a sibling coefficient in Model 2 substantially improves the fit, as does inclusion of education (and the religious commitment variable from the ISSP data) in Model 3. However, including an interaction term for siblings by sex in Model 4 does not significantly improve the fit in any of these cases, suggesting that the relationship of parents' fertility with that of their sons and daughters is similar. The coefficients for the preferred Model 3, apart from the country coefficients, are shown in The first set of analyses aims to identify the average contribution of family size and other variables in each of the datasets using an increasingly comprehensive series of models in The coefficients for education level vary between countries, so in order to estimate their maximum contribution to the explanation of differences in fertility, separate models were fitted to each country, as set out in the Methods section. Sibling coefficients were estimated from quasipoisson models for each country, (a) including age and sex only (unadjusted for socioeconomic factors) and (b) also including education level and—in the case of the ISSP—religious attendance (adjusted models); see Values for the four datasets in A limitation of an analysis such as this is that it cannot satisfactorily address the question of the pathways for transmission. For example, age at partnership has been found to be an important proximate determinant of the fertility correlation between generations, and the intergenerational relationship of age at first birth tends to be rather stronger than it is for overall fertility. However, early age at partnership is also associated with higher parental partnership breakdown, and, for example, higher proportions of couples in developed countries such as Britain now divorce as have three or more children, so there is scope for partnership as well as fertility history to become an increasingly important factor, even though high proportions of births now take place outside formal marriage in many developed countries.“Socialization” is widely cited as the primary mechanism for intergenerational fertility transmission in contemporary societies, although the empirical basis for doing so is weak, as alternative explanations are rarely investigated. Since children are usually socialized by their biological parents over a long and formative period, it is difficult to establish the relative contribution of attitudes, preferences, and social learning. There may be a genetic inherited propensity for continuities in family size , but oncContemporary data show that intergenerational transmission of health status has only a small role to play , given the similarity of patterns for men and women. Transmission of poor obstetric practices and short breastfeeding periods in some families would be expected to lead to intergenerational fertility correlations in overall fertility  in high-mortality societies. However, current low levels of early age mortality mean that such factors have little impact.Therefore, for contemporary industrialized country populations, biological ability is a less relevant mechanism, at least outside of specialized groups such as women who choose to start childbearing at older ages, when interactions between behavioral factors and reduced fecundability may be important. In contrast to earlier studies that assumed that any relationship was only likely to be found in noncontracepting populations, it is more plausible that intergenerational fertility continuities will be manifested in post-transitional ones, and that any likely genetic mechanism will be behavioral rather than physiological.Analysis of large numbers of countries with coordinated datasets provides considerable additional insight into levels, trends, and the contribution of alternative covariates. A number of studies have shown that persistent intergenerational fertility correlations have substantial impacts on the distribution of genetic characteristics, especially those that are important for inherited disease  and overerational fertility transmission over time. This is in contrast to many of the “traditional” fertility differentials, such as religion or urban/rural residence, which have declined or disappeared over time. Fertility analysis is often concerned with identifying variables associated with fertility decisions and outcomes, and intergenerational continuities at the individual family level retain a strong influence.Since existence of wider kin networks has been identified as relevant to childbearing in both historical and contemporary developed and developing countries e.g., , the imp