This study applies survival analysis to the birth histories from 317 national surveys to model pathways to low fertility in 83 less-developed countries between 1965 and 2014. It presents period measures of parity progression, the length of birth intervals and total fertility that have been standardized fully for age, parity, and interval duration. It also examines parity-specific trends in the proportion of women who want no more children. Outside sub-Saharan Africa, fertility transition was dominated by parity-specific family size limitation. As the transition progressed, women also began to postpone their next birth for lengthy periods in many countries. During the first half of the fertility transition in much of sub-Saharan Africa and in some other countries, however, women stopped childbearing without targeting particular family sizes. Moreover, birth intervals in sub-Saharan Africa have been lengthening since the onset of the transition. Birth control is not restricted to a dichotomy between limitation and spacing. Other reasons for curtailing childbearing and postponing having another birth also shape countries’ pathways through fertility transition.The online version of this article (10.1007/s13524-019-00848-5) contains supplementary material, which is available to authorized users. Much of the demographic literature on fertility transition and fertility intentions is phrased in terms of limitation and spacing, which are viewed as contrasting modes of birth control that reflect women’s preferences about the size of their families and the intervals between their births, respectively  are as central to their fertility decision-making as they are to the research agenda of demography. Other factors that might affect these decisions are conceptualized either as operating via women’s preferences for a particular family size or interval length, or as idiosyncratic and unimportant. However, women may also seek to avoid childbearing for reasons that are unrelated to their reproductive histories, such as difficulties in their relationship with their husband or partner, perceived economic hardship or insecurity, or ill health. Such concerns may lead women to avoid childbearing even if they would want another birth in more favorable circumstances. Postponing having another birth for such reasons has different demographic consequences from the spacing of births until the previous child reaches a particular age. Spacing of the latter type is inherently self-limiting—that is, once the preferred interval has been achieved, the motivation to avoid conception disappears—and can have only a moderate impact on overall fertility. In contrast, postponement of the next birth for nondemographic reasons can continue indefinitely and may have a major impact on overall fertility  surveys, Demographic and Health Surveys (DHS), and Reproductive Health Surveys (RHS) from 83 countries to investigate trends and patterns in family formation over the course of their fertility transitions. The WFS was conducted in the late 1970s and early 1980s. It collected full birth histories, allowing investigation of fertility during the previous 15 years. The analysis therefore covers the period from 1965 to 2014. The database of demographic surveys includes every DHS in the public domain in early 2019 and all the RHS that collected birth histories. It excludes nine WFS surveys conducted before 1985 in countries that have not undertaken a RHS or DHS since.Almost one-half the surveys were conducted in sub-Saharan Africa, but the database includes surveys from most parts of the world other than its high-income countries , and her parity and five-year age group at the start of each episode are calculated. Figure We conduct a period-based analysis of parity progression and interval dynamics  in this last regard; (2) as it estimates fertility by five-year period from a data set including all available surveys rather than for a five-year period before each survey; and (3) as fertility at all parities is estimated from a single model including interactions with parity. Each of these differences serves to increase the robustness of the estimates. First births are modelled separately as a function of quinquennial period and women’s age in years using a similar, but simpler, Poisson regression model.The output from the regression model is a complete series of fitted age-order-duration–specific fertility rates for each five-year period covered by the fertility survey data. From these, one can construct summary life table estimates of fertility by birth order. Using the same methods as Retherford et al. , which wThe analysis proceeds by using several heuristic diagnostics calculated from the life tables for duration-specific fertility according to parity to assess the features of the fertility transition in each country. Timæus and Moultrie  suggeste0 and PPR4 on the one hand, and PPR4 and PPR8 on the other, where PPRn denotes the proportion progressing from the nth to the n + 1th birth.The first two diagnostics are based on the series of period PPRs for each quinquennium. These PPRs measure the proportion of women of each parity who have another child within 12 years. One can infer that parity-specific stopping occurs if plots of progression across parities become increasingly concave over time ) had dropped by more over time than the probability of closing the birth interval at 30–60 months (30b30).60b60) in a country dropped compared with the probability of closing the interval within five years (B(60)). We classify countries in terms of each of the indices by estimating the regression of the first measure on the second and calculating the residuals. Countries in which both residuals are small or the median closed birth interval rose by less than three months are classified as neither spacing nor postponing by contraceptive means.Spacing and postponement produce different patterns of change in the schedule of duration-specific hazard rates (Timæus and Moultrie B0) had drThe pattern of fertility decline in each of the 83 countries is classified according to these measures; the results are assessed to examine diversity and regional clustering in the pathways by which different countries have progressed through the fertility transition and to identify countries that have undergone exceptional transitions. To avoid producing overly cluttered figures, we present results for alternate quinquennia, terminating in 2005–2009, together with estimates for 2010–2014 in countries for which they are available.Last, we examine the trend across successive surveys in the proportion of currently married women by parity who report that they want no more children in order to assess whether the evolution of women’s fertility preferences mirrors trends in parity progression. The measure is calculated for reportedly fertile married women who either gave birth in the previous year or were yet to start childbearing.4World Population Prospects . The drop in the higher-order progression ratios was so large, though, that it could be due only to the widespread use of birth control to stop childbearing. However, women cease childbearing at a wide range of family sizes. No evidence exists that their final outcomes converged on small desired family sizes. Mirroring this, the proportion of the women who want no more children rose steadily with parity rather than rising sharply after they had two children, as in India.Panel a of Fig. Most countries outside mainland sub-Saharan Africa exhibit the opposite pattern of change, with larger reductions in the period probability of progressing to the fourth birth than in that of progressing from the fourth to eighth birth. A few non-African countries, however, followed an “African” pattern of decline in parity progression, with relatively large reductions among higher-parity women and little evidence of parity-specific limitation. These countries include Afghanistan, Pakistan, Jordan, Yemen, Ecuador, and several countries in which the most recent surveys date back to the 1990s . Table 4 and PPR8 equals that between PPR0 and PPR4; countries above this line have convex curves. The lower line identifies countries in which the difference between PPR0 and PPR4 is at least three times the difference between PPR4 and PPR8; countries below this line have highly concave curves. Most African countries have convex curves or fairly straight ones. The two outlying African countries are Lesotho and South Africa. In contrast, most countries in other regions have concave or highly concave curves, with parity progression falling away rapidly between the first and fourth births. The exceptions are Afghanistan; Pakistan; the Middle East and North Africa (MENA) region, except Egypt and Turkey; Mexico prior to 1990; and East Timor. As in India and Kenya, plots of the proportion of women by parity who want no more children generally mirror those in parity progression. Thus, in countries in which the curves for parity progression have become highly concave, those for women wanting no more children are strongly convex; in countries where the former have remained more linear, so have the latter .Whereas panel a of Fig. Figure Except in Southern Africa, the rate of increase in the median length of birth intervals in sub-Saharan Africa was similar to that in other regions. However, outside sub-Saharan Africa and the Caribbean, the latter region being one in which intervals were very short initially, the lengthening of birth intervals was concentrated in the second half of the fertility transition. Thus, controlling for total fertility, virtually no overlap exists between median birth intervals in African countries and those elsewhere.A few countries saw no increase in the median length of closed birth intervals. The list includes several countries in the Sahel and Central Africa in which fertility changed little, but also Sierra Leone and Ethiopia, which are countries that saw a significant drop in total fertility. This group of countries also includes India . The data on most African countries and the few other countries where the most recent total fertility rate exceeded four children were unaffected by this.In most countries in which parity-specific limitation has played a part in the fertility transition, this pattern was already evident before total fertility fell to four children. Only a few countries experienced a period dominated by parity-independent curtailment followed by one dominated by parity-specific limitation. In addition to South Africa and the five relatively low fertility sub-Saharan African countries with a mixed pattern of parity progression at the end of the study period, these countries are Bangladesh, Egypt, and Indonesia. Turning to postponement, much of the lengthening of birth intervals in countries outside sub-Saharan Africa occurred during the second half of their fertility transition. Nevertheless, most countries characterized by substantial postponement at the end of the study period saw an initial shift in this direction before total fertility fell to four children per woman.The concept of postponement originated in the discovery that very long birth intervals had emerged during the course of fertility transition in Southern Africa  map that we draw of fertility transition across what was once termed “the developing world” is a complex one. Africa is not unique: a few other countries have experienced “African” transitions. Nevertheless, the overall picture is clear and spatially coherent. The initial stages of fertility transition in sub-Saharan Africa have followed a different track from that taken by almost all the rest of the world. The region has been characterized by the curtailment and (in most countries) postponement of childbearing, without the development of clear-cut preferences for small desired family sizes. The pace of fertility decline in Africa will remain slow until large numbers of African women start limiting their families to only a few children. Gaining a better understanding of the Developing typologies is ultimately an arid exercise if it fails to point the way to explanations. The pathways through fertility transition documented here suggest that in many countries, rather than having a master schedule for their reproductive lives, most women plan their families as they go. If they have enduring quantitative fertility preferences, these are probably numerically imprecise, such as “at least two” or “fewer than my mother.” Wanting to have a(nother) child now, later, or not at all, together with being unsure whether one wants a child later, are an exhaustive and mutually exclusive set of possibilities. Parity-specific family size limitation and birth spacing are not. The terms usefully encapsulate the two main motivations for practicing birth control that relate to women’s reproductive histories. However, characterizing all birth control as either limitation or spacing systematically diverts attention away from nondemographic reasons for intentionally stopping childbearing or postponing having another birth. Changes across the less-developed world during the last half-century in patterns of parity progression by birth order, the length of birth intervals, and interval-duration–specific fertility demonstrate that limitation and spacing are not the only important motivations for adopting birth control. In many countries, large numbers of women practice birth control to stop childbearing for reasons other than limiting their families to some desired size or to postpone having another birth for reasons unrelated to the age of their youngest child.ESM 1(PDF 345 kb)


Wnt signaling plays an important role in the self-renewal, fate-commitment and survival of the neural stem/progenitor cells (NS/PCs) of the adult central nervous system (CNS). Ischemic stroke impairs the proper functioning of the CNS and, therefore, active Wnt signaling may prevent, ameliorate, or even reverse the negative effects of ischemic brain injury. In this review, we provide the current knowledge of Wnt signaling in the adult CNS, its status in diverse cell types, and the Wnt pathway’s impact on the properties of NS/PCs and glial cells in the context of ischemic injury. Finally, we summarize promising strategies that might be considered for stroke therapy, and we outline possible future directions of the field. In the central nervous system (CNS), Wnt signaling plays a pivotal role in embryogenesis, but it is also decisive postnatally, especially in regulating the proliferation, differentiation and migration of adult neural stem/progenitor cells (NS/PCs). In recent years, new links between Wnt signaling and a multitude of neuronal impairments affecting the CNS have emerged. Among others, schizophrenia , Alzheim2+) pathway [Wnt proteins act as important morphogens during embryogenesis, and their impact was also proven in postnatal ontogenetic stages, where they influence stem cell survival, proliferation, differentiation, and cell migration and polarity . Before  pathway ,15. Vari pathway . However pathway ,18. In a pathway .The canonical Wnt pathway regulates the stability of its key mediator β-catenin, which has a dual role in the cell. In epithelial cells, it plays a structural role in cell–cell adhesion. As a component of a protein complex with cadherins, the protein forms adherent junctions , while iUnlike the canonical Wnt pathway, the non-canonical PCP pathway orchestrates the orientation of the cells that reside in the tissue ,31. At t2+ pathway primarily regulates cell adhesion and motility. Upon Wnt signaling activation, the signal is relayed through phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers Ca2+ release from the endoplasmic reticulum. Elevated intracellular Ca2+ concentration together with DAG activate Ca2+-dependent enzymes, such as protein kinase C (PKC), calcineurin, and Ca2+/calmodulin-dependent protein kinase II (CaMKII). While PKC downstream signaling promotes cytoskeleton remodeling in the PCP pathway [The Wnt/Ca pathway , calcine pathway . The maiAdditionally, Wnt signaling may be suppressed or regulated by a plethora of extracellular proteins. Secreted FZD-related proteins (sFRPs) and Wnt inhibitory factor (WIF) both bind to Wnt proteins, and thus chelate the ligands that may potentially activate Wnt signaling. Therefore, they act as antagonists for both the canonical and non-canonical pathways . AnotherThe Wnt signaling pathway in the adult brain, as in many other tissues, is indispensable in the regions where new cells are generated. In the healthy CNS, Wnt signaling plays multiple roles in the self-renewal, neurogenesis and homeostasis of neural stem cells (NSCs). Adult NSCs undergo asymmetric division to maintain a pool of pluripotent cells, while providing progenitor cells capable of differentiating to neuronal and glial cell populations. Recent studies provide evidence that these processes require precise regulation of molecular signals in order to prevent the gradual loss of postnatal NSCs, and ensure continuous neurogenesis in the brain (reviewed in Reference ).In the mammalian brain, adult neurogenesis is limited to two distinct regions where doublecortin (DCX)-positive putative neuroblasts occur—the dentate gyrus (DG) of the hippocampus   and the disrupted in schizophrenia 1 gene (Disc1) has been shown to reduce the proliferation of adult NPCs in the hippocampus through Wnt/β-catenin inhibition [Adult neurogenesis is a process including the proliferation and specification of neural progenitors, their differentiation to neurons, and neuron migration and integration into the existing circuits. The active canonical Wnt pathway, together with the removal of Sox2-mediated repression, leads to the activation of quiescent NSCs and drives their proliferation, most likely through the expression of the gene encoding transcription factor neurogenic differentiation 1 (NeuroD1) and through the activation of long interspersed nuclear elements 1 (LINE1) . Wnt sighibition . Besideshibition . In addihibition . Moreovehibition , indicathibition . The agehibition  and sFRPhibition  were exphibition . Strikinhibition . On the hibition . Notablyhibition .+ transporting accessory protein 2 (ATP6AP2) was identified as a regulator of the cell response from Wnt/β-catenin to Wnt/PCP signaling in adult NSCs isolated from the hippocampus [The non-canonical Wnt signaling pathway also plays a role in adult neurogenesis, as evidenced by behavioral changes and reduced long-term memory in mice deficient of JNKs ,68. Conspocampus . In addipocampus . All of Considering the activity of the Wnt signaling pathways in the neurogenic regions of the adult brain, it is necessary to distinguish the Wnt-signal-producing cell types from the Wnt-receiving cells in the region. As in other adult tissues with homeostatic capacity , NSCs iAstrocytes are present in all parts of the brain and provide many supporting functions, such as the maintenance of tissue homeostasis and the blood–brain barrier (BBB), but they also participate in the tripartite synapses (reviewed in Reference ). A subsNG2-expressing glial cells, also called polydendrocytes, are the most recently discovered type of glial cells . NG2 gliOlig2 in cell cultures isolated from the adult SVZ without affecting the neurogenesis from these cells [The expression of the platelet-derived growth factor receptor α (PDGFRα) is essential for the differentiation of NG2 glia to oligodendrocytes , and thrse cells . Consistse cells ,113. In se cells .Axin2 [NS/PCs in both neurogenic regions—the SVZ and the SGZ of adult mice—were shown to express the Wnt/β-catenin-responsive gene Axin2 , and manAxin2 ,56,58,59Axin2 ,116,117.Axin2 ). In addAxin2 ,53, and Axin2 . This isAxin2 . On the Axin2 . In the Axin2 . As showAxin2 ,123. HowAxin2 revealed the active Wnt/β-catenin pathway in adult ependymal cells residing in the SVZ [Ependymal cells are present only in the SVZ, constituting a multiciliated lining of the LVs. They form pinwheel-like structures in order to surround the apical processes of NSCs attached to the basal lamina of the LVs . Their s the SVZ . In addi the SVZ  is invol the SVZ .Stroke is a major cause of death and disability worldwide, and based on its etiology, it can be either ischemic or hemorrhagic. Ischemic stroke is caused by a reduction in the cerebral blood flow, whereas hemorrhagic stroke is caused by intracerebral bleeding. In this review, we focus on ischemic stroke, since it is the predominant form, accounting for approximately 80% of all stroke cases . IschemiBased on its extent and origin, ischemic brain injury may be subdivided into global cerebral ischemia (GCI) and focal cerebral ischemia (FCI). While FCI only affects a certain region of the brain, GCI is caused by decreased blood flow in the whole brain. Common causes of FCI are thrombosis and embolism, whereas GCI occurs after systemic hypoperfusion or asphyxia ,132. In 2+, together with elevated intracellular concentrations of sodium (Na+) and chloride ions (Cl−), followed by a passive influx of water and edema formation [2+ leads to the activation of Ca2+-dependent enzymes, such as phospholipase A2 and cyclooxygenase. This results in the production of reactive oxygen species (ROS) and the subsequent damage of genomic DNA, proteins, lipids and the cell membrane [The reduction in the blood flow leads to a deficit in glucose and oxygen in the brain tissue, thus damaging cellular energy metabolism. A quick depletion of ATP follows, and leads to the loss of the membrane potential and depolarization due to the failed maintenance of ionic gradients by ion pumps, followed by glutamate release from neurons. This ischemic pathway thus leads to the process known as glutamate excitotoxicity (reviewed in Reference ). The acormation . Simultamembrane . One of membrane . Ischemimembrane . Moreovemembrane .In reaction to FCI, a structure known as the glial scar is formed. Within hours after ischemic injury, macrophages and microglia appear, and subsequently, NG2 glia emerge in the tissue surrounding the injured region within a few days after the insult. A permanent glial scar consists predominantly of astrocytes, but it also contains microglia and NG2 cells ,139. TheWnt signaling has been shown to play an important role after ischemic injury in the brain. The current treatment of ischemic stroke is limited to restoring the blood flow through the occluded artery ; howeverOne such process might be the regeneration of brain vascularization. The activation of Wnt signaling by Wnt3a caused an increase in the BBB-like function of cultured human brain endothelial cells . The WntWnt signaling does not only impact the restoration of the blood flow after ischemic injury, but it has also been described as a neuroprotective agent. The concentration of the Wnt signaling pathway antagonist Dkk1 rose in the rat hippocampus after transient GCI, while restoration of the pathway protected hippocampal neurons against the damage caused by ischemia . A similApart from its function in neuroprotection, Wnt signaling has been reported to play a role in nervous tissue regeneration after stroke. Many studies have reported that ischemic injury leads to increased neurogenesis in the SVZ and SGZ . Since WCELSR1 mRNA was observed in the rat SVZ after ischemic stroke, and it led to increased neurogenesis and angiogenesis, mainly via the Wnt/PKC pathway [The neuroprotective and regeneration-stimulating effects of Wnt signaling after ischemic injury were also evidenced by the promotion of neurogenesis mediated by cadherin epidermal growth factor laminin G seven-pass G-type receptor 1 (CELSR1). The upregulation of  pathway . Neurogl pathway .Ppm1d gene encoding Wip1 phosphatase. Animals of this mouse strain exhibited worsened functional recovery after ischemia, which was compensated for by pharmacological activation of the Wnt signaling pathway. This provided further understanding of Wnt-related mechanisms occurring in the nervous tissue after stroke [An insight into a native mechanism by which the damaged tissue increases Wnt signaling was obtained in in vitro cultures of adult rat NSCs under hypoxic conditions. Under these circumstances, increased levels of β-catenin and increased neurogenesis were observed. Simultaneously, hypoxia-inducible factor 1α (HIF1α) expression was upregulated, and the inhibition of this factor counteracted hypoxia-induced expression of β-catenin . A furthr stroke . A differ stroke . Additior stroke . Interesr stroke . This suTaken together, there is strong evidence that Wnt signaling is crucial in stimulating NS/PC proliferation after ischemic injury. Strikingly, Morris and co-workers showed that the expression of Wnt genes was not upregulated in the SVZ of rats after ischemic injury . ContrarAlthough NSCs benefit from the activation of the Wnt pathway, the situation is more intricate when it comes to astrocytes. It has been suggested that canonical Wnt signaling is increased in this cell type after ischemic injury. However, its role was linked to post-ischemic seizures, in which astrocytes participate. Interestingly, the inhibition of Wnt signaling in these cells after ischemic injury decreased the otherwise elevated susceptibility to seizures, indicating a possibly detrimental role of Wnt signaling in astrocytes . Additio2+ pathway, independently of β-catenin. It is evident that these results provided useful insights into the pro-inflammatory activation of microglia; however, the relevance of the Wnt pathway–microglia crosstalk in the injured brain has not been investigated [Wnt signaling also influences the reaction of microglia to ischemic conditions. Oxygen-glucose deprivation caused a decrease in the Wnt1 expression in microglial cells. Remarkably, treatment with Wnt1 had a beneficial effect on microglial survival, while the inhibition of Wnt1 increased their apoptosis . Additiostigated .The beneficial effects of Wnt signaling were also observed in the cells of oligodendroglial lineage. Myelin repair in the ischemic penumbra was increased after treadmill exercise, and this effect was abolished after the treatment with Wnt signaling inhibitor XAV939, suggesting a possible role of active Wnt signaling in NG2 glia—cells known for their role in remyelination . The difIn an effort to find a new, more effective treatment for ischemic stroke that would improve the patients’ long-term prognosis and reduce the adverse health consequences, many experimental approaches “came into play”. Some of these approaches, such as treatment with allogeneic mesenchymal bone marrow cells or hyperbaric oxygen therapy, are already being tested in clinical trials .4 receptor agonists in neurodegenerative disorders, including ischemic stroke , usually derived from the bone marrow. The disadvantage of these approaches lies in the induction of an immune response in the recipient. A possible solution to this obstacle seemed to be the use of NSCs derived from iPSCs. However, the generation of iPSCs is time-consuming, and their usage is associated with a risk of cancer; both phenomena greatly complicate this therapeutic approach (reviewed in Reference ). Since eference ), was lieference . The croeference , or witheference . The lattability . Moreovetability . Interespathways . Similarpathways , and incpathways , respectpathways . The effpathways ). It is Interestingly, several Wnt inhibitors, including Dkk proteins or miRNAs, appear to be produced in the adult brain after stroke, and contribute to neuronal degradation ,161. TheAberrant Wnt signaling underlies many types of human diseases, including cancer, and therefore a great effort has been made to identify the negative regulators of the signaling. On the other hand, a number of synthetic activators of the Wnt pathway were also (co)discovered (reviewed in Reference ). Since Although a large amount of experimental data has already been obtained, it is clear that further experiments will be needed to be able to fine-tune the signaling level of the Wnt signaling pathway. The results of these experiments should maximize the regeneration of the damaged brain, without any adverse effects on the patient’s body.


Consequently, FSCJ specified the ADI for betamethasone at 0.01 μg/kgbw per day.The Food Safety Commission of Japan (FSCJ) conducted a risk assessment of betamethasone(CAS No. 37-44-9), a synthetic adrenocortical hormone, based on the documents includingassessment reports from the European Medicines Evaluation Agency (EMEA). Among results ofvarious studies, a no-observed-adverse-effect level (NOAEL) of betamethasone (asbetamethasone dipropionate) was shown as 0.02 mg/kg bw per day in a fertility and earlyembryonic development study in rats. FSCJ concluded that it is appropriate to set anacceptable daily intake (ADI) of betamethasone by applying the same ADI as dexamethasone.The ADI for dexamethasone at 0.01 μg/kg bw per day (0.00001 mg/kg bw/day) was specifiedbased on the NOAEL of 0.001 mg/kg bw per day of the endocrine toxicity study inrats The Food Safety Commission of Japan (FSCJ) conducted a risk assessment of betamethasone(CAS No. 37-44-9), a synthetic adrenocortical hormone, based on the documents includingassessment reports from the European Medicines Evaluation Agency (EMEA).invitro, tyrosine aminotransferase (TAT) activity in rat liver, titer comparisonfor pharmacological effects among major corticosteroids, and observation in humans.The data used in the assessment include Absorption, Distribution, Metabolism, Excretion(ADME) , residues (cattle and pigs), genotoxicity, acutetoxicity , subacute toxicity , carcinogenicity(mice and rats), as well as reproductive and developmental toxicity . Other data include pharmacological effects, immunotoxicity in vitro gene mutation, betamethasone showed negative resultsfor bacteria and mammalian cells. A positive result was obtained in an invitro chromosomal aberration assay, whereas an in vivomicronucleus test was negative. These data indicated no genotoxicity relevant to humanhealth. It was thus able to specify the acceptable daily intake (ADI) for betamethasone.In assays of Major adverse effects of betamethasone including suppressed body weight as well as atrophyof thymus and spleen were attributable to glucocorticoid activity of betamethasone.In a 104-week carcinogenicity study in rats by gavage, none of effects of betamethasone wasobserved on tumor incidence. Although the route of administration was not oral, notumorigenicity was observed on a 104-week dermal carcinogenicity study in mice. Thus,betamethasone was judged to be non-carcinogenic.Malformations, including cleft palate observed in developmental toxicity studies in ratsand rabbits, are possible to relate to the glucocorticoid activity. None of the adverseeffect was detected after the subcutaneous injection of 0.003 mg/kg bw or less per day in adevelopmental study in rabbits. It is reasonably assumed that no-observed-effect level(NOEL) of the oral administration was higher than the level of subcutaneous route.Among results of various studies, a no-observed-adverse-effect level (NOAEL) ofbetamethasone (as betamethasone dipropionate) was shown as 0.02 mg/kg bw per day in afertility and early embryonic development study in rats. Toxicities including suppressedbody weight, decrease in feed consumption and reduction of the thymus weight were, however,observed at this dose in a 4-week subacute toxicity study and in the 104-weekcarcinogenicity study in rats. Thus FSCJ considered it not appropriate to set an ADI basedon this NOAEL, 0.02 mg/kg bw per day.NOEL of 0.004 mg/kg bw per day was identified based on increased TAT activity in rat liver.However, FSCJ judged this endpoint not appropriate for establishment of an ADI. IncreasedTAT activity is rather physiological but not adverse reaction to glucocorticoids.1). The adverse effects of dexamethasone were attributable toglucocorticoid actions of dexamethasone. Taken together, FSCJ concluded that it isappropriate to set an ADI of betamethasone by applying the same ADI as dexamethasone. TheADI for dexamethasone at 0.01 μg/kg bw per day (0.00001 mg/kg bw/day) was specified based onthe NOAEL of 0.001 mg/kg bw per day of the endocrine toxicity study inrats1).FSCJ recognizes that betamethasone has similar toxicological properties and equivalentglucocorticoid activity to dexamethasone, an epimer, which was evaluated byFSCJConsequently, FSCJ specified the ADI for betamethasone at 0.01 μg/kg bw per day.


To understand the dynamics of malaria transmission, membrane feeding assays with glass feeders are used to assess the transmission potential of malaria infected individuals to mosquitoes. However, in some circumstances, use of these assays is hindered by both the blood volume requirement and the availability of fragile, specially crafted glass feeders. 3D printed plastic feeders that require very small volumes of blood would thus expand the utility of membrane feeding assays.Anopheles stephensi mosquitoes and cultured Plasmodium falciparum gametocytes. In addition, the optimum number of mosquitoes that can feed on the nano-feeder was determined by evaluating fully fed mosquitoes visually and by assessing blood- meal volume with a colorimetric haemoglobin assay.Using two 3D printing production methods, MultiJet (MJ) and Digital Light Processing (DLP), we developed a plastic version of the most commonly used standard glass feeder (the mini-feeder) with an improved design, and also a smaller feeder requiring only 60 µl of blood (the nano-feeder). Performance of the 3D printed feeders was compared to standard glass mini-feeders by assessing infectivity of gametocytes to mosquitoes in standard membrane feeding assays with laboratory reared An. stephensi mosquitoes can take a full blood-meal (median volume 3.44 µl) on a nano-feeder.The 3D printing methods allowed quick and inexpensive production of durable feeders. Infectivity of gametocytes to mosquitoes was comparable for MJ and DLP 3D printed feeders and glass feeders, and the performance of the 3D printed feeders was not influenced by repeated washing with bleach. There was no loss in transmission efficiency when the feeder size was reduced from mini-feeder to nano-feeder, and blood-meal volume assessment indicated ~10 Here we present 3D printed mini- and nano-feeders with comparable performance to the currently used glass mini-feeders. These feeders do not require specialized glass craftsmanship, making them easily accessible. Moreover, the smaller nano-feeders will enable evaluation of smaller blood volumes that can be collected from finger prick, thus expanding the utility of membrane feeding assays and facilitating a more thorough evaluation of the human infectious reservoir for malaria. Plasmodium falciparum. Efforts to further reduce malaria burden would benefit from an improved understanding of malaria transmission dynamics  or in prevalence of infected mosquitoes [glass mini-feeders = 85% (range 80–95) and 3D plastic feeders = 83% (range 71–88)].To determine if comparable mosquito infection rates can be achieved using the smaller sized 3D plastic nano-feeders, they were compared to the standard glass mini-feeders in SMFAs. Cultured gametocytes were fed to mosquitoes (six replicate feeds) and infection prevalence and intensity were compared Fig. . There wAn improved understanding of malaria transmission dynamics would enable better design and implementation of interventions that aim to reduce transmission and ultimately decrease the burden of (severe) malaria in endemic areas. In clinical and epidemiological studies, DMFAs are often performed to assess an individual’s infectivity to mosquitoes using venous blood and glass mini-feeders. However, infectivity assessments on small blood volumes for populations such as infants, where phlebotomy is challenging , or methIn this study, plastic feeders were produced using two 3D printing techniques. 3D printing allows custom feeder optimization. We optimized feeder design by (i) adding a vent tube to allow more efficient feeder filling with blood, (ii) miniaturizing the mini-feeder design to produce a nano-feeder with a 60 µl capacity, and (iii) widening the injection duct of the nano-feeder to fit a p200 pipette tip, avoiding the need for blunt needles for feeder filling . 3D prinPlastic feeders were compared with the standard glass mini-feeders in SMFAs with cultured gametocytes. In these experiments, there was no difference in infection efficiency, with highly similar mean oocyst intensity and prevalence of mosquito infection. Moreover, performance of the plastic feeders was not influenced by repeated washing using bleach. These findings using two different 3D printing materials and technologies are in line with findings from Witmer et al.  who repoAnopheles mosquitoes filter and concentrate erythrocytes during feeding and excrete excess fluid, known as prediuresis . Nextdent SG  was used as resin, no support material was used. Each layer of printed liquid resin was hardened by UV beamer projection (xyz resolution 50 µm). In 30 minutes, post printing (B), feeders were two times ultrasonic cleaned in 96% ethanol and flushed with 96% ethanol before they were dried with compressed air and post-cured for 10 min in a UV box .Additional file 4: Figure S2. Feeder temperature of glass mini-feeders and 3D MJ-printed nano-feeders. Two probes were used to simultaneously measure the temperature of the first (P1) and last (P2) feeder in a row of four. At 0 s, the water bath was turned on  and the blood-meal was injected in the feeder cavity of glass mini-feeders (circles) and nano-feeders (triangles). Both plastic and glass feeders reached a stable temperature quickly within 4.5 min after attaching to the water bath. Plastic feeders remained at a stable temperature on average 1.8 °C lower (mean = 35.9 °C) than glass feeders (mean = 37.7 °C).Additional file 5: Figure S3. Mosquito blood-meal size determined by eye. Mosquitoes were selected by eye and determined unfed (UF), partially blood-fed (PBF) or fully blood-fed (FBF) (from left to right).Additional file 6: Figure S4. Mosquito feeding performance on 3D MJ-printed nano-feeders. In three independent experiments with three different blood donors, plastic cages with 5, 10, 15, 20 or 50 mosquitoes were fed on a 60 µl blood-meal to determine the feeding performance on the nano-feeder. Data from independent experiments were pooled per group for analysis; pie charts present the mean feeding rate in percentages of fully- (FBF), partially- (PBF) or unfed (UF) mosquitoes. In all conditions a number of mosquitoes were PBF or UF. No blood material in the feeder cavity was left for cups with 20 or 50 mosquitoes. For 10 mosquitoes per feeder, all visually FBF mosquitoes had a blood-meal > 2.5 µl, with a mean volume of 3.52 µl  and feeding prevalence of 65% FBF.Additional file 7: Table S1. Mosquito feeding rates after protocol optimization. To increase mosquito feeding rates the original nano-feeder protocol was optimised by (i) extension of the starvation period prior to feed (+ 12 h), (ii) stimulation of mosquitoes prior to feeding by placing a culture flask (canted neck 25 cm2) filled with warm water for 1 minute on top of cup, and (iii) plastic cages were changed to small paper cups to reduce distance between the mosquitoes and the feeder.


HTLV-1-infected cells rarely express viral antigens in vivo; however, they easily express the antigens after short-term culture. Therefore, such virus-expressing cells may lead to activation and expansion of antigen-specific T cell responses. Infected T cells with HTLV-1 and HTLV-1-specific CD8+ cytotoxic T lymphocytes invade the central nervous system and produce various proinflammatory cytokines and chemokines, leading to neuronal damage and degeneration. Therefore, cellular immune responses to HTLV-1 have been considered to play important roles in disease development of HAM/TSP. Recent studies have clarified the viral strategy for persistence in the host through genetic and epigenetic changes by HTLV-1 and host immune responses including T cell function and differentiation. Newly developed animal models could provide the opportunity to uncover the precise pathogenesis and development of clinically effective treatment. Several molecular target drugs are undergoing clinical trials with promising efficacy. In this review, we summarize recent advances in the immunopathogenesis of HAM/TSP and discuss the perspectives of the research on this disease.Human T-lymphotropic virus type 1 (HTLV-1) is associated with adult T cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is an inflammatory disease of the spinal cord and clinically characterized by progressive spastic paraparesis, urinary incontinence, and mild sensory disturbance. The interaction between the host immune response and HTLV-1-infected cells regulates the development of HAM/TSP. HTLV-1 preferentially infects CD4 HTLV-1-infected cells rarely express viral antigens in vivo; however, they easily express antigens after short-term culture. Therefore, such virus-expressing cells may lead to activation and expansion of antigen-specific T cell responses. The virus-host immunologic interaction plays a pivotal role in the pathogenesis of HAM/TSP. HTLV-1-infected T cells and CD8+ cytotoxic T lymphocytes (CTL) against HTLV-1 invade the central nervous system (CNS) and release proinflammatory cytokines and chemokines, resulting in the tissue damage. These immune responses are considered to underlie the pathogenesis of HAMTSP. Both HTLV-1 viral regulatory proteins, Tax and HTLV-1 basic leucine zipper factor (HBZ), play critical roles in immune dysregulation in HAM/TSP  is the human retrovirus firstly discovered in 1980  with the antigen-major histocompatibility complex (MHC) molecule. The diversity of TCR repertoires is central components of adaptive immune system function and can be altered in the context of infections, malignancies or immunological disorders. Analyzing TCR repertoires may help to gain a better understanding of immune-mediated responses in neuroinflammatory diseases. In HAM/TSP patients, a previous report on the TCR analysis showed a direct demonstration of clonal expansions within both CD4+ and CD8+ cells are evenly distributed in active inflammatory lesions, while the predominance of CD8+ cells and high levels of IFN-γ are detected in the chronic stage (Umehara et al. + T cells in the spinal cord, but not in neural cells using in situ PCR technique (Moritoyo et al. + T cells are infected with HTLV- 1, but the neural cells are not.The characteristic pathology of HAM/TSP consists of a chronic inflammation with diffuse degeneration throughout the central nervous system (Izumo et al. + T cells infiltrate the spinal cord and express the TIA-1 molecule, which is a CTL marker (Umehara et al. + T cells infiltrating the CNS. In addition, HTLV-1 proteins are not detected in the CNS-resident cells, but they were identified in more than 60% of the infiltrating CD4+ T cells. Although neurons were generally preserved, approximately 40% of CD4+ T cells and some oligodendrocytes underwent apoptosis. Apoptotic oligodendrocytes were frequently in contact with CD8+ T cells, resulting in demyelination. These findings indicate that the immune responses between HTLV-1-specific CTL and HTLV-1-infected CD4+ T cells could cause apoptosis in surrounding neural cells (bystander damage; Fig. HTLV-1-specific CTL infiltrate the CNS and play a role in HAM/TSP pathogenesis Kubota . Activat+ human umbilical cord stem cells, generate human lymphocytes (Niewiesk −/−γc−/− and bone marrow-liver-thymic mouse showed establishment of peripheral infection that led to lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption within the CNS of infected mice (Ginwala et al. + T cells in various organs including the CNS of infected hu-mice. This humanized mouse model could be a suitable model to evaluate the imunopathogenesis and develop a novel treatment for HAM/TSP.Animal models have been widely used to study HTLV-1 viral transmission, disease pathogenesis, and treatment Niewiesk . HoweverNiewiesk . Inocula+ T cell function in HAM/TSP patients although no clinical efficacy was observed (Enose-Akahata et al. In HAM/TAP, most therapeutic trials have aimed to inhibit or regulate the immune response, or to reduce the HTLV-1 proviral load in an attempt to decrease the risk or alter the course of the disease. Corticosteroids are most widely used to decrease the inflammation in the CNS, particularly in the early stage. Motor disability in some patients could be improved with steroids (Nakagawa et al. + T cells and establish infected T cell clones in the host, regulating the balance between proviral latency and reactivation. Clonal expanded T cells infected with HTLV-1 dysregulate T cell function and differentiation and subsequently induce HTLV-1-specific T cell responses. These cell-mediated immune responses and released proinflammatory cytokines could cause neural damage, which is thought to play a central role in the development of HAM/TSP. Recent advances in research provide a better understanding of mechanisms associated with HTLV-1-infected cells and cellular immune responses in HAM/TSP. Further research will be required to develop strategies to eliminate HTLV-1-infected cells or enhance cellular immunity for the development of effective treatments in patients with HAM/TSP.HTLV-1 preferentially infects CD4


Despite known associations of lower serum uric acid (UA) with Alzheimer’s disease (AD) dementia or AD-related cognitive impairment, little is known regarding the underlying patho-mechanisms. We aimed to examine the relationships of serum UA with in vivo AD pathologies including cerebral beta-amyloid (Aβ) and tau deposition, AD-signature region cerebral glucose metabolism (AD-CM), and white matter hyperintensities (WMH). We also investigated the association between serum UA and cognitive performance, and then assessed whether such an association is mediated by the brain pathologies.A total of 430 non-demented older adults underwent comprehensive clinical assessments, measurement of serum UA level, and multimodal brain imaging, including Pittsburgh compound B-positron emission tomography (PET), AV-1451 PET, fluorodeoxyglucose (FDG)-PET, and magnetic resonance imaging scans. Mini-Mental State Examination (MMSE) and word list recall (WLR) test scores were used to measure cognitive performance.Serum UA level was significantly associated with AD-CM, but not with Aβ deposition, tau deposition, or WMH volume. Serum UA levels also had significant association with WLR and marginal association with MMSE; such associations disappeared when AD-CM was controlled as a covariate, indicating that AD-CM has a mediating effect.The findings of the present study indicate that there is an association of low serum UA with AD-related cerebral hypometabolism, and whether this represents a causal relationship remains to be determined. Uric acid (UA) is a naturally produced water-soluble antioxidant, which contributes more than half of the free radical scavenging activity in the peripheral nervous system . UA is aAn emerging body of epidemiological studies have indicated that a lower serum UA level is associated with poorer cognitive function and increased risk of mild cognitive impairment (MCI) or overall dementia , althougin vivo neuropathological changes that could link lower serum UA and AD-related cognitive decline.Despite such associations of lower serum UA with AD dementia or AD-related cognitive impairment, little is known regarding the underlying patho-mechanisms. Some in vivo animal cell culture and postmortem human brain cell studies suggested that UA may act as an antioxidant to mitigate Aβ-induced neuronal injury  or to rein vivo AD pathologies including cerebral Aβ and tau deposition, AD-signature region cerebral glucose metabolism (AD-CM), and white matter hyperintensities (WMH) in non-demented older adults. Secondly, we investigated the association between lower serum UA and cognitive impairment, and then assessed whether such an association is mediated by brain pathologies that had significant relationships with lower serum UA.In this context, we first aimed to investigate the relationships of serum UA with z-score was <−1.0 for at least one of four episodic memory tests: Word List Memory, Word List Recall (WLR), Word List Recognition, and Constructional Recall; these are included in the Korean version of the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD-K) neuropsychological battery (This study was part of the Korean Brain Aging Study for Early Diagnosis and Prediction of Alzheimer’s Disease (KBASE), which is an ongoing prospective cohort study . As of F battery . All MCIThe exclusion criteria were as follows: (1) presence of a major psychiatric illness; (2) significant neurological or medical conditions, or comorbidities that could affect mental function; (3) contraindications for a magnetic resonance imaging (MRI) scan ; (4) illiteracy; (5) the presence of significant visual/hearing difficulties and/or severe communication or behavioral problems that would interfere with clinical examinations or brain scans; (6) use of an investigational drug; and (7) pregnancy or lactation. Further information regarding the recruitment of the KBASE cohort was described in a previous report by our research group . The stuz-score indicates poorer cognitive function  was assessed based on data collected by trained nurses during systematic interviews of participants and their informants; a vascular risk score (VRS) was calculated based on the number of vascular risk factors present and reported as a percentage . SmokingAPOE) genotyping was performed as previously described (APOE4) positivity was defined as the presence of at least one ε4 allele.After an overnight fast, blood samples were obtained via venipuncture in the morning (8–9 a.m.). Serum levels of UA were measured using a colorimetry method . The normal ranges for serum UA are 3.7–9.2 g/dL in men and 3.1–7.8 g/dL in women. Serum albumin was also measured using the same method to evaluate a general nutritional state. Additionally, genomic DNA was extracted from whole blood and apolipoprotein E (escribed . APOE ε411C] Pittsburgh compound B (PiB)-positron emission tomography (PET) and 3D T1-weighted MRI scan using a 3.0T Biograph mMR (PET-MR) scanner (Siemens), in accordance with the manufacturer’s guidelines. The details of the PiB-PET imaging acquisition and preprocessing were described previously  [eviously . An autoeviously  were appeviously . Each paeviously .n = 107) underwent [18F] AV-1451 PET scans (Siemens) using a Biograph True point 40 PET/CT scanner (Siemens), in accordance with the manufacturer’s guidelines. While all the other neuroimaging scans were performed during the baseline visit, AV-1451 PET imaging was performed at an average of 2.6 (standard deviation 0.3) years after the baseline visit. The details of AV-1451 PET imaging acquisition and preprocessing were described previously  using the abovementioned 3.0T PET-MR scanner in a validated automatic procedure that has previously been reported . BrieflyAPOE4 positivity, VRS, clinical diagnosis [CN vs. MCI], serum albumin, body mass index, smoking status, and alcohol intake status) that might affect the relationship between serum UA and neuroimaging biomarkers  interaction term, as well as serum UA and age  as independent variables; the neuroimaging biomarker was used as a dependent variable, and the analyses were controlled for age, sex, education, APOE4, VRS, and clinical diagnosis as covariates. Additionally, to investigate the association between serum UA and cognitive performance, the z-score differences of the MMSE and WLR were tested among the three UA strata by GLM analysis with post hoc tests using the LSD method. The same GLM analyses were performed with control of AD neuroimaging biomarkers (those that showed significant associations with UA) as covariates, to investigate whether the associations between UA and cognitive function measurements by MMSE or WLR are mediated by those AD biomarkers. All statistical analyses were performed using IBM SPSS Statistics software . The level of statistical significance was set as a two-tailed p-values < 0.05.To examine relationships between serum UA and neuroimaging biomarkers, multiple logistic regression analyses, linear regression analyses, or general linear model (GLM) analysis with post-hoc tests using the least significant difference (LSD) method were performed as appropriate. Serum UA, as an independent variable for each analysis, was first analyzed as a continuous variable, and then as a stratified categorical variable; subjects were divided into three strata  based on the tertiles of the serum UA level. For each analysis of associations between UA and AD neuroimaging biomarkers, three models were tested for stepwise control of potential confounders. The first model did not include any covariates, the second model included age and sex as covariates, and the third model included all potential covariates  analysis .In the present study of non-demented older adults, lower serum UA was associated with decreased AD-CM, but not with other AD neuroimaging biomarkers or WMH. There was also a significant positive association between serum UA and cognitive performance, which was mediated by AD-CM. To the best of our knowledge, this is the first study to reveal a relationship between serum UA and AD-CM, as well as between serum UA and cognitive performance.We found a strong positive association of serum UA with AD-CM. Consistent with this result, previous animal cell culture and postmortem human brain cell studies showed that UA reduced Aβ-induced neuronal injury  and synaNotably, we did not find any associations of serum UA with amyloid or tau pathologies. This indicates that the protective effect of UA against AD is not directly associated with the deposition of the two core AD proteins. Additionally, the presence of WMH, as a measure of cerebrovascular injury, was not associated with serum UA in the present study. This finding is not consistent with the results of a previous human study, which showed that elevated serum UA was associated with increased WMH volume . This diWe examined the relationships of serum UA with MMSE, a measure of global cognition, and with WLR, a measure of episodic memory. Serum UA showed a significant positive association with WLR score and tended to show a positive association with MMSE score. Given that episodic memory decline is the earliest and most prominent change in AD , the assThere were a few limitations in the present study. First, because this was a cross-sectional study, the association may not represent causality. For example, it is possible that the low serum UA may be the consequence of the cerebral glucose hypometabolism, or a sign of worse disease. It is known, for example, that weight loss often precedes the development of clinical dementia in AD patients , and thaThe findings of the present study indicate that there is an association of low serum UA with AD-related cerebral hypometabolism, and whether this represents a causal relationship remains to be determined.kbasecohort@gmail.com).The data of the current study can be available from the independent data sharing committee of the KBASE research group on reasonable request. Requests for data access can be submitted to the administrative coordinator of the KBASE group by e-mail  and SNU-SMG Boramae Center (IRB No: 26-2015-60), Seoul, South Korea, and was conducted in accordance with the recommendations of the current version of the Declaration of Helsinki. All subjects or their legal representatives gave written and informed consent.JK and DL conceived and designed the study. MB, DY, JL, SJ, KK, GJ, HL, J-YL, C-HS, Y-SL, SS, YK, and DL were involved in acquisition, or analysis and interpretation of the data and helped to draft the manuscript. JK, MB, DY, JL, and DL were major contributors in writing the manuscript and critically revising the manuscript for intellectual content and served as principal investigator and supervised the study. All authors read and approved the final 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.


Brassinosteroids, the steroid hormones of plants, control physiological and developmental processes through its signaling pathway. The major brassinosteroid signaling network components, from the receptor to transcription factors, have been identified in the past two decades. The development of biotechnologies has driven the identification of novel brassinosteroid signaling components, even revealing several crosstalks between brassinosteroid and other plant signaling pathways. Herein, we would like to summarize the identification and improvement of several representative brassinosteroid signaling components through the development of new technologies, including brassinosteroid-insensitive 1 (BRI1), BRI1-associated kinase 1 (BAK1), BR-insensitive 2 (BIN2), BRI1 kinase inhibitor 1 (BKI1), BRI1-suppressor 1 (BSU1), BR signaling kinases (BSKs), BRI1 ethyl methanesulfonate suppressor 1 (BES1), and brassinazole resistant 1 (BZR1). Furthermore, improvement of BR signaling knowledge, such as the function of BKI1, BES1 and its homologous through clustered regularly interspaced short palindromic repeats (CRISPR), the regulation of BIN2 through single-molecule methods, and the new in vivo interactors of BIN2 identified by proximity labeling are described. Among these technologies, recent advanced methods proximity labeling and single-molecule methods will be reviewed in detail to provide insights to brassinosteroid and other phytohormone signaling pathway studies. Brassinosteroids (BRs) are plant steroid hormones critical in numerous developmental and physiological processes, such as stem elongation, vascular differentiation, seed size, male fertility, flowering time, senescence, cell division, and resistance to biotic and abiotic stresses ,2. BecauThe development of the understanding of the BR signaling pathway has progressed with the advancements in biotechnology. Molecular genetics provides the fundamentals for the study of signaling pathways. In particular, the combination of forward and reverse genetics facilitated the identification of BR signaling components. Here, we summarize traditional and new methods used. Conventional methods, including EMS mutant screening, activation tagging, yeast two-hybrid (Y2H) screening, two-dimensional (2D) difference gel electrophoresis (DIGE), and liquid chromatography (LC)–tandem mass spectrometry (MS/MS), the applications of which have laid the foundation of our current understanding of the BR signaling pathway. New technologies, such as bioinformatics, clustered regularly interspaced short palindromic repeats (CRISPR) system, proximity labeling (PL), and single-molecule technology, have considerably enhanced this understanding. In this review, we illustrate how these technologies have been used in BR network studies, with emphases on PL and single-molecule technology. We review the development of these two methods, highlighting their application in planta. The coverage of these methods can help researchers in the field gain new insights for their own studies.Forward genetics is the screening of phenotypes of interest under different backgrounds and conditions to identify the gene that causes a certain phenotype. It laid the foundation for our understanding of the BR signaling pathway in the early days. The application of forward genetic screening usually begins with naturally or artificially generated mutations.−6 or 10−7 M final concentration of eBL, the synthetic most active brassinolide ..72]. By TIRFM is uniquely suited to visualize processes that occur in and around the membrane of living cells. However, plant cells have a rigid cell wall surrounding the plasma membrane, which restricts the penetration of the evanescent field and scatters the illumination. In 2000, Rohrbach developed a multiangle evanescent-wave microscope, referred to as VA-TIRFM, which overcame this shortcoming . By contFRET is a popular tool to measure the dynamic activities of biological molecules at the nanoscale ,77. In FCoSMoS is a powerful method to investigate biomolecule reaction mechanisms of multiple components, including assembly pathways , composiDuring single-molecule experiments, fluorophores usually photobleach (become nonfluorescent due to photodamage) in seconds and exhibit a stepwise intensity trajectory d. Photob−15 L defined by a tightly focused laser beam  BRI1-GFP particles exhibited multiple diffusion types in the membrane; (2) BRI1 endocytosis is clathrin-dependent and regulated by BR; and (3) membrane microdomains contribute to BRI1 partitioning and endocytosis and even affect BR signaling. By counting the photobleaching steps of BRI1-GFP, BRI1 molecules on the membrane were found to function as monomers or dimers .A. thaliana [Song et al. were the first to use CoSMoS based on a multicolor TIRFM and a homemade flow cell to perform an in vitro investigation of the signaling timescales of the major BR signaling components BES1 and BIN2 of thaliana ,91. By pthaliana ,91.2O2 induces the oxidative modification of BZR1 by traditional biological methods [Compared with findings that H methods , single-Various methodologies can be applied to facilitate the identification of new BR members and foster our comprehension of BR signaling and other signaling networks. In this review, we introduce several recent developments of technologies and illustrate how they contribute to our current understanding of the phytohormone BR. The method used in BR signaling is also available for other signaling pathways, particularly PL and single-molecule technologies. Notably, not many applications of these novel methodologies exist in the field of BR research, indicating that the usage of these methods is still at its budding stage, and many opportunities await. We believe that these methods will develop into powerful plant science research tools.In addition, there are still many unanswered questions in BR signaling pathway research such as how the tissue specific development is regulated and whether non-coding RNA functions in BR signaling regulation. Besides phosphorylation and acetylation, does another modification exist and how does it regulate the BR signal? How do the redundant genes work? How does the BR signal affect modifications of the whole plants? Therefore, we still need to develop new methods for different conditions in BR signaling research, such as tissue specific signal-cell sequencing, next generation sequencing, and proteomics of post-translational modifications. Moreover, almost all of these methods are based on bioinformatics. Furthermore, comparative studies of BR signal pathway across different plant species will lead to a better understanding of the evolution and better application of BR signal pathway.


In vitro data suggest that myoendothelial coupling underlies EDH-type dilations as evidenced by blocking experiments as well as simultaneous, merely identical membrane potential changes in endothelial and smooth muscle cells (SMCs), which is indicative of coupling through ohmic resistors. However, connexin-deficient animals do not display any attenuation of EDH-type dilations in vivo, and endothelial and SMCs exhibit distinct and barely superimposable membrane potential changes exerted by different means in vivo. Even if studied in the exact same artery EDH-type dilation exhibits distinct features in vitro and in vivo: in isometrically mounted vessels, it is rather weak and depends on myoendothelial coupling through connexin40 (Cx40), whereas in vivo as well as in vitro under isobaric conditions it is powerful and independent of myoendothelial coupling through Cx40. It is concluded that EDH-type dilations are distinct and a significant dependence on myoendothelial coupling in vitro does not reflect the situation under physiologic conditions in vivo. Myoendothelial coupling may act as a backup mechanism that is uncovered in the absence of the powerful EDH-type response and possibly reflects a situation in a pathophysiologic environment.The endothelium controls vascular tone adopting blood flow to tissue needs. It releases chemical mediators  and exerts appreciable dilation through smooth muscle hyperpolarization, thus termed endothelium-dependent hyperpolarization (EDH). Initially, EDH was attributed to release of a factor, but later it was suggested that smooth muscle hyperpolarization might be derived from radial spread of an initial endothelial hyperpolarization through heterocellular channels coupling these vascular cells. The channels are indeed present and formed by connexins that enrich in gap junctions (GJ).  Apart f+-channels, which induce relaxation likely through closure of voltage-gated Ca2+-channels representing a rather speedy signaling pathway. Since the dilaton is depending on the presence of ECs and characterized by smooth muscle hyperpolarization, this obscure mediator has been termed endothelium-derived hyperpolarizing factor (EDHF) assuming that also this mechanism communicates in the same language, i.e., release of a mediator as for the classical autacoids, NO, and PG. However, this view was challenged and a direct electrotonic way of communication was suggested. Charge is transferred from ECs to smooth muscle through intercellular channels  driven by an initial endothelial hyperpolarization that spreads radially into the vessel wall  that modulate vascular responses  or different cell types  and allow transfer of small molecules (< 1 kDa) and ions. The latter equals current flow along an electrochemical potential difference and, ultimately, permits the transfer of membrane potential changes between interconnected cells. Hundreds of such channels are located at GJ where adjoining plasma membranes are only 2 nm apart . GJ can The family of connexin genes is comprised of ~20 members , and the proteins are named according to their theoretical molecular mass; for example, connexin40 (Cx40) has a predicted molecular mass of about 40 kDa . In the in vitro co-culture system, myoendothelial junctions were composed of Cx40 and Cx43 provided by both cell types. This occured despite the expression of Cx37 in endothelial and SMCs in this artificial system but this connexin was excluded from these junctions  by inhibition of the protein tyrosine phosphatase SHP-2. This effect prevented in isolated vessels the Ca2+ drainage from the endothelium into the smooth muscle and enhanced thereby Ca2+ levels in ECs promoting vasodilation induced by vasoactive agonists  in murine small skeletal muscle arteries. Functionally, the authors observed in a culture system, a modulation of Cx37 permeability by NO specifically for this connexin (compared to Cx40 and Cx43) and conclude from these findings that NO exerts a specific modulatory effect on myoendothelial junctions due to the fact that Cx37 is the main Cx at this strategic location, and NO preferentially affects its function . The inhagonists . Taken tvia GJs  to subjacent SMCs and, conversely, action potentials originating in the arterial media were rapidly conducted to the endothelium . The sigvia GJs .3 and/or Ca2+ ions in either direction  is a useful strategy that provides even certain specificity for different connexin subtypes depending on the amino acid sequences of the peptide. In fact, numerous studies have shown that they are able to suppress EDHF-type smooth muscle hyperpolarizations and dilations in vitro . In somein vitro , whereasin vitro .2+ transfer from smooth muscle to endothelium as outlined above. The latter opposing mechanism was abrogated in mice carrying a mutant Cx40 protein, which also impaired chemical coupling  seem to be invoked and appear to be a required brick of the intercellular signaling pathway.Taken together, there is compelling evidence that myoendothelial coupling through connexins provides a functional pathway that allows electrotonic spread of membrane potential changes . Blockade of this pathway attenuates or even abrogates EDH-type dilations in vivo is more cumbersome; however, an efficient strategy is the use of mice deficient for connexins. We published the first study on vascular connexin function by using Cx40-deficient mice in which we demonstrated a crucial role for Cx40 to conduct endothelium-dependent dilations longitudinally along the vessel wall, thereby coordinating vascular cellular behavior  than arterioles accessible for intravital microscopy examined in vivo (20–50 μm). The latter arterioles are those that provide the vascular resistance, and their behavior is revealed by pressure drop or resistance measurements in intact organs or animals.Collectively, these results argue against the hypothesis that Cx40 is universally required for EDH-type signaling in vitro as well as in vivo examination because the artery is located at a spot that is accessible for intravital microscopy. This small artery exhibits in vitro an acetylcholine-induced dilation that is mediated to a considerable part by an EDH-type mechanism . Thus, the EDH-type dilation is larger than in a conducting artery , which relies mostly on NO but it is smaller than in arterioles. Arteriolar responses in the microcirculation in vivo are nearly exclusively mediated by EDH-type dilation  that exhibits a diameter (~130 μm) allowing dilation .in vitro using wire myography. Blockade of NO-synthase and COX nearly abrogated the dilation in Cx40-deficient arteries but uncovered EDH-type dilations in wildtype mice. This demonstrates that EDH-type dilations require Cx40 in this setting and suggest that myoendothelial coupling requiring Cx40 supports this pathway  and the sodium pump reduced the responses in both, wildtype and Cx40-deficient mice  that were studied isometrically ent mice . Althougent mice  indicatiSince mice globally deficient for Cx40 are hypertensive due to an abrogated feedback of pressure on renin release , hypertein vivo, is independent of Cx40. Furthermore, this powerful response is even capable to replace the dilatory function of other endothelial mediators (mainly NO). Importantly, EC-specific Cx40-deficient mice are not hypertensive  conditions. These different experimental setups have also shown to modulate the sensitivity of rat mesenteric arteries for vasoconstrictors  that initiate a Ca in vivo . Similar in vivo . This vein vivo that acts independent of Cx40. We suggest that we need to reconsider the “sticky” business, which may not be so sticky at all in search for this mediator . At such conditions, myoendothelial coupling may take over and provide a remaining, less powerful EDH-type dilation that is, in fact, dependent on Cx40.Taken together, a strong powerful EDH-type dilation exists mediator . Importain vivo it remains to be considered if myoendothelial coupling exists in vivo at all. It may well be that heterocellular coupling is turned off during physiologic vascular control and is only activated during special (pathophysiologic?) conditions. In contrast to the findings with isolated arteries and arterioles described above, a number of studies have failed to demonstrate tight electric myoendothelial coupling in arterioles in vivo  leaves EDH-type dilations fully intact  in vivo . For exaterioles . Additiorization . Conversothelium ; whereasvia MEGJ . Also inand SMCs . Again, and SMCs . Finallyand SMCs .in vivo. The data rather suggest that the membrane potential of these distinct cell populations is regulated independently reflecting their specific functions. Interestingly, connexins at the myoendothelial junctions are subject to functional regulation (in vivo findings. Although significant charge transfer through MEGJ was not convincingly demonstrated in vivo, significant contributions of myoendothelial coupling to vascular function may well be of importance, for example the transfer of intracellular messengers (These experimental findings do not provide evidence for functional myoendothelial coupling that aligns the membrane potential of endothelial and SMCs gulation , which mssengers .in vitro studies demonstrate an important role for myoendothelial coupling by transferring charge from endothelial to SMCs to elicit EDH-type dilations. However, in vivo a distinct, more powerful mechanism is responsible for such dilations that easily overcome the lack of myoendothelial coupling. The evidence for efficient charge transfer through myoendothelial coupling in vivo is scarce. This is not related to a lack of data, in fact, many laboratories have attempted to tackle this important question, but failed to find solid evidence documenting myoendothelial coupling. In fact, these efforts have accumulated strong indications for the opposite, i.e., endothelial and SMCs “do their own thing” with respect to membrane potential. However, under certain experimental conditions, myoendothelial coupling supports EDH-type dilation, but this EDH-type response is rather weak compared to the EDH-mechanism acting in vivo.Many All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide. One of the most critical signaling pathways in HNSCC is the Epidermal Growth Factor Receptor/ Phosphatidylinositol 3-Kinase (EGFR/PI3K) pathway. IQ motif-containing GTPase- activating protein 1 (IQGAP1), a protein upregulated in multiple types of cancer, acts as a scaffold for this pathway and others implicated in cancer. IQGAP1 is overexpressed in HNSCCs, and its overexpression correlates with poorer prognosis in HNSCC patients, indicating that IQGAP1 might be important in HNSCC development. Here, we summarized our recent demonstrating a role of IQGAP1 in promoting HNSCC, at least in part, by scaffolding the EGFR/PI3K signaling pathway. PIK3CA, which encodes for the catalytic subunit of PI3K, is amplified in > 40% of HNSCCs, and contains gain-of function mutations in about 20% of HNSCCs [PIK3CA mutations correlate with advanced-stage HNSCCs, promoting HNSCC cell growth, tumor progression, invasion and metastasis [HNSCCs, which arise in the mouth and throat region, are the sixth most frequent cancer worldwide, with approximately 53,000 new cases and 11,000 associated deaths in the United States in 2019 [tastasis -10. Unfotastasis .-/-Iqgap1, [+/+Iqgap1) mice, when stimulated with EGF [in vivo.IQ motif-containing GTPase-activating protein 1, (IQGAP1), is a scaffolding protein that speeds up the efficiency of intracellular signaling by assembling multiple factors that mediate these signaling pathways. IQGAP1 affects multiple cellular activities such as cytoskeletal dynamics, cell-cell adhesion, cell proliferation, cell motility and invasion -16. IQGAgap1-/-, ) showed with EGF , demonst-/-Iqgap1 mice developed significantly lower cancer incidences, lesser disease severity, and fewer cancer foci per mouse, when compared to the +/+Iqgap1 mice [-/-Iqgap1 mice showed significantly lower levels of PI3K signaling than those in +/+Iqgap1 mice, indicating that IQGAP1 contributes to HNSCC, at least in part, through PI3K signaling [Considering the importance of PI3K signaling in HNSCCs, we explored whether IQGAP1 plays a role in head and neck carcinogenesis using a well-validated mouse model that drives HNSCC using a synthetic oral carcinogen, 4-nitroquinoline 1-oxide . After +/+ mice . Tumors ignaling . In humaignaling .Other than increasing PI3K signaling, there are other possible mechanisms by which IQGAP1 may drive HNSCCs. In skin, IQGAP1 promotes tumorigenesis by scaffolding the Ras-MAPK signaling pathway . HoweverPIK3CA mutations or wild-type PIK3CA respond to IQ3 peptide treatment [PIK3CA mutation also correlates with shorter time to disease recurrence in a subset of HNSCC [IQGAP1 binds to both wild type and mutated PI3K . Both brreatment , 21. Thiof HNSCC . These H


Limits of subsistent studies and further work are also discussed, providing a novel vision for the study and application of tantalum.Despite the brilliant bioactive performance of tantalum as an orthopedic biomaterial verified through laboratory researches and clinical practice in the past decades, scarce evidences about the essential mechanisms of how tantalum contributes to osteogenesis were systematically discussed. Up to now, a few studies have uncovered preliminarily the biological mechanism of tantalum in osteogenic differentiation and osteogenesis; it is of great necessity to map out the panorama through which tantalum contributes to new bone formation. This minireview summarized current advances to demonstrate the probable signaling pathways and underlying molecular cascades through which tantalum orchestrates osteogenesis, which mainly contain Wnt/ Tantalum (Ta), a refractory metal, was well-known for its excellent biocompatibility, corrosion resistance, and bioactivity, making Ta a desirable biomaterial for medical applications –4. Sinceβ-catenin signaling pathway [β) and bone morphogenic proteins (BMPs) signaling pathway [Despite these inspiring advantages of Ta, orthopaedic implants made of titanium (Ti) and its alloys are still the prominent choice for orthopedists , 9. As s pathway , transfo pathway –19, mito pathway , 21, and pathway , 23. The pathway . Howeverβ superfamily consists of BMPs subgroup, TGF-βs, activin, osteoprotegerin (OPG), receptor activator for nuclear factor-κ B ligand (RANKL), and receptor activator for nuclear factor-κ B (RANK) [β superfamily members could bind the transmembrane heteromeric receptor complex comprising two kinds of receptors (type I and type II), thereby modifying intracellular proteins and molecules, the classical small mothers against decapentaplegic (Smad) complex pathway and MAPK cascade, to regulate gene expression [β superfamily not only plays an important role in the intramembranous and endochondral ossification during the embryonic period but also orchestrates osteogenesis and osteolysis through regulating osteoblast and osteoclast [TGF-B (RANK) , 34. TGFpression . TGF-β steoclast , 36, 37.βs, namely, TGF-β1, TGF-β2, and TGF-β3, and there are two kinds of TGF-β receptors, namely, type I and type II TGF-β receptor  [β signaling pathway, the TGF-βs firstly interact with TGF-β receptor complex comprising two TβRIs and two TβRIIs, and then, the TβRII phosphorylates the TβRI, activating the receptor-activated Smads (R- Smads), Smad2 and Smad3 successively [βs can also act through the nonclassical TGF-β signaling pathway, also known as non-Smad-dependent pathway, in which binding of TGF-βs with the receptor complex results in phosphorylation of TGF-β activation kinase1 (TAK1), thereby activating the MAP kinase kinase 3/6 (MKK3/6)/p38 signaling pathway, or extracellular signal-regulated kinase (ERK) signaling pathway [There are three conformations of TGF-, TβRII) . In the essively . Smad2/3 pathway , 39.β/Smads signaling pathway, was significantly higher in the Ta group after 21 and 28 days of culturing. As shown in β/Smad signaling pathway. Interestingly, the productions of ALP and calcium nodules reduced gradually with the increase of SIS3, which demonstrated that Ta might stimulate osteogenesis through activating the TGF-β/Smad signaling pathway. Similarly, Hefni et al. implanted porous tantalum trabecular metal (PTTM)  and Ti cylinders into bilateral mandibles of osteopenic patients [β3 and TGF-β2 in the Ta group at mRNA level, as compared to the Ti group. These results indicated synergistically that Ta could promote osteogenesis through the TGF-β signaling pathway [Shi et al.  coculturpatients , finding pathway –43.β superfamily, and the BMP signaling pathway is one of the most famous signaling pathways related to osteogenesis [BMPs account for the largest subgroup of the TGF-ogenesis . The binogenesis , 46, theogenesis .Lu et al.  coculturβ superfamily [μm-scale tantalum powder could inhibit preosteoclast cell proliferation and differentiation remarkably, as compared with Ti powder [The OPG/RANKL/RANK regulatory system is a coin orchestrating osteogenesis and osteolysis, and all of the three critical molecules belong to the TGF-erfamily . The comerfamily . As sucherfamily , 51. Lu erfamily  found thi powder . These sβ-catenin pathway is involved in body development and growth, especially bone metabolism [β-catenin connects with glycogen synthase kinase 3β (GSK3β), adenomatous polyposis coli (APC), and axis inhibition protein (Axin) to form a degradative complex, which is degraded through the targeting effect of Axin to the proteasome. When the Wnt ligands bind the transmembrane receptors, low-density lipoprotein receptor-related proteins 4/5/6 (LRP4/5/6) and Frizzled, the sensitized LRP4/5/6 recruits Axin derived from the degradative complex. The absence of Axin results in the disassembly of the degradative complex under the influence of disheveled (DVL) and gives rise to the release and accumulation of β-catenin in the cytoplasm. Accumulative β-catenin gets into the nucleus and regulates transcription of osteogenesis relevant genes [Wnt/tabolism , 52, 53.nt genes .β-catenin, as compared to Ti discs with the same surface topography. Meanwhile, they also found a higher expression of downstream genes of the Wnt/β signaling pathway in the Ta group, including secreted phosphoprotein 1 (Spp1), alkaline phosphatase, liver/bone/kidney (ALPL), Runx2, Axin2, and C-myc. However, it deserves consideration that the upregulation was also observed in the expression of Smad6 which was reported to compete with Smad1/5/8 to suppress the formation of Smad4-Smad1/5/8 complex [μm-scale tantalum powder was evaluated through DNA microarrays, and the result suggested that Ta enhanced the expression of Wnt1 signaling pathway protein 3 (WISP3) which plays an important positive role in Wnt/β-catenin signaling pathway [β-catenin signaling pathway [Shi et al.  found th complex , 54. Add pathway . These s pathway .α/β heterodimer and is responsible for mediating the cell-matrix and cell-cell interaction [αv/β1, α2β1, and α5β1, have been reported to play a critical role in osteogenesis [Integrin, a transmembrane adhesion protein, consists of eraction . The bineraction . Integriogenesis , 56–59.α5, integrin β1, ERK1/2, and Runx2 of rBMSCs at both protein and mRNA level, as compared with that of Ti. And they also found that inhibition of integrin α5, β1, and ERK 1/2 by siRNA resulted in down-regulation of downstream genes and impaired osteogenesis; overexpression of integrin α5 and β1 resulting from coding sequence could promote osteogenesis, and this effect could be subsequently counteracted by inhibition of ERK1/2. Analogously, Ta coating on Ti was reported to be able to enhance the expression of FAK which plays an important role in the integrin signaling pathway [As such, some researchers have tried to explore whether Ta could promote osteogenesis through the integrin signaling pathway. Lu et al.  found th pathway . Increas pathway . All of Besides the above-mentioned signaling pathways, Ta was reported to promote osteogenic differentiation and osteogenesis via some other mechanisms, such as the MAPK signaling pathway mediated by oxidative stress , 30, actReactive oxygen species (ROS), also known as free radicals, are small molecules derived from oxygen and are involved with various biological processes, for instance, the differentiation of BMSCs , 61. WanhiEmcnhi) [Osteogenesis was coupled with the angiogenesis of a specific vessel subtype CD31iEmcnhi) –64. When1iEmcnhi iEmcnhi) . And theiEmcnhi) . Moreoveβ-catenin, BMP, TGF-β, and integrin signaling pathways. The integration of these mechanisms would provide a novel insight into the study and application of Ta as orthopedic implants. To our best knowledge, this is the first review summarizing the mechanisms of Ta in osteogenesis.Porous Ta has been extensively applied as orthopaedic implants due to its excellent biocompatibility, corrosion resistance, and osteogenesis capacity. The focus of this review is to sketch out an osteogenesis panorama of Ta on the basis of current publications. At present, the underlying signaling pathways through which Ta induced osteogenesis were preliminarily discovered, such as Wnt/It is well-known that the biological performance of orthopedic implants mainly depends on the inherent properties and surface topography (structures and morphologies) , 67; theβ-catenin activated by Ta not only enhanced the transcription of Runx2 but also induced the expression of Smad6 which was reported to be a negative feedback regulator of BMP signaling pathway [There were complex cross-talking and interactions throughout different signaling pathways, and the involved pathways regulated each other positively or negatively. Similarly, there was cross-talking in the signaling pathways triggered by Ta. For example, osteogenic differentiation was involved in different signaling pathways activated by Ta, and  pathway . Ta may  pathway .Of course, we must acknowledge that there are some discrepant reports. Chen et al.  demonstrEven though several studies illuminated the underlying osteogenesis mechanisms of Ta applying some classical signaling pathways, some limits are also worth pondering. First of all, subsistent studies explored the signaling pathways triggered by Ta just in preliminary, and some mechanisms were not stated in detail; further in-depth researches for specific mechanisms remain to be done. Secondly, there were no publications investigating whether Ta promotes osteogenesis through other classical pathways associated with bone remolding, such as insulin signaling pathway or NOTCH signaling pathway, because there are complex cross-talking between these signaling pathways , 78, 79.In the future, studies about mechanisms of Ta on osteogenesis should be more comprehensive, in detail and in-depth, and the influence of Ta on osteolysis deserves attention. It is of great significance to investigate the transducing from the mechanical signal of Ta to biological signal, which has been a hot topic in the field of biomaterials currently , 80, 81.β-catenin signaling pathway, BMP signaling pathway, TGF-β signaling pathway, and integrin signaling pathway. These studies give essential explanations to the phenomenon that Ta induces osteogenesis.In recent years, our understanding of the osteogenic action of Ta has remarkably advanced. Current evidence from published studies revealed that Ta modulates osteogenesis through regulation of the Wnt/