As Gymnadenia R.Br. (Gym) has an obvious uric acid-lowering effect, but its specific bioactive substances and mechanism are still unclear. The key metabolites and pathways used by Gym to reduce uric acid (UA) were identify.in vitro; then, the Ultra-high-performance liquid chromatography and Q-Exactive mass spectrometry (UHPLC-QE-MS) based on non-targeted metabolomics analysis of Traditional Chinese Medicine were performed for comparison of Gym with ethanol concentration of 95% (low extraction rate but high XOD inhibition rate) and 75% (high extraction rate but low XOD inhibition rate), respectively; finally, the protective effect of ethanolic extract of Gym on zebrafish with Hyperuricemia (referred to as HUA zebrafish) was explored.An optimized extraction process for urate-lowering active substances from Gym was firstly been carried out based on the xanthine oxidase (XOD) inhibition model We found that the inhibition rate of Gym extract with 95% ethanol concentration on XOD was 84.02%, and the extraction rate was 4.32%. Interestingly, when the other conditions were the same, the XOD inhibition rate of the Gym extract with 75% ethanol concentration was 76.84%, and the extraction rate was 14.68%. A total of 539 metabolites were identified, among them, 162 different metabolites were screened, of which 123 were up-regulated and 39 were down-regulated. Besides significantly reducing the contents of UA, BUN, CRE, ROS, MDA, and XOD activity in HUA zebrafish by Gym and acutely reduce the activity of SOD.Along with the flavonoids, polyphenols, alkaloids, terpenoids, and phenylpropanoids, the ethanolic extract of Gym may be related to reduce the UA level of Gym. Gymnadenia conopsea (L.) R.Br. , it grows widely in temperate and subtropical regions of Asia as well as throughout Europe. Traditionally, its tuber is applied to Traditional Chinese Medicine, Tibetan Medicine, Mongol Medicine, and other medicines to treat numerous symptoms of health problems in China  is caused by a disorder of uric acid (UA) metabolism or abnormal purine metabolism. It occurs when the serum UA level is higher than 420 μmol/L –28. The  methods . Chen etC on XOD . Park etC on XOD . Le et aC on XOD  found thC on XOD . ScienceTherefore, whether the ethanolic extract of Gym can reduce the production of UA by inhibiting XOD activity, thereby alleviate HUA. In this work, we will focus on this hypothesis. To this end, specific ethanolic extractions based on different concentrations of Gym should be carried out first to ensure the representativeness of the samples. First, we optimized the extraction process of UA-reducing active substances of Gym and analyzed the metabolomics of the ethanolic extract of Gym by UHPLC-Q-Exactive mass spectrometry (UHPLC-QE-MS). Finally, the protective effect of the ethanolic extract from Gym on HUA zebrafish was explored. Consequently, it can well explore the possible metabolic effect of ethanolic extract of Gym on the reduction of UA and its protective effect on HUA zebrafish.Together with bioinformatics, UHPLC-QE-MS was applied for chemical identification, profiling, and quantitation. UHPLC-QE-MS used a high pressure of more than 1,000 bars and worked at a tolerable flow of up to 5 mL/min. These properties allowed it to analyze slighter particles less than 2.2 mm. Besides the traditional HPLC method, UHPLC-QE-MS detection was an ideal estimating method for pharmaceutical formulations or drugs in bulk and analyzing the metabolites in biological fluids . The tecXanthine sodium salt , bovine milk XOD (CAS:9002-17-9), APL (CAS:315-30-6), and formic acid  were provided by Sigma Chemical Co., LTD . Methanol (CAS:67-56-1) and acetonitrile (CAS:75-05-8)  were offered by CNW Technologies . The 2-chloro-L-phenylalanine  was bought from Shanghai Hengbai Biotechnology Co., LTD . Ethanol absolute was purchased from Chengdu Jinshan Chemical Reagent Co., LTD . 10 × PBS buffer (pH 7.4) was offered by Shanghai Thermo Fisher . The gym was obtained from the Tibet specialty warehouse naicang supermarket .in vitro was established and further optimized  test design was carried out according to The 4-factors and 3-levels orthogonal LThe samples of Gym with 95 and 75% ethanol concentration were extracted and analyzed by LC/MS. Briefly, the Waters UPLC BEH C18 column was used on Agilent 1,290 UPLC system  for LC-MS/MS analysis –64. The n = 20/group), respectively, and the volume of each well was 2 mL. Each culture dish was marked with the group and treatment. Except for the blank group, the other 10 groups were treated with PO and XSS, pre-incubated at 28°C for 1 h, and the corresponding dosages of APL and Gym extraction aqueous solution were added. All treatment groups were cultured in a 28°C incubator for 24 h. The contents of UA, Blood Urea Nitrogen (BUN), Creatinine (CRE), Malondialdehyde (MDA), and the enzyme activity of XOD and Superoxide Dismutase (SOD) were measured according to the kit’s instructions. The content of Reactive Oxygen Species (ROS) in 48 h was also determined by model reference in the meantime . In all statistical comparisons, P < 0.05 was considered significant, and P < 0.01 was extremely significant. Besides, principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) was conducted to process the metabolomics analysis using SIMCA software . This was of importance when Variable Importance in the Projection (VIP) > 1 while P-value < 0.05.The GraphPad Prism software  was used to conduct one-way ANOVA analysis through a The extraction rate of Gym ethanolic extract was greatly affected by different liquid–solid ratios, and they were positively correlated . HoweverSubstances have different solubility and content in different ethanol concentrations , 77. TheUltrasonic power is one of the main factors affecting the extraction rate of effective components. If the power is not selected properly, the components contained in the sample will be extracted incompletely –80. Within vitro  of QC and 95 and 75% ethanolic extract of Gym under positive and negative ions (We want to know why the XOD inhibition rate of 95% ethanolic extract of Gym was significantly higher than that of 75% ethanolic extract ive ions . The expR2Y (cum) = , Q2 (cum) = ] were detected in our experiment. Moreover, the OPLS-DA model was not overfitted according to the cross-validation and response permutation test (RPT)  . Therefot-test is required to examine the characteristics of metabolites among groups. And the P-value is usually used to evaluate the likelihood of differences between groups he world . About 2he world . The reshe world .Based on the HUA zebrafish model, the UA-lowering effect of the Gym ethanolic extracts of two different ethanol degree groups was evaluated. Correspondingly, the results are presented in Additionally, we also measured the ROS content of 2 dpf zebrafish embryos stripped of the membrane after 1 h of administration and the contents of MDA and SOD in zebrafish at 5 dpf. It can be seen that the levels of ROS and MDA were substantially increased in the model group. Nevertheless, they could be significantly reduced by APL and different dosages of two ethanolic extracts of Gym groups . HoweverThe abnormal activity of liver xanthine oxidase or the decreased excretion of UA will lead to an increase in UA accumulation, which is the main cause of HUA –96. Therin vitro, the extraction process of UA-reducing active substances in ethanolic extract of the Gym was optimized. When the ethanol concentration was 95%, the ratio of solid to liquid was 1:40, the ultrasonic power was 500 W, the extract time was 80 min, the ultrasonic temperature was 70°C, the inhibition rate of ethanolic extracts of Gym on XOD was 84.02%, and the extraction rate was 4.32%. Interestingly, we found that when the ethanol concentration was 75%, and other conditions were the same, the inhibition rate of Gym on XOD was 76.84% and the extraction rate was 14.68%. In other words, the extraction rate of 75% ethanol is much higher than that of 95% ethanol, and the inhibition rate of 95% ethanolic extract on XOD is significantly higher than that of 75% ethanolic extract.In our study, through the XOD inhibition model To clarify this phenomenon, we further analyzed the metabonomics of two ethanolic extracts of Gym based on UHPLC-QE-MS. A total of 539 metabolites were detected, including 129 terpenoids, 69 alkaloids, 57 polyphenols, 48 flavonoids, 3 quinones, 36 phenylpropanoid, 24 organic acids, 16 amino acids, 13 sugar and alcohol, 42 lipids and aromatics, 12 coumarins and lignans, 11 carboxylic acid and organic oxygen, and 79 miscellaneous. Of the 162 different metabolites screened, 123 were upregulated, such as Scopoletin, Artemisinin, Methyl jasmonate, Benzoic acid, Racanisodamine, P-hydroxybenzaldehyde Phenylpropanolamine, Cholic acid, Meperidine, Ligustilide, and 3,4,5-trihydroxystyrene. Meanwhile, 39 metabolites showed a downward trend, such as Paracetamol, L-proline, Heptadecanoic acid, Paregoricin E, Cinnamaldehyde, Cinnamamide, Nortriptyline, and D-gluconic acid. These differential metabolites were classified into 13 species, including 29 polyphenols, 28 flavonoids, 17 phenylpropanoids, 16 alkaloids, 13 terpenoids, 11 lipids, 7 amino acid derivatives, 6 benzenes and derivatives, 4 organic and aromatics, 5 organic acids and derivatives, 4 coumarins and derivatives, 3 carbohydrates, and 19 miscellaneous. There are also various metabolite pathways highlighted in the metabolite pathway enrichment analysis. They are the aminoacyl-tRNA biosynthesis pathway, phenylalanine, tyrosine and tryptophan biosynthesis pathway, glucosinolate biosynthesis pathway, and phenylalanine metabolism pathway. The study of Yao et al.  also shoIn vivo, UA is proven to be the final product of human purine metabolism. Adenosine is oxidized to inosine by adenosine deaminase, which is further broken down to hypoxanthine, then converted to xanthine by XO, and xanthine is then converted to UA by XO. The whole process is catalyzed by xanthine oxidase (P < 0.05), which was consistent with a study of Zhao et al. , while the content of SOD (the antioxidant enzyme) also decreased stress. This suggests that the ethanolic extract of Gym can attenuate the oxidative stress induced by hyper UA by promoting the activity of antioxidant enzymes and inhibiting lipid peroxidation.The available evidence reveals that the formation of UA is catalyzed by XOD, which is a purported source of ROS, and the changes in XOD and UA are biomarkers of oxidative stress. Additionally, XOD may provide an important source of nitric oxide (NO) that quenches the injurious effects caused by ROS –110. Li ed to XO . Wu et aed to XO . The resIn conclusion, the ethanolic extracts of Gym can significantly improve the HUA of zebrafish. On the one hand, it may be related to its reduction of UA production. On the other hand, it may be related to its significant reduction of BUN and CRE levels, reversing the damage of PO to the kidney, thereby promoting UA excretion, and it may also be related to its antioxidant protection by reducing the production of ROS and MDA, reducing the oxidative stress response.The ethanolic extract of Gym can effectively improve the HUA of zebrafish. It can reduce the UA, BUN, CRE content, and XOD level of the HUA zebrafish, suggesting that the ethanolic extract of Gym causes a certain inhibitory effect on the XOD activity of the HUA zebrafish, and then affects the entire UA production pathway, thereby reducing the production of UA. In addition, the ethanolic extract of Gym can substantially reduce the levels of ROS and MDA of HUA zebrafish, and could reduce the SOD level, indicating that the ethanolic extract of Gym can alleviate the oxidative stress reaction caused by hyper UA by improving the activity of related antioxidant enzymes and inhibiting lipid peroxidation. The results of metabonomics showed that its effect on reducing UA might be related to its flavonoids, polyphenols, alkaloids, terpenoids, and phenylpropanoids. And aminoacyl-tRNA biosynthesis pathway, phenylalanine, tyrosine, and tryptophan biosynthesis pathway; glucosinolate biosynthesis pathway, phenylalanine, and other metabolic pathways deserve further study.The original contributions presented in this study are included in the article/This animal study was reviewed and approved by Guangdong Laboratory Animal Monitoring Institute.TC: optimization of extraction process experiment, animal experiment, and article writing. DP: preliminary drug screening and experimental optimization. WZ: project advancement. SM and CY: literature search. XY: data processing of orthogonal experiment. JL: literature compilation. All authors contributed to the article and approved the submitted version.


The signaling inputs that function to integrate biochemical and mechanical cues from the extracellular environment to alter the wound-repair outcome to a fibrotic response remain poorly understood. Here, using a clinically relevant post-cataract surgery wound healing/fibrosis model, we investigated the role of Phosphoinositide-3-kinase (PI3K) class I isoforms as potential signaling integrators to promote the proliferation, emergence and persistence of collagen I-producing alpha smooth muscle actin (αSMA+) myofibroblasts that cause organ fibrosis. Using PI3K isoform specific small molecule inhibitors, our studies revealed a requisite role for PI3K p110α in signaling the CD44+ mesenchymal leader cell population that we previously identified as resident immune cells to produce and organize a fibronectin-EDA rich provisional matrix and transition to collagen I-producing αSMA+ myofibroblasts. While the PI3K effector Akt was alone insufficient to regulate myofibroblast differentiation, our studies revealed a role for Rac, another potential PI3K effector, in this process. Our studies further uncovered a critical role for PI3K p110α in signaling the proliferation of CD44+ leader cells, which is important to the emergence and expansion of myofibroblasts. Thus, these studies identify activation of PI3K p110α as a critical signaling input following wounding to the development and progression of fibrotic disease. Matrix proteins, growth factors and cytokines, all important factors in the reparative response to injury, are also linked to the induction of fibrosis ,4,5,6,7.The initial appearance of myofibroblasts following wounding is often to promote the reparative process. These myofibroblasts do not persist after the wound is healed. Instead, they become senescent, and are typically programmed to undergo apoptotic cell death . HoweverIn studies with ex vivo mock cataract surgery explant cultures that mimic the in vivo fibrotic outcome of cataract surgery known as PCO, our studies show that the wound edge is rapidly populated by lens resident immune cells that guide the migration of the wounded lens epithelium ,30. The Much remains to understand about the signaling pathways that promote the persistence of myofibroblasts following a wounding and the development of fibrosis. Our studies show a specific link between activation of the PI3K p110α subunit and the appearance and proliferation of myofibroblasts following a wounding.Ex vivo post-cataract surgery chicken explant lenses were prepared as previously described ,32. PleaFor immunolabeling studies, ex vivo post-cataract surgery explant cultures were fixed in 4% formaldehyde for 15 min. Subsequently, cultures were permeabilized in 0.25% Triton-X-100 for 10 min, blocked in 5% goat serum for 30 min, incubated with primary antibodies for 30 min to 1hr followed by incubation with fluorescent-conjugated secondary antibodies . For immunolabeling we used the following primary antibodies αSMA , Fibronectin EDA , active RAC  and a vimentin polyclonal . The following primary antibodies were obtained from the Developmental Studies Hybridoma Bank (DSHB) created by the NICHD of the NIH : pro-collagen I antibody (SP1.D8) deposited to the DSHB by Furthmayr, H. (DSHB Hybridoma Product SP1.D8) and CD44 antibody (1D10) deposited to the DSHB by Halfter, W.M. (DSHB Hybridoma Product 1D10). Explant cultures were counterstained with DAPI  to identify nuclei.Proliferation was analyzed using an EdU click it assay kit . These post-cataract surgery cultures then were immunolabeled with CD44 to identify resident immune cells at the leading edge and nuclei were co-stained with DAPI. Labeled cultures were examined by confocal microscopy.Apoptosis was analyzed in the post-cataract surgery explant cultures using a TUNEL assay kit  that detects double strand DNA breaks, which were co-stained with DAPI and examined by confocal microscopy.Western Blot: This immunoblot analysis was performed as described previously . BrieflySimple Western system by Protein Simple : Post-cataract surgery wounded explant cultures were extracted in RIPA buffer. Samples were prepared for WES immunoblot as previously described . BrieflyConfocal imaging was performed using either the Zeiss 510 or Zeiss 800 confocal microscopes. Z-stacks were collected with a 40× objective, each optical plane at either 0.33 µm or 0.49 µm, as indicated. For z-stacks collected with a 20× objective each optical plan was at 0.6 µm. Images are shown as either single optical planes from the z-stack or as projected images, as indicated.p-value of 0.05 or less was considered statistically significant. Quantification of protein expression for all Western blots was performed using ProteinSimple analysis software. For WES analysis, signal intensity is obtained using COMPASS software to obtain signal intensity by quantifying the area under the curve of the acquired graph. The % of CD44+ cells that are EdU+ were calculated over 3 independent experiments.Data are shown as the mean +/− SEM and analyzed with unpaired Student’s t-test or by ANOVA using Prism . PI3K is a major downstream effector of multiple receptor inputs linked to driving fibrosis, such as TGFβ and integrins ,39,40,41Our findings show that class I PI3Ks serve as crucial intracellular signaling coordinators of the fibrotic response to lens cataract surgery wounding. The catalytic subunit of class I PI3K is found in four different isoforms, of which p110α, p110β, and p110γ have been shown to be expressed in the lens , and eacAs Akt is a major downstream effector of PI3K, we examined whether its activation was responsible for inducing the resident immune cells at the leading wound edge to transition to an αSMA+ myofibroblast fate. Ex vivo post-cataract surgery wounded lens explant cultures were treated with vehicle or the Akt specific inhibitor MK-2206 at both 1 µM or 3 µM from D1 through D3 post-injury. Both inhibitor concentrations effectively suppressed activation of Akt A. While Another major downstream effector of PI3K is the small GTPase Rac ,46, a weTo determine whether Rac is involved in mediating the transition of the leader cells to αSMA+ myofibroblast phenotype, ex vivo post cataract surgery lens wounded explant cultures were treated from D1 through D3 with vehicle (DMSO) or with the Rac 1 inhibitors NSC23766 or EHop-016. Western blot analysis showed that the inhibition of Rac activation using either NSC23766 or EHop-016 resulted in a significant suppression of αSMA expression G. ConsisOur findings above revealed roles for both p110α and Rac signaling in promoting the transition of mesenchymal leader cells to αSMA+ myofibroblasts . To deteRac is known to act both upstream and downstream of PI3K signaling . TherefoPI3K signaling is a critical regulator of both cell proliferation and survival ,52, bothThere is a critical need to elucidate the pathobiological signaling mechanisms that drive the proliferation, differentiation, and persistence of the αSMA+/collagen I-producing myofibroblasts that are responsible for the progression of fibrotic disease. In this study we sought to understand the function of class I PI3K p110 isoforms activated in response to cataract surgery wounding in the integration of signaling inputs from the environment that induce myofibroblast emergence and persistence, leading to the development of fibrosis. This study reveals a requisite role for PI3K, specifically the PI3K p110α isoform, in signaling the mesenchymal resident immune leader cells that rapidly populate the wound edge to produce and organize a FN-EDA provisional matrix and their subsequent transition to collagen I producing αSMA+ myofibroblasts. Importantly, our studies show that injury-induced signaling through the PI3K p110α isoform is required for the proliferation, expansion, and persistent activation of this leader cell population, and the progression of fibrosis..Our findings reveal that activation of the PI3K p110α isoform impacts many different aspects of the fibrotic process, each of which could be regulated through the engagement of distinct upstream receptor inputs and/or downstream signaling effectors. Our studies also demonstrate that other PI3K isoforms expressed by the lens, PI3K p110β or p110γ  were aloAt the membrane, PI3K serves as a sensor of changes that occur in the extracellular environment, with the ability to integrate biochemical and mechanical cues to signal alteration in cell function . MembranIt is well established that PI3K is a signaling intermediate in the induction of collagen synthesis ,62,63,64Importantly, we have linked PI3K p110α induced proliferation of activated lens resident immune cells at the wound edge to their transition to a myofibroblast phenotype and to the expansion of the myofibroblast population. Typically, cell proliferation is a prerequisite for differentiating cells to acquire a new phenotype ,69. TherOverall, our studies show an important link between activation of both PI3K p110α and Rac and the production of a pro-fibrotic matrix environment following a wounding, as well as the emergence and proliferation of myofibroblasts. These findings support the therapeutic targeting of PI3K p110α to treat lens fibrotic disease.


The hedgehog (HH) family of secreted proteins, and the signaling pathway they trigger, play crucial roles in cell proliferation, differentiation and migration, not only in development, but also in many types of cancers. It is thus not surprising that it has been the focus of intense investigation by biochemists, geneticists and clinical doctors. Four decades of studies have revealed many levels of regulation, from the production, modification, release and degradation of the ligands, to the roles of various receptors, coreceptors, downstream kinases, phosphatases, and effector transcription factors. Strikingly, HH signaling in vertebrates has been associated with the primary cilium, a unique cell surface organelle whose critical roles in development and diseases were only appreciated in the past two decades. The importance of this pathway is further exemplified by the FDA approval of HH/SMOOTHENED (SMO)-blockers as a stand-alone anti-cancer therapy. To date, novel regulators and effectors of HH signaling continue to be identified, adding to the complexity of the molecular interaction network.The seven articles in this Research Topic reflect on the recent advances and provide perspectives in the molecular mechanisms of HH pathway regulation, the crucial roles of HH signaling in development and pathology, as well as new strategies for treating HH signaling related diseases including cancers.Nguyen et al. provide an overview on ciliary lipid compositions and their impact on HH signaling throughout evolution . In addition to ciliary membrane-specific phosphoinositides, this signaling pathway is stunningly dependent on many other lipids and sterols, functionally affecting HH ligand transport as well as intracellular signal transduction—a truly unique feature of the HH pathway.Given its significance for correct embryonic development and its involvement in the patterning of various tissues and organs, HH signaling is subject to tight regulation. One such layer of regulation is imposed by distinct lipid compositions specifying selected membrane regions as found in the primary cilium of vertebrates. Drosophila and mammals. Jia and Jiang outline the regulation of SMO trafficking by these post-translational modifications and hereby compare mechanistic concepts which arose in flies and humans (Jia and Jiang).One example for the intricate relationship between lipids and signaling is the SMO protein, which can be covalently and non-covalently associated with sterols. However, SMO is also subject to ubiquitination and SUMOylation, both of which constitute evolutionarily conserved control mechanisms of HH signaling in Zhou and Jiang provide a review on phosphorylation as a major mechanism of GLI regulation (Zhou and Jiang). They introduce our current understanding of cAMP-dependent protein kinase (PKA), Casein kinase 1 (CK1) and Glycogen synthase kinase 3 (GSK3)-mediated phosphorylation in the processing and activation of GLI proteins in vertebrates and invertebrates. They also discuss the latest research findings, some of which are still being debated, e.g., on the roles and regulations of ciliary cAMP in HH signaling. They also discuss the discovery of new GLI regulators, such as the Fused family of kinases, Polo-like kinase 1 (PLK1) and CK2, which catalyze the phosphorylation of GLI proteins, and contribute to their functional regulation.The Glioma associated oncoprotein (GLI) family of transcription factors are the effectors of the HH pathway, converting the HH signal into transcriptional responses leading to cellular outputs. He et al. report a study carried out in Drosophila, in which they generated a transgenic toolbox of in vivo miRNA sensors for the core components of the HH pathway, and performed a genome-wide in vivo miRNA overexpression screen in the developing wing imaginal disc . Out of 12 miRNAs identified through this screening, they report that miR-10 and miR-958 attenuate HH signaling by directly targeting fu and smo, respectively. The functional relevance of the newly discovered miRNAs was validated by overexpression experiments that altered HH’s role in cell fate specification during testis development.Micro RNA (miRNA) regulation of HH signaling has been an intense ResearchTopic over the past decade; however, the preferred genetic loss of function approach often yields dismally little useful information, possibly due to functional redundancy and contextual dependence of this class of regulators as a whole or each miRNA in particular. Here, Wang et al. review the roles of HH signaling in cerebellar development and cancer . They review classical genetic loss-of-function studies establishing SHH-mediated interaction between Purkinje cells (PC) and granule cell progenitors (GCP), two major neuronal cell types of the cerebellum. They focus on GLI targets that directly regulate cell proliferation, including Cyclin D (CycD), Cyclin-dependent kinase 4 (Cdk4), MYCN, and their roles in the pathogenesis of medulloblastoma (MB), a pediatric cerebellar tumor. Finally, they discuss the use of inhibitors for SMO, GLI and downstream CDKs as therapeutic strategies for MBs. Some of the inhibitors are already approved or in clinical trials for MB treatment, attesting to the clinical importance of understanding the roles of HH signaling in cerebellar development and MB etiology.HH signaling plays essential roles in the development of many organs, and when abnormally activated, leads to malignancies. Tesanovic et al. present another example of the importance for well-orchestrated HH signaling in normal developmental processes, and how its dysregulation leads to malignancies such as acute myeloid leukemia (AML), one of the most lethal types of blood-borne cancers . HH signaling is critical for the persistence of therapy-refractory leukemic stem cell pools and a SMO inhibitor has recently been approved for clinical use in AML, significantly expanding the application repertoire of this class of compounds beyond that of basal cell carcinoma.Ma et al. studied the roles of parathyroid hormone (PTH) in fracture healing using a mouse model . They show that endogenous PTH is required for efficient fracture healing, and a shortened exogenous PTH fragment (PTH 1–34) can rescue the fracture healing defects in Pth mutant mice. Importantly, they find that PTH 1–34 promotes cAMP response element binding protein (CREB) phosphorylation and subsequent activation of Ihh expression. This work suggests that PTH 1–34 can be an effective treatment for fracture, and points to an important role of IHH signaling as the underlying mechanism.Another HH family member, Indian hedgehog (IHH), plays a critical role in chondrogenesis in development. In summary, the seven articles, authored by experts at the forefront of the HH signaling field, cover a wide range of research areas, from basic mechanisms of HH signal transduction and pathway regulation, to the connection between HH signaling and various diseases. Finally, current and potential treatments of various cancers as well as of bone fracture repair are presented. Hence this Research Topic provides a timely update on the latest progress in the study of HH signaling in development and disease, and points out pressing questions and future directions.


The biological clock as an idea and idiom is closely associated with the emergence and impact of new reproductive technologies. Fertility treatments have contributed to the popular and medical discourse of the biological clock by making it a signature image of declining and resurgent fertility . The incBy definition, the idea of the biological clock promotes a futuristic outlook: seeking to arrest reproductive decline, or anticipate its outcomes , is the This Symposium issue aims to highlight the tensions in these widespread imaginings around the biological clock as it is made, remade and unmade in the context of reproductive biomedicine. In this attempt, we are interested in looking at how technology facilitates particular notions of controlling the reproductive body, while simultaneously highlighting the risks of both technological assistance and embodied temporality. Taking into account several important strands of emerging research, we offer a critical engagement with the notion of the biological clock as it becomes enmeshed in the conceptualization of the body and time within assisted reproduction.Lucy Van de Wiel and Heather Jacobson’s contributions. Van de Wiel’s research on the ‘financialization of infertility’ explores how venture capitalists have begun to invest in proactive fertility insurance through ‘company-sponsored fertility benefits’. This new financialization of fertility risk promotes new engagements among women working in global corporations, which encourage ‘proactive’ management of one’s fertility while thus enforcing the need for fertility insurance to safeguard the fulfilment of reproductive hopes, plans and desires. At the same time, for the American surrogates in Heather Jacobson’s article, the amount of time spent in each IVF-surrogacy arrangement means a negotiation of other ‘life clocks’. The money spent on managing what Jacobson calls the ‘ART clock’ weighs heavily not only intended parents – who may have to pursue multiple IVF cycles until their surrogate becomes pregnant—but on surrogates as well. Here, time is money as surrogates must learn to navigate childcare and emotional investment in the intended parents' lives with the uncertainty of IVF cycles, as seen in repeated embryo transfers and hormone cycles, miscarriages and other exigencies.The ticking clock is more than a metaphor in both Nolwenn Bühler’s analysis of fertility preservation and reproductive technologies. Bühler embarks on an archaeology of cellular manipulation within reproductive science in the desire for longevity. In order to subvert reproductive decline, Bühler suggests that age and ageing become more complicated as well. At the same time, in Anindita Majumdar’s study, assisted reproduction becomes an important node of conceptualizing post-menopausal reproduction, especially in facilitating reproduction amongst those past their reproductive prime. In rural North India, assisted reproductive technology is part of the attempt to overturn the narrative of declining reproduction, by facilitating pregnancies amongst ageing women, while at the same time reinforcing socially debilitating practices of son preference in the name of intergenerational longevity.Eggs and oocytes become markers of ageing and decline in Together, our biological clock articles reveal the importance of continuing to engage critically with this ubiquitous, complicated and highly paradoxical idiom, which, having become an emblem of our time, is also a measure of its mixed messages about the future of fertility care, preservation and hope.The author reports no financial or commercial conflicts of interest.


Studies support that elevated concentrations of GHGs trigger similar signaling pathways to that observed in commonly studied abiotic stresses. In the process, NO plays a forefront role, in which the nitrogen metabolism is tightly related. Regardless of their beneficial roles in plants at a certain level of accumulation, high concentrations of CO2, CH4, and N2O-mediating stress in plants exacerbate the production of reactive oxygen (ROS) and nitrogen (RNS) species. This review assesses and discusses the current knowledge of NO signaling and its interaction with other signaling pathways, here focusing on the reported calcium (Ca2+) and hormonal signaling, under elevated GHGs along with the associated mechanisms underlying GHGs-induced stress in plants.Nitric oxide (NO), an ancient molecule with multiple roles in plants, has gained momentum and continues to govern plant biosciences-related research. NO, known to be involved in diverse physiological and biological processes, is a central molecule mediating cellular redox homeostasis under abiotic and biotic stresses. NO signaling interacts with various signaling networks to govern the adaptive response mechanism towards stress tolerance. Although diverging views question the role of plants in the current greenhouse gases (GHGs) budget, it is widely accepted that plants contribute, in one way or another, to the release of GHGs (carbon dioxide (CO Howeverytokinin . NO alsoytokinin  and auxiytokinin . In the ytokinin . Availabytokinin .2), methane (CH4), nitrous oxide (N2O), and ozone (O3) play important roles in plant physiology. CO2 is required for photosynthesis and is fixed to store energy in the form of carbohydrates  or nitrogen (RNS) species during abiotic or biotic stress events   . However2 , superoxide anion (−), singlet oxygen (1O2), and RNS , etc.). In the process, NO signaling has proven essential and plays a central role. NO interacts with other signaling pathways, and the results of this interaction confer beneficial outcomes for plants. Generally, ROS and RNS are produced by plants under normal conditions and are harmless at low concentrations. However, upon stress induction by either abiotic stimuli or living organisms, the production of ROS and RNS increases up to the point of causing oxidative or nitro-oxidative stress, which may result in oxidative damage and culminate in cell death. To alleviate the detrimental effects of ROS or RNS overproduction, plants activate antioxidant (enzymatic and non-enzymatic) systems and induce several stress-responsive genes as part of the adaptive response mechanisms toward stress tolerance. This review assesses the current knowledge of the regulatory role of NO in plants under elevated CO2, CH4, N2O, or O3. This work also highlights the crosstalk between NO signaling and the above-mentioned potent GHGs, as well as the causative effects of elevated CO2, CH4, N2O, or O3 on NO production and signaling events. Likewise, we discuss the possible interplay between CO2, CH4, or N2O-induced stress with NO and other stress signaling pathways in plants to maintain a balanced reduction-oxidation status.Studies show that high amounts of atmospheric CO2 , CH4  emitted globally through human activities  and NH4, with NO3 being the major form of nitrogen taken up by plants. The efficiency of nitrogen use by plants is mediated by several genes encoding nitrate reductase (NR) or belonging to five distinct high-, dual-, or low-affinity NO3 transporters protein families, including NRT1, NRT2, chloride channel (CLC), and slow anion channel-associated/slow anion channel-associated homologs (SLAC/SLAH) (3 assimilation pathway (2 as reported by 2 increases the use efficiency of organic nitrogen (NUE)  . In high pathway . NR undeen (NUE) . This cocontents .2 recorded differential patterns, including an increase , some amount of NO2 reduced from NO3 in the nitrogen metabolism is transported to the chloroplast. There, NO2 is converted to NH4 for further incorporation into amino acids, as part of the assimilation process. Another fraction of NO2 is converted to NO in the cytosol  is the major cellular reservoir of NO, and its accumulation is controlled by GSNO reductase . GSNOR catalyzes the reduction (irreversible reaction) of GSNO to glutathione disulfide  and ammonia (NH3)   . It was athione) . Accordi2, which in turn mediates the induction of flavonoid biosynthesis in tea (Comellia sinensis L.). The authors observed that SA, NO, and flavonoid contents increased in plants exposed to elevated CO2.Plant hormones are key players in plant growth and development, operating either antagonistically or in synergy with one another. Plant hormones are also well-known for their involvement in signaling events during abiotic or biotic stress. For instance, salicylic acid (SA) signaling regulates plant response to stress . Other s2 assimilation. Several studies support that the regulation of stomatal aperture size is achieved through a complex sensory and signaling network . It is widely known that plant guard cells are essential for photosynthesis and transpiration, and the stomata aperture is sensitive to environmental stimuli. Recent progress in stress signaling revealed that high levels of CO2-induced stomata closure suggest ROS such as hydrogen peroxide (H2O2), as key factors. ROS are major regulators of stomatal conductance in response to internal or external stress inducers  and NO-mediated signaling pathways is inhibited by ABA. The authors suggested that NO and Ca2+ are active components of ABA-induced stomatal closure. Similarly, 2-induced stomatal regulation.Stomata are epidermal pores through which gas exchange is regulated, including CO network . Stomatainducers . Pharmacoxidases . From an2-mediated stomatal closure via an ABA-independent pathway would involve the OST1/SnRK2 (OPEN STOMATA 1/SNF1-related protein kinases 2) kinases family. In addition, studies have suggested that the signaling pathway for elevated CO2-induced stomatal closure shares several elements with but does not overlap with (3 transporter protein families), ROS and NO production, enhanced Ca2+ level (2 would cause a reduction in photosynthetic physiology in plants. A study conducted by 2 levels (720 ppm) increased photosynthesis in sugarcane plants compared to those grown under ambient (370 ppm) CO2 level. This evidence was supported in several CO2-enrichment studies  signaling, phytohormone signaling, etc.  provides mechanical support to the cell and enables the cell to execute essential functions . The actng, etc. . Evidentng, etc. . As indiignaling . Stomataignaling  revealedignaling .S-nitrosation previously referred to as S-nitrosylation , including tyrosine nitration or sylation , thus afar level . Given tar level , includi2+ signaling  during episodes of elevated CO2, which led to enhanced lateral root development. The authors equally indicated that a CO2-mediated increase in NO production triggers the accumulation of cytosolic Ca2+ that acts as a co-factor for NO. In a converse approach, 2 signaling in guard cells and indicated that low–high CO2 transitions modulate the cytosolic Ca2+ transient patterns.Depicting the mechanisms underlying plants response to abiotic stress helps improve the understanding of genetic factors associated with stress tolerance in plants. Although investigating the role of each signaling molecule independently provides some useful information on their level of implication in the regulatory mechanisms during abiotic stress events, a genome-wide approach gives new insights on the possible interactions between signaling molecules and their impact at the whole plant level. Generally, signaling pathways do not operate solo. Rather, they are activated along with other signaling networks with which they may establish a certain level of interaction. This interaction may occur in a balanced way since it is a high-energy demanding process consisting of antagonistic or synergetic relationships. This may result in what could be referred to as signaling cascades, allowing the induction or suppression of downstream adaptive response mechanisms. Just like other stress-related conditions where NO plays an active role , a study4) was previously regarded as a physiologically inert gas but is now emerging as a possible signaling molecule in plants   Figure 2activity . CH4 exee stress . Similarreported . From an2) availability in plants. It is believed that NH4 and NO3 are the major sources of nitrogen for plants, with NO3 being the most abundant. O2 differentially influences GHGs emission patterns, acting on the activity of specific soil microorganisms, such as methanogens and methanotrophs. In addition, as indicated in previous paragraphs, the influential role of NO on nitrogen assimilation events positions this molecule at the core center of interest in various stress-related studies. It was reported that NO contributes to CH4-mediated induction of osmotic stress tolerance in mung bean (4-rich water via heme oxygenase/carbon monoxide and Ca2+ pathways.Studies aiming at elucidating the unexplored facets of NO in plants discuss the possible interplay between NO and oxygen (Oung bean . Cucumbe2O) is a potent GHG with a GWP much higher than CO2 and CH4. N2O is emitted by diffusion from soil or via plant transpiration  occurs during the photosystem I (PSI)-dependent photoreduction of the photosynthesis. This was similarly observed by 2O and NO are known as NOx, and react with volatile organic compounds and hydroxyl, resulting in organic NO3 and nitric acid (HNO3) formation , they undergo various physiological and biochemical changes. In the process, various signaling pathways are activated and act as messengers to trigger an adequate adaptive response mechanism towards stress tolerance, along with the induction or suppression of stress-related genes  in Arabidopsis, followed by O3-mediated induction of SA biosynthesis and signaling pathway genes, and ethylene accumulation. Interestingly, 3 reductase is responsible for O3-triggered NO generation and secondary metabolites in Ginkgo biloba plants. Moreover, useful information that would allow enhancing our understanding of the molecular mechanism underlying O3-mediating induction of various signaling pathways and ROS in plants is well summarized by 3-induced stress signaling in plants.Furthermore, a study revealed that dual application of NO and Onterplay . In the 3 layer in the stratosphere helps absorb UV-B radiation. However, in modern times, exposure to UV-B radiation increases gradually due to the thinning of the protective O3 layer caused by human activities , and observed the induction of an oligonucleosomal DNA fragmentation, which characterizes apoptotic-like changes in the nucleus, similar to that observed in human cells (Arabidopsis plants to UV-C radiation induced programmed cell death (PCD), the latter being suggested to be mediated by Caspase-like activities, which in turn would modulate DNA fragmentation. In the process, Caspase inhibitors suppress DNA fragmentation and cell death, where two AtDAD1 and AtDAD2, earlier identified as homologs of Defender against Apoptotic Death-1, are proposed to suppress the onset of DNA fragmentation while supporting an involvement of the endoplasmic reticulum in this form of the plant PCD pathway.Moreover, land plants rely on light for photosynthesis, and leaves serve as light-capturing organs. However, as an undesired corollary, they also absorb damaging light, especially ultraviolet B (UV-B) radiation (280–320 nm waveband of the solar irradiation) , a kind tivities . Plants tivities . Reportstivities . In addian cells . Reportsan cells ; howeverArabidopsis involves the inositol-3-phosphate synthase (IPS1), a key enzyme for biosynthesis of myo-inositol and its derivatives. Other studies showed that the role of ROS are important elements of a wide signaling web that composes with other signaling mediators to activate cellular protective mechanisms in response to UV-B radiation. For instance, Arabidopsis plants treated with the NO scavenger PTIO  and/or with L-NAME (NG-monomethyl-L-arginine), an NOS (NO synthase) inhibitor, suppressed the induction of chalcone synthase (CHS)-encoding gene; therefore suggesting that UV-B-triggered the expression of CHS would require NO  resulting in a downstream increase in flavonoid and anthocyanin contents capable of absorbing UV-B radiation and scavenging ROS.On the other hand, UV radiation triggers the production of free radicals, including ROS and NO , as wellquire NO . One sho2, CH4, N2O, O3 or UV light. This review also gives insights into the interaction of NO with other signaling pathways and highlights the involvement of the nitrogen metabolism and the flavonoid pathway genes in NO-mediated stress signaling during elevated GHGs. These GHGs present both beneficial outcomes and detrimental effects to the plant, depending on their level of accumulation in the atmosphere. Therefore, taking advantage of the current understanding of NO in its diverse dynamic roles in various stressful conditions, NO can be regarded as a game changer in the efforts towards the mitigation of the impact of climate change, while providing a novel path to enhancing the resilience of agricultural and food production systems. In the context of climate change, depicting the molecular basis of NO-mediated plants’ response to elevated CO2, CH4, N2O, or O3 would help elucidate the regulatory mechanisms underlying plants’ response to these GHGs. In addition, exploring the interplay between NO-mediated nutrient acquisition and use efficiency, and the associated defense mechanisms against GHGs would provide more insights towards a paradigm shift for a more resilient agriculture.Nitric oxide (NO) is at the core center of interest in many biosciences and environmental-related research programs, mainly due to its involvement in almost all physiological and biological processes in plants. Since the initial report of NO in plants about four decades ago, our understanding of the molecular mechanism underlying NO biosynthesis and signaling in plants increased during the last two decades, and the physiology of NO has been widely investigated under both normal and stressful conditions. In this work, we assessed and presented the current knowledge on the regulatory network involving NO and its derived molecules in the adaptive response mechanism of plants towards elevated CONK and B-GM designed the work, conducted the literature mining, and drafted the paper, S-ML, YK, DL, and G-ML contributed to the literature review and manuscript preparation. B-WY and J-HL conceived, revised and edited the manuscript and supervised the project. All authors contributed to the article and approved the submitted version.This work was supported by the Cooperative Research Program for Agriculture, Science and Technology Development (Project No. PJ01707301) and “2019KoRAA Long-Term Training Program” of the Rural Development Administration, Korea, and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant No.2020R1I1A3073247).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The last decade has seen adoptive cell therapies, such as chimeric antigen receptor T-cell (CART-cell) therapy, gain FDA approval against specific cancers. Additionally, there are numerous clinical trials ongoing investigating additional designs and targets. Nevertheless, despite the excitement and promising potential of CART-cell therapy, response rates to therapy vary greatly between studies, patients, and cancers. There remains an unmet need to develop computational frameworks that more accurately predict CART-cell function and clinical efficacy. Here we present a coarse-grained model simulated with logical rules that demonstrates the evolution of signaling signatures following the interaction between CART-cells and tumor cells and allows for  I.Chimeric antigen receptor T-cell (CART-cell) therapy is an adoptive cell therapy in which a patient’s T-cells are genetically modified to express chimeric antigen receptor (CAR) constructs. The CAR is a fusion of protein domains consisting of extracellular antigen targeting and intracellular signaling and costimulatory domains. The targeting domains are antibody-derived single chain variable fragments (scFv). The signaling domain is T-cell receptor (TCR)-derived CD3ζ with CD28 or 4-1BB typically used as costimulatory domains [Therapy dynamics involve interconnected activation, signaling, inhibitive, cytotoxic pathways including crosstalk and feedback between and within effector and target cells. Differences in any one variable  can lead to non-intuitive effects on CART function and impact therapy dynamics. It is not feasible to test clinically or experimentally the vast range of perturbations which may influence therapy responses, but these perturbations can offer significant insight on how CART products may be improved.Computational modeling of molecular signaling networks is a powerful tool for understanding the dynamics of complex systems. These models can generate testable hypotheses and predict different possible non-intuitive outcomes of the system. Creating clinically valuable differential equations-based models requires gathering highly specific and robust datasets which are currently experimentally cost-prohibitive. In contrast, logic-based approaches allow the creation of networks based on understanding of biological functions  of individual components to study the behavior of the system under different perturbations.While literature on CART-cell computational modeling is sparse, logic models of T-cell activation, cytokine and inhibitory signaling have been previously investigated –4. MotivII.A.A minimal model  of the spdcd1 gene expression (PDCD1ge) [The CAR considered for the signaling network consists of CD3ζ activating and CD28 costimulatory domains. Ligation of the CAR by tumor antigens (TA) through an active immune synapse (IS) leads to LCK facilitated activation of CD28, CD3ζ, and ZAP70 . ActivatPDCD1ge) . In the PDCD1ge) .Cytotoxicity in T-cells is mediated through the release of perforin/granzyme B (PERGRZB) and surface molecules such as Fas ligand (FASL) from cytotoxic granules (CYTOGRAN). This process requires actin cytoskeleton reorganization supported by WASP and cdc42 (WASPCDC42), polarizing microtubules organizing centers toward the IS (PKCθ), and granule secretion  . FASL siPDCD1ge [IL2 signals through IL2R leading to the activation of kinases JAK1 and JAK3 (JAK13), which together activate PI3K-AKT-MTORC1, and MAPK pathways. IL2R, and JAK13 activate TF STAT5. STAT5 and NFAT promote PDCD1ge . For simInhibitory signaling is mediated by engagement of ligands PDL1 or PDL2 (PDL1L2) with PD1 and CD80 or CD86 (CD8086) with CTLA4 through an active IS. Engagement of these receptors activates the phosphatase SHP2 which counteracts the kinase activity of LCK, ZAP70, PKCθ, JAK1, JAK3, PI3K, and ERK. In the absence of SHP2, PD1 is also able to signal through SHP1 which has similar activity . PD1 is Activated LCK and CD3ζ initiate negative feedback of LCK to dampen activation signaling through SHP1 . ActivatThe complied network is a coarse-grained curated construction of the biological network which can be modularly extended to include additional factors and pathways contributing to network regulation not considered here.B.a and pd, which respectively set the probability that a given update function will update during a given iteration based on its current state (either ON or OFF). This is done to account for inherent randomness in the system where not all process (reactions or sets of reactions) can be assumed to occur during each iteration. For similar approaches to dynamic modeling of Boolean and logic based networks, see [The rules governing the nodes and edges of the constructed model and corresponding literature references are detailed in rks, see , and [19a = 0.5. In the absence of continuous activating signals, activated signaling proteins and molecules in cells are degraded through a variety of processes . To account for this each update function is assigned a pd = 0.05. High probabilities of node activation alongside low probabilities of node degradation are assumed to match kinetic data on T-cell activation responses showing bimodal all-or-none responses and degradation processes slowly attenuating these signals thereafter [Each update function considers edges (connections between nodes) which participate in processes to turn on a node  or inhibit these processes . Default probabilities for each update function are set to pereafter .Three readout nodes are included which capture the trajectories of signaling signatures . The CARThe simulation is cast as an enclosed loop with the intention to model the evolution of signaling signatures through the continuous activation of CART-cells by interactions with tumor cells. This is intended to be representative of the initial evolution of signaling events in CART-cell therapy.A random order asynchronous update scheme is used for all nodes. This assumes all nodes are updated rapidly but may update in varying order and leads to stochastic (as opposed to deterministic) trajectories which is better representative of the heterogenous biological system that consists of multiple CART-cell types  and different states of differentiation. The model does not explicitly model or differentiate between these states.github.com/MCWComputationalBiologyLab/Shah_2023_IEEE.All results are generated from averaging 10,000 simulations. Initial states of input nodes are randomized when activated. Input nodes are held at a constant 0 when inactive. Initial states of all other nodes set to 0 except LCK which is assumed to be active at basal states . The modIII.We first consider an example of the dynamic behavior of the network following engagement of the CAR . The selcfunc), CINHIB (pcihib), and TAPOP (ptapop) signaling signatures. Addition of IL2 leads to increased functional signaling and comparable cytotoxic signaling  and additional signaling through modeled pathways  would both impact the presented model dynamics and need to be further investigated.in vitro assays of CART and tumor cell interactions to define necessary parameters, this approach would facilitate analysis of CART and tumor cell population dynamics in continuous time.The presented approach is limited to demonstrating how signaling dynamics in CART cells may sequentially evolve. Transitioning the model to a semi-quantitative approach that links signal transduction events with proliferative and apoptotic events on appropriate time scales is a reasonable next step. Supported with data from single cell


Fertility preservation is an increasingly important topic in adolescent and young adult cancer survivorship, yet treatments remain under-utilized, possibly due to lack of awareness and understanding. The internet is widely used by adolescents and young adults and has been proposed to fill knowledge gaps and advance high-quality, more equitable care. As a first step, this study analyzed the quality of current fertility preservation resources online and identified opportunities for improvement.We conducted a systematic analysis of 500 websites to assess the quality, readability, and desirability of website features, and the inclusion of clinically relevant topics.The majority of the 68 eligible websites were low quality, written at college reading levels, and included few features that younger patients find desirable. Websites mentioned more common fertility preservation treatments than promising experimental treatments, and could be improved with cost information, socioemotional impacts, and other equity-related fertility topics.Currently, the majority of fertility preservation websites are about, but not for, adolescent and young adult patients. High-quality educational websites are needed that address outcomes that matter to teens and young adults, with a priority on solutions that prioritize equity.Adolescent and young adult survivors have limited access to high-quality fertility preservation websites that are designed for their needs. There is a need for the development of fertility preservation websites that are clinically comprehensive, written at appropriate reading levels, inclusive, and desirable. We include specific recommendations that future researchers can use to develop websites that could better address AYA populations and improve the fertility preservation decision making process. Every year, more than 85,000 adolescents or young adults  are diagnosed with cancer in the United States . The AmeAlthough AYA’s participation in treatment decisions is essential , provideAYA knowledge and empowerment related to fertility preservation decision making is critical and could potentially be aided through the use of relevant, accessible, accurate and trustworthy online resources. Providing high-quality patient education resources online can improve access to trusted information between visits. Online resources are widely used among AYA patients ,23, withBuilding on past research examining internet-based fertility preservation information for adult cancer patients ,29, thisIn collaboration with clinicians, decision scientists, health literacy experts, and a clinical librarian, we followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. To mimic AYA patients’ and caregivers’ internet search behavior, we used multiple patient-friendly search phrases in the two most-used search engines: Google and Yahoo –32. We dBefore searching each term, reviewers cleared their cache, cookies, and search and usage history to depersonalize the search and reduce bias. We reviewed the first 50 results generated on each search engine for each phrase. After obtaining 50 results for each search phrase, we cross-referenced the results to remove any duplicate websites. We then excluded websites that were not intended to provide patient education for the general public, such as academic publications, resources for medical professionals, treatment guidelines, and information on clinical trials. In addition, we excluded advertisements, websites not written in English, news articles from popular media sources  or about academic articles or treatment guidelines, blogs with personal narratives written in the first or third person with fertility preservation, social media posts, podcasts, presentations, PDFs or documents, and content “tags” used to filter posts on larger websites. We excluded PDFs, documents, and presentation because we felt that they could not be evaluated against interactive websites and would be unfairly penalized for lacking features like that are not possible to include in these forms of online resources.We also excluded websites that did not include key terms relevant to AYAs and fertility preservation. Sites were considered relevant to AYAs if they referred to patients between the ages of 12–25 years old or contained the words “child/children”, ““adolescent,” “young adult” or its abbreviation YA, “teen,” “pediatric,” “girl” or “boy” in the site title or the text of the website. We only considered the terms “child/children,” “girl,” and “boy” relevant if they referred to the patient potentially undergoing fertility preservation, not the patient’s ability to have a child. We also considered sites relevant if they included discussions of puberty or menarche, or words related to fertility or fertility preservation .All eligible websites were recorded in an internal data extraction sheet using REDCap ,34 hosteTo evaluate each website, we used a combination of validated measures and team-created evaluation tools to characterize the website’s quality, readability, desirability, and inclusion of topics recommended by National Comprehensive Cancer Network (NCCN) practice guidelines for AYA oncology patients.To measure the quality of websites on fertility preservation for AYA patients, we used DISCERN, a validated measure of quality for online health information . DISCERNNext, we used the Simple Measure of Gobbledygook  readability formula due to its ability to measure reading levels below the 12th grade reading level. To minimize human error, we used the automatic Uniform Resource Locator (hereafter referred to as URL) scoring feature. This online readability calculator automatically excludes headers, coding, and sidebar text and extracts only the relevant text from the websites to determine readability scores. Coders reviewed the automatically generated scores by examining the online calculator’s extracted text of every website for any extraneous errors . If the text that the calculator scored contained errors, a coder copied and pasted text from a “reader view” of the web page (which only shows the main text with no headers or sidebars) into the manual text scoring function of the online readability calculator. Then the team compared the manual score of the text to the original online calculator score with errors, and recorded the manual score if there was a difference greater or equal to 0.5 . For ease of analysis, we labeled scores above the 12th grade as College Level.To assess the desirability of the websites, we used Schiffman et al.’s desirability measure . SchiffmTo record websites’ inclusion of topics recommended by practice guidelines for patient-provider discussions on fertility preservation, we created a checklist of topics based on the NCCN Clinical Practice Guidelines in Oncology Adolescent and Young Adult (AYA) Oncology Version 2.2022 . Each suFor a table of all of the measures we used for this research, please see Appendix 1.Our search strategies yielded 500 sites for evaluation. After excluding 227 duplicate websites, we assessed 273 websites for eligibility. Following eligibility screening, we determined 68 websites to be eligible for review  or cancer-related non-profits . Other websites were NCI Designated Comprehensive Cancer Centers , fertility centers and sperm banks , independent  patient-facing websites or publications , academic institutions , and practitioner or research-based societies and institutions .On average, these websites appeared as the 28th search result. Thirty-one websites (45.6%) were intended for AYA patients, 28 (41.2%) were intended for both patients and parents, and nine websites (13.2%) were solely aimed at parents or caregivers of AYA patients. Less than half  of websites were solely dedicated to AYA patients.According to the DISCERN measure of quality, the websites scored as Very Poor , Poor , Fair , Good , Excellent  quality.In terms of DISCERN questions on sites’ content, 57 websites (83.8%) scored low  to the question, “Does it refer to areas of uncertainty?”, because they contained little information on uncertainty related to the experimental nature of certain treatment choices, unclear success rates for fertility preservation procedures in terms of successful collection of biological samples or a successful future attempt at pregnancy, or unknown benefits and risks of various procedures. For the question, “Is it clear what sources of information were used to compile the publication (other than the author or producer)?, the majority of sites scored low , meaning they had few or no references to sources other than the author or producer. In response to the question, “Is it balanced and unbiased?”, nearly three quarters of sites  scored low because they did not clearly indicate whether they were written from a personal or objective point of view, there was no evidence that a range of sources were used to inform the content, and there was no evidence of external review.All websites were at or above a 9th grade reading level, and the average reading level for all websites was 12th grade. About half  were at a college reading level (grades 13–14). Less than 10%  were at a 9th grade reading level, 10.3% (n = 7) were at a 10th grade reading level, 10.3% (n = 7) at an 11th grade reading level, and 25% (n = 17) at a 12th grade reading level.The websites included an average of two out of the 21 AYA-generated desirable features. About a third of websites had one feature  and  had no features. The most common feature included was facts about fertility preservation, with almost all websites containing that information . Almost half  of websites included links to other fertility preservation sites or resources. Less than 20% of websites  included survivor stories, had a question and answer section , or contained animations or videos .Of the 68 eligible sites, 13.2% (n = 9) were written solely for male patients, 29.4% (n = 20) were written for female patients, and 57.4% (n = 39) included both male and female content. Of the 48 websites with content for male patients, 89.5% (n = 43) mentioned sperm banking, 16.7% (n = 8) mentioned cyclic guanosine monophosphate-specific phosphodiesterase type 5 (PDE5) inhibitors or electro-ejaculation for patients with erection or ejaculation issues, and 14.6% (n = 7) mentioned testicular sperm extraction (TESE). Of the 59 sites with content for female patients, 88.1% (n = 52) mentioned oocyte or embryo cryopreservation, 59.3% (n = 35) mentioned ovarian tissue cryopreservation, 28.8% (n = 17) mentioned menstrual suppression through medications like GnRH agonists, and 39% (n = 23) mentioned oophorexy or ovarian transposition.Related to socioemotional topics recommended by NCCN, 39.7% (n = 27) of the 68 websites discussed costs related to fertility preservation, and few  mentioned psychological or emotional considerations of fertility preservation. Notably, only 5.9% (n = 4) addressed additional complications for fertility preservation for transgender patients.Overall, websites dedicated to fertility preservation for AYA patients were of lower quality and written at a high reading level, with few features that AYA patients find desirable and few topics recommended by NCCN practice guidelines. To address gaps in knowledge and foster engagement in shared decision making, high quality, accessible, and inclusive fertility preservation resources are needed that are attractive and engaging for AYA populations.Only 46% of websites were solely dedicated to AYA patients. While discussion of fertility preservation undoubtedly involves both AYA patients and caregivers, there is also a need for patient-centered communication designed specifically for the needs of AYA patients, especially given that they are the patients that may undergo the treatments and will live most directly with the impacts of such treatments. Since our analysis revealed that many websites were not solely for AYA patients, and that websites were generally written at a high reading level with few features AYAs might find desirable, it appears that the sites in this search were functionally about, not designed for AYA patients. While most websites had facts about fertility preservation, few included desirable elements for adolescents and young adults, which may make fertility preservation information appear uninteresting or irrelevant. Furthermore, most sites would appear on the second or third page of search results and may be overlooked by AYA patients who typically consider the first or second search results the most trustworthy .Low DISCERN scores indicate that much of the online information for AYA patients may have significant quality limitations that could exacerbate knowledge gaps . Many weThe high reading levels (average 12th grade) of these websites underscore the inaccessibility of online content on fertility preservation, for adults and for AYAs. The average reading level for adults is 8th grade ,42, and The results of this study also align with the extant literature showing that the majority of fertility preservation websites largely focus on common fertility preservation treatments like egg or embryo freezing and sperm banking and mostly focus only on the procedure. This gap observed in the results of our review suggests a lack information on promising emerging treatments, socioeconomic issues, psychological or emotional experiences, or gender identity. Notably, few websites discussed PDE5 inhibitors or electro-ejaculation, potentially overlooking patients that are peri-pubertal or that have physical or psychological difficulties with sample production . In the As an initial step in a focused program of research, several limitations should be considered when interpreting the results. Our search terms and inclusion criteria focused on specifically identifying public education websites for AYA cancer patients considering fertility preservation; however, AYAs may choose to view clinical guidelines, online brochures, or other resources or websites that include information in narrative form. Similarly, focusing on general terms such as “teen” and “adolescent,” may have missed sites aimed at older AYA patients. However, these criteria add strength to the search for materials that AYA patients would likely encounter as our team approached our search using patient-friendly search terms and publically available records..In terms of methods, the NCCN guidelines were also created to inform clinical encounters, not online media. Notably, we did not measure how many websites discussed testicular cryopreservation since the NCCN considers this treatment experimental at the time of our review. The NCCNotably, it was not within the scope of this review to assess whether patients or survivors were engaged in the development of currently-available websites. A key tenet of high-value health care is ensuring care is meaningful, accessible, and equitable by learning from patients about the full spectrum of outcomes that matter to each, and all, patients . FurtherStrengths of our approach include that it balanced evidence-based systematic evaluation of multiple clinically- and AYA-relevant topics and features with AYA-derived criteria and a real-world focus on publicly-available websites used by patients and families. Results can inform and guide ongoing and future work designing high-quality online resources to address the gaps in AYA fertility preservation knowledge.To address these gaps in health education for adolescent and young adult cancer patients, research is needed to develop and evaluate high-quality patient education websites tailored to young adults’ cognitive and communication needs. Higher quality content can be achieved through clear citations, unbiased writing, addressing a broad range of fertility preservation treatment options, and by including the financial, psychological, and emotional impacts of fertility care. Care should be taken to test and ensure appropriate reading levels, using clear definitions for complex medical terms. Additionally, incorporating desirable features such as videos or survivor narratives could increase engagement, and specifying terms related to sex and gender could improve inclusivity. Importantly, high-value health care approaches such as engaging diverse AYAs in multidisciplinary design teams should be used to ensure fertility preservation education materials focus on the health and psychological outcomes that matter to each and all adolescents and young adults. Finally, search engine optimization approaches can be used to ensure high-quality medical websites appear at the top of patients’ search results. For specific opportunities to consider incorporating these recommendations into AYA fertility preservation educational websites, see While online patient education materials show much promise for improving younger patients’ awareness and education, there currently exists several significant gaps in the quality, comprehensiveness, readability, desirability, and inclusiveness of publicly-available fertility preservation websites for adolescents and young adults. Clinicians and researchers have a timely opportunity to engage AYAs to co-design high-quality, innovative patient education websites that foster improved patient knowledge and ability to engage in shared decision making about their oncofertility care.


Broomcorn and foxtail millet were the most important crops in northern China during the Neolithic period. Although the significance of broomcorn millet in human subsistence exceeded that of foxtail millet during the early Neolithic, this pattern was reversed by the end of Neolithic period. However, the process underlying this shift remains unclear. The recent excavation of the Gedachuan (GDC) in Zhangjiachuan county has revealed an abundance of relics including millet crop remains from relatively continuous strata of the Yangshao and Qijia cultures, and therefore provides a unique opportunity to examine how and when foxtail millet replaced broomcorn millet as the dominant crop in the western Loess Plateau during the Neolithic period. In this study, we identify 1,738 and 2,686 broomcorn and foxtail millet remains, respectively, from 74 flotation samples, accounting for 38.81% and 59.98% of total plant remains, respectively. Compared with 23 direct dates of carbonized crop grains in GDC, we propose that the weight of foxtail millet in plant subsistence of GDC first exceeded that of broomcorn millet as early as ∼5,500 BP, filling an important gap in the archaeobotanical record from the western Loess Plateau. Further comparative analysis of multidisciplinary data suggests the shift in significance of these two millet crops during the late Neolithic may have been triggered by variations in human settlement intensity and climate change in the western Loess Plateau. The results of this study also suggest that the Banpo Phase of Yangshao Culture survived in the western Loess Plateau as late as ∼5,600 BP. Broomcorn and foxtail millet are among the oldest domesticated cereals in the world, first used in northern China around 10,000 BP . The culThe western Loess Plateau (WLP) has been identified as a key area for the development of rain-fed agriculture during the Neolithic Age . RemainsNearly continuous stratum spanning the early, mid and late phases of Yangshao Culture  and Qijia Culture  have been uncovered from the excavation of the GDC site, providing a rare opportunity to explore the trajectory of plant subsistence in the WLP during the mid-late Neolithic period. Using identified plant remains, radiocarbon dating of crop remains unearthed from the GDC site, and their comparison to published data in the WLP, we aim to study the history of the transformation in cropping patterns, especially the shift in crop dominance from broomcorn millet to foxtail millet during the Yangshao and Qijia periods. Moreover, the chronology of the Banpo Phase in the WLP is vague due to the limited radiocarbon dates and “old carbon effect” of radiocarbon dating for charcoals. The GDC site is currently the largest Banpo Phase site in the WLP, and therefore, we also examined the chronology of the Banpo Phase of Yangshao Culture in this work.The western Loess Plateau (WLP) is situated to the west of Liupan Mountain, East of Wushaoling, North of Qinling, and is mainly situated in the middle of Gansu province . The mai2,which preserved many rich cultural relics, including a complete Yangshao settlement. Neolithic relics include ash pits, houses, burials, kitchen pits, ditches, and kilns, and the relationship between the archeological strata is complex. According to the most recent excavations, the Neolithic settlement site stratums can be roughly divided into four periods: Banpo Phase of early Yangshao Culture, Miaodigou Phase of middle Yangshao Culture, late Yangshao Culture and Qijia Culture. Recently, the larger and well-preserved ring trench settlement in the Banpo Phase of Yangshao Culture was found , and finally were tested using a compact Accelerator Mass Spectrometer, Mini Carbon Dating System (MICADAS). These experiments were completed in the Radiocarbon Chronology Laboratory of Lanzhou University. Bayesian modeling of the 0 curves , using tsurement , to ensusurement . The Baysurement .Setaria italica), 1,738 broomcorn millet (Panicum miliaceum), 2 rice (Oryza sativa) and 2 wheat (Triticum aestivum) seeds (Avena sativa), Green foxtail (Setaria viridis), Milkvetch Root , Daghestan Sweetclover (Melilotus suaveolens), Threehorned Badstraw , and Garden Sorrel (Rumex acetosa).The plant remains unearthed from the GDC site are listed in m) seeds . A smallDuring the Banpo Phase, 138 foxtail millet seeds (13.28% of the plant assemblage), 895 broomcorn millet seeds (86.14% of the plant assemblage) and 2 wheat seeds (0.19% of the plant assemblage) were identified from 28 samples . During the Miaodigou Phase, 115 foxtail millet seeds (23.23% of the plant assemblage) and 373 broomcorn millet seeds (75.35% of the plant assemblage) were identified from 32 samples . Here, only one rice seed was identified. During the period of the Qijia Culture, 2,433 foxtail millet seeds (83.49% of the plant assemblage) and 470 broomcorn millet seeds (16.13% of the plant assemblage) were identified from 14 samples . As in the Miaodigou period, only one rice seed was identified during the period of the Qijia Culture. The ratio of the number of identified foxtail and broomcorn millet seeds increased from 0.15 (Banpo Phase) and 0.31 (Miaodigou Phase) to 5.18 (Qijia Culture), indicating an increasing trend of foxtail millet cultivation importance in subsistence agriculture over time. In addition, a few weed seeds were identified in the Banpo, Miaodigou and Qijia Culture periods, accounting for 0.38%, 1.21% and 0.34% of the identified plant seeds, respectively.14C dates of charred seeds and charcoal with 95.4% age range are displayed in The 23 new According to the archaeobotanical evidence identified from the GDC site , the sigth millennium BP (th millennium BP (The utilization of broomcorn millet in the nearby Dadiwan site can be traced back to the 8nnium BP , while nnnium BP , as wellnnium BP . Intensinnium BP , and thennium BP , which innium BP , while fnnium BP . The hydnnium BP . Only a nnium BP , suggestnnium BP .The proportion of foxtail millet in cereal count overtook that of broomcorn millet in the GDC site after ∼5,500 BP based on the direct dates of the millet remains in the GDC site . This paThe percentage of foxtail millet in plant remains in the GDC site was clearly higher than broomcorn millet after ∼5,500 BP , indicatBesides changes in the environment and survival pressure, the assemblage formation process might influence cropping patterns. For instance, grain ubiquity could be driven by crop processing with variations in post-harvest labor or the scale of processing could influence crop percentages e.g., . HoweverThe 11 radiocarbon dates of millet remains from the Banpo cultural layers in the GDC site ranged from 5,997 to 5,581 BP , which iThe new archaeobotanical and radiocarbon dating data presented in this study suggest that the shift in crop dominance from broomcorn millet to foxtail millet in the WLP occurred following ∼5,500 BP, with broomcorn millet becoming the primary subsistence crop during the period of ∼6,000-5,500 BP, and foxtail millet becoming the most important crop in the subsequent period of ∼5,500-4,000 BP. The relative importance of foxtail millet to broomcorn millet increased in a stepwise fashion in the WLP from the Banpo, MiaoDigou, late Yangshao phases into the period of Qijia Culture, which was likely triggered by the aggravation of human survival pressure in relation to human settlement intensity and climate change. Based on the analysis of new radiocarbon dates of millet remains from the Banpo cultural layers at the GDC site, we estimate the chronology of the Banpo Phase of Yangshao Culture in the WLP to ∼6,000-5,600 BP, although additional investigation is required in the future.The original contributions presented in this study are included in the article/GD, GC, and MM designed the study. YY, JW, GL, and JD conducted the field surveys and sample collection. YY, JW, HC, GL, and JD completed the experiments and data correction. YY, JW, HC, and MM analyzed the data and designed the figures. YY, JW, and MM wrote the manuscript in consultation with all authors. All authors discussed the results and commented on the manuscript.


Alternatively, promiscuity in signaling may also offer evolutionary advantages and enable different CLEs to work in combination to activate or switch off different RLK signaling pathways.CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) signaling through receptor-like kinases (RLKs) regulates developmental transitions and responses to biotic and abiotic inputs by communicating the physiological state of cells and tissues. CLE peptides have varying signaling ranges, which can be defined as the distance between the source, i.e., the cells or tissue that secrete the peptide, and their destination, i.e., cells or tissue where the RLKs that bind the peptide and/or respond are expressed. Case-by-case analysis substantiates that CLE signaling is predominantly autocrine or paracrine, and rarely endocrine. Furthermore, upon CLE reception, the ensuing signaling responses extend from cellular to tissue, organ and whole organism level as the downstream signal gets amplified. CLE-RLK-mediated effects on tissue proliferation and differentiation, or on subsequent primordia and organ development have been widely studied. However, studying how CLE-RLK regulates different stages of proliferation and differentiation at cellular level can offer additional insights into these processes. Notably, CLE-RLK signaling also mediates diverse non-developmental effects, which are less often observed; however, this could be due to biased experimental approaches. In general, CLEs and RLKs, owing to the sequence or structural similarity, are prone to promiscuous interactions at least under experimental conditions in which they are studied. Importantly, there are regulatory mechanisms that suppress CLE-RLK cross-talk  Thus, the ensuing cell to organ level effects vary as the downstream signal gets amplified and spreads through intercellular communication . This seProliferation is the increase in the number of cells with the same identity as a result of continuous symmetric cell divisions, whereas differentiation involves symmetric or asymmetric formative divisions resulting in cells with different identities. CLE-RLK signaling regulates proliferation and differentiation of meristems, the region that embodies pluripotent stem cells, which continuously supply proliferating cells in the plant body. Plant and non-plant CLE peptides also regulate primordium formation in the root during bacterial symbiosis and nematode infections. Such CLE-RLK mediated regulation of proliferation and differentiation have been reported largely at the organ and tissue levels . HoweverThere are several stages that lead to cell division and differentiation or proliferation at cellular level. When the need for proliferation and differentiation is evaluated, CLE peptides, in addition to other signaling factors, can be secreted to signal the decision. Then the stem cell that will undergo formative division stage 1— receivesMarchantia polymorpha encodes two CLE peptides: (1) MpCLE1, a TDIF-like peptide and (2) MpCLE2, a CLV3-like peptide, and two RLKs: MpTDIF RECEPTOR (TDR) and MpCLAVATA 1 (CLV1). The apical meristem, hosted by the apical notch, is located at the growing tip of the gametophyte body . The apical notch, apart from being responsible for the radial expansion of the thallus, also produces gametangiophores, which are the reproductive structures developed from the thalli  was resistant, implying that PpCLEs regulate proliferation through PpRPK2 (CLE peptides regulate both differentiation and proliferation in moss. h PpRPK2 . This inArabidopsis inflorescence meristem (IFM), two key CLE peptides, CLV3 and CLE40, are expressed in complementary regions: CLV3 is expressed in the central zone (CZ), where stem cells proliferate at a very slow rate. Stem cell daughters can shift to the peripheral zone (PZ) where the cells proliferate at a higher rate and start acquiring organ identities  in the PZ , thus indirectly promoting stem cell proliferation at the CZ  and its descendant cells in the PRM, and it has been shown to mediate both auto and paracrine regulatory signaling  and phloem pole pericycle (PPP) cells from differentiating into PSE likely through paracrine signaling. Cell files surrounding PSE have the ability to reprogram and acquire PSE identity. CLE45 from PSE cell file signals to the neighboring CC and PPP cell files expressing RPK2  to preveCLE25 is expressed in sieve elements of roots . This further led to defects in PSE differentiation during early seedling development. However, at later stages, these earlier PSE defects resulted in significant accumulation of starch in leaves due to compromised starch remobilization caused by defective phloem transport  have been shown to diffuse from phloem to procambium and promote proliferation of procambial/cambial cells by inducing WUSCHEL-related HOMEOBOX4 (WOX4) expression; in parallel, they also inhibit differentiation of cambial cells into xylem (PttPHLOEM INTERCALATED WITH XYLEM (PttPXY). PttCLE41 or PttPXY in hybrid poplar resulted in taller trees with increased stem diameter due to higher cambial cell proliferation and xylem cell formation  and suppresses these divisions by binding BAM1, which is expressed widely across the vascular zone including xylem precursors . BAM2 anvia the RLK HAESA-LIKE 1 (HSL1) expressed in MMCs, meristemoids and PCs likely in an autocrine manner (Meristemoid mother cells (MMC) are the stomatal lineage stem cells which give rise to the majority of the pavement cells (PC) in leaves by undergoing proliferative divisions and subsequent differentiation. An MMC first divides asymmetrically into a large stomata lineage ground cell (SLGC) and a smaller meristemoid cell, which in turn may undergo further rounds of asymmetric divisions forming more meristemoid cells. SLGC and meristemoid cells then differentiate into PCs and stomatal guard cells (GC), respectively . CLE9, ee manner . CLE9-HSe manner . Thus, Ce manner .Medicago truncatula, MtCLE12,-13 and -35 are expressed in the root in response to rhizobium and they mediate AON by signaling (by possibly binding) via the CLV1-like RLK SUPER NUMERIC NODULES (MtSUNN) expressed in the shoot. Thus, AON is likely the result of endocrine signaling from root to shoot , which is likely expressed in the leaf phloem  also resulted in hypernodulation. Moreover, LjKLV and LjHAR1 interact with each other , which suppresses excessive nodulation . In Medito shoot . Similarf phloem . Observaf phloem . Moreovedulation . Similarch other . This inMtCLE13 is already expressed in initial stages of nodule development. In the incipient and developing nodule primordium, MtCLE13 is expressed strongly in the dividing cortical cells and mildly in pericycle cells around the regions of bacterial infection. But in the later young, round nodule stage, it is expressed throughout. However, MtCLE12 is not expressed in the developing nodules, but in the later young, round nodule stage where, like MtCLE13, it is also expressed throughout the mature primordium  invade plant roots. The juvenile nematodes typically select a pericycle or procambial cell and inject several effector proteins including CLE peptide mimics into the cells through their stylet. The CLE mimics then reach the apoplast  and maniHsCLEB  length and LR numbers. It is hypothesized that CLE26 controls auxin transport, thus modulating the auxin distribution during LR initiation  fungi and rhizobia . Araya ear level  to influar level . Dong etondition . In summCLE6 is expressed at the bases of young rosette leaves and of floral organs, whereas CLE5 is expressed at the bases of young rosette leaves and of cauline leaves. CLE5 and 6 act downstream of leaf patterning factors in the leaf primordium to fine tune final leaf morphology. The mutants cle5 and cle5;cle6 show mild defects in leaf width, curvature and symmetry . It functions in parallel with CLAVATA signaling in mediating these processes. Several CLE peptides, including CLV3, and the RLKs BAM1, -2 and CLV1 are involved in this CLAVATA signaling pathway although how they interact to regulate flower primordium outgrowth and complete flower formation is not yet characterized. At higher temperatures, these processes are controlled by the transcriptional repressor EARLY FLOWERING 3 (ELF3) that bypasses CLV2/CRN signaling  and via other downstream regulators, including ABA. CLE9 overexpressing plants generally had smaller stomatal apertures and the leaves exhibited lower rate of water loss. Notably, unlike in the case of CLE25, BAMs are not the mediators of CLE9 signaling. Although the two CLEs might share the same downstream signaling components, the RLK that responds to CLE9 is not yet identified  and suppress strigolactone levels, thus reducing the rate of colonization  expressed in pollen. CLE45 signaling via SKM1 and SKM2, which are both expressed in pollen, have been shown to prolong pollen tube viability at high temperatures by maintaining its mitochondrial activity until it reaches the ovules  will provide a greater prospect of defining the ancestral role.2. The spatial dimension: Who initiates the CLE signal, who receives it and how?Understanding the spatial aspect of a signaling process is essential as it enables us to delineate the cells, tissue or organs that are in direct communication. For example, induction of stem cell proliferation in a given tissue could be a consequence of CLE peptide produced by the neighboring tissue  where rapid differentiation takes place.On the other hand, how the CLE signal reaches the destination is an essential part of the signaling process. The overall analysis of CLE signaling range show that auto- and paracrine CLE signaling is more common than endocrine. It is likely that establishing long-distance communication is challenging as it depends on several factors such as (1) controlling the expression level and concentration of the peptide which further depends on its stability , dilution during transport through xylem, duration, consistency and level of expression of the peptide (2) post-translational modification of the peptide (3) access to the transport system which includes expression pattern of both the peptide and RLKs, as they should be expressed in the vicinity of vascular tissue: CLE peptide for its extensive transport and RLK for conveniently binding the transported peptide molecules (4) conditions in the destination cells or tissue where further dilution or concentration of the peptide molecules can take place , 2016. HAn important attribute of the CLE-RLK interaction is promiscuity. Promiscuity is a broad term used in two main contexts :“Broader specificity”—This is when a protein can bind multiple ligands or interact with different proteins to mediate different effects. For example, “hub” proteins that contain disordered regions, which offer them conformational plasticity to serve as centers of signaling networks .“Physiologically irrelevant interactions”—This is when a protein interacts with a substrate in an artificial test scenario, which will not occur in the natural system. For example, a group of enzymes that are expressed as a part of an operon and function together will perform a particular process in the natural system. But one isolated enzyme, in a new environment, might perform an entirely new function owing to its active binding site that could bind another substrate .Promiscuous interactions among proteins in a given system could be influenced by: (a) Sequence and/or structural similarity among the possible binding partners, which decides the binding affinity to a specific interacting partner compared to other proteins , (b) Co-Promiscuity has been observed among CLEs and RLKs during their interactions—be it natural or forced, such as in experimental simulations. Here, we have illustrated some of the common observations with examples.Several CLE peptides (from the same or different species) causing the same effect—Examples: Exogenous application of CLE25, CLV3, CLE46, and TDIF upregulate NCED3 expression in leaves  CLV1 and BAM family of RLKs  and of its closest paralogue SlCLE9 in tomato SAM  and internal developmental signals trigger CLE/RLK expression, thus controlling their expression timing—CLE40 expression zone in the PZ in response to the proliferation/differentiation status of the SAM  controlled? Can it be achieved solely by regulating the CLE-RLK expression pattern and levels?What are the downstream signaling components of CLE-RLK and, by extension, the downstream effects that are conserved across clades? Understanding the differences in closely related non-angiosperm vascular plants will already be insightful.MN and RS conceived the idea. MN wrote the manuscript and made the figures. All authors contributed to the article and approved the submitted version.MN was supported by the DFG through a grant within the CRC1208 to RS.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.