Serum uric acid (UA) has been reported to be associated with ischemic stroke and inflammation. However, whether or not UA is related to the recurrence of ischemic stroke, and whether inflammation plays a role in the relationship between them remain inconclusive.We sought to explore the relationship between UA and the recurrence of ischemic stroke and to define the role of neutrophil-to-lymphocyte ratio (NLR) in the aforementioned relationship.A total of 8,995 patients were included in this study. Basic information and blood samples were collected, and whether or not each participant experienced ischemic stroke recurrence within 3 years was documented. Patients were stratified into three groups according to their UA level, as follows: ≤ 266, 267–339, and ≥ 340 μmol/L. COX regression and restricted cubic spline regression models were used to evaluate the clinical correlation between UA and ischemic stroke recurrence, mediation analysis and interaction and joint analysis were used to evaluate the role of NLR in the association of UA and ischemic stroke recurrence, and sensitivity and subgroup analyses were performed to test the robustness of the data.Ischemic stroke recurrence was related to male sex, older age, higher UA level, higher NLR, hypertension, diabetes, and cardiovascular disease. Following adjustment for potential confounders, a high level of UA (≥ 340 μmol/L) increased the risk of recurrence by 92.6% in patients with previous ischemic stroke. We also found that NLR affects the association between UA and the recurrence of ischemic stroke in older adults, suggesting that patients with high NLR and high UA levels are at greater risk for ischemic stroke recurrence.UA level is non-linearly associated with recurrence, and NLR has an additive interaction between UA and ischemic stroke recurrence. Stroke is the second most common cause of death and the third leading cause of disability, with an increasing incidence in developing countries . The GloSurvivors of stroke and transient ischemic attack are at risk for a recurrent stroke, which is often more severe and disabling than the index event. Recurrent strokes continue to account for 25–30% of all strokes and represent unsuccessful secondary prevention . With reUric acid (UA), the final product of purine degradation in humans, can induce oxidative stress and promote the development of atherosclerosis and inflammation . StudiesAn inflammatory response, the exact nature of which is suggested by blood inflammatory markers can occur in many diseases . InflammThis study therefore examine whether ischemic stroke recurrence is associated with the UA level and considered whether NLR plays a role in the relationship.We collected baseline data of patients who were hospitalized in the neurology department of the First Affiliated Hospital of Wenzhou Medical University from 2015 to 2017. Patients with acute cerebral infarction confirmed by magnetic resonance imaging during hospitalization were included. In contrast, those who met any the following criteria were excluded: (1) A history of malignant tumors, autoimmune diseases, gouty arthritis, kidney disease, or systemic infection; (2) recent use of drugs related to UA metabolism; and (3) missing data. Each patient was followed up with for 3 years after the baseline period, and the recurrence of cerebral infarction was recorded. Demographic data, including age, gender, hypertension, diabetes, atrial fibrillation, coronary heart disease, valvular disease, homocysteine (HCY) concentration, carotid atherosclerosis, intracranial arteriosclerosis, peripheral arteriosclerosis, and TOAST were collected. Indicators of inflammation, such as white blood cell count (WBC), absolute neutrophil count (ANC), absolute lymphocyte count (ALC), NLR and UA level were collected. Finally, magnetic resonance imaging data were collected. Patients were stratified into three groups according to UA level, as follows: ≤ 266, 267–339, and ≥ 340 μmol/L . All datThe serum levels of UA were measured using an automatic biochemical analyzer  in the laboratory of our hospital. Serum ALC and ANC values were also measured using an automatic blood cell analyzer  in the laboratory of our hospital. And the NLR value is the ratio of ANC to ALC.U-test, as appropriate.Data were described using mean and standard deviation values for normally distributed continuous variables and median and interquartile range values for non-normally distributed continuous variables. Frequencies with percentages were used to describe categorical variables. Baseline characteristics were compared using the chi-squared test, analysis of variance, or Mann-Whitney To examine the association between UA level and the incidence of ischemic stroke recurrence, Cox proportional hazards models were used to calculate hazard ratios with 95% confidence intervals (CI). Then, 3 models were estimated, as follows: in model 1, no potential confounders were adjusted; in model 2, age, gender, hypertension, diabetes, atrial fibrillation, coronary heart disease, HCY concentration, carotid atherosclerosis, and TOAST were adjusted; and in model 3, the variables in model 2 and NLR were adjusted. In addition, we explored the potential non-linear associations using 3-knotted  restricted cubic spline regression.To further explore the associations of UA and NLR with the recurrence of ischemic stroke, we calculated the mediation proportion by the mediator (NLR) for the association of UA and the recurrence of ischemic stroke. We also performed an interaction and joint analysis of UA level and NLR with ischemic stroke recurrence.To test the robustness and potential variations in different subgroups, we repeated the analyses stratified by age, gender, hypertension, diabetes, atrial fibrillation, coronary heart disease, and intracranial atherosclerosis.We conducted several sensitivity analyses. First, we chose ANC and ALC to replace NLR for repeated analysis. Second, WBC was chosen to replace NLR to analyze the association between inflammation indicators and UA level in ischemic stroke recurrence. Third, we excluded participants with cardiac disease or arteriosclerosis because both UA level and NLR may be affected by these diseases.N = 1,145; mean age, 66 years; 62.89% men) were more likely to be male and older; have a higher WBC, higher ANC, lower ALC, and higher UA level; and have hypertension, diabetes, atrial fibrillation, and atherosclerosis. Gender, hypertension, diabetes, atrial fibrillation, carotid atherosclerosis, HCY concentration, TOAST, WBC count, and NLR were significantly different  among the 3 groups grouped by UA level. Patients with high UA levels tended to have high WBCs and high NLRs.p for trend < 0.0001). As shown in In To explore whether NLR is a mediating factor in the relationship between the increase in UA level and ischemic stroke recurrence, a mediating effect analysis was performed. As seen in To further explore the relationship between NLR and UA in the recurrence of ischemic stroke, an interaction and joint analysis was performed. We also divided the NLR level into 3 categories. As seen in First, our research showed that UA is a risk factor for the recurrence of ischemic stroke in older adults. Second, UA is non-linearly associated with ischemic stroke recurrence. Third, there is an impact of NLR on the association between UA and ischemic stroke recurrence. In other words, there is a correlation between UA level and stroke recurrence, and NLR has an additive role in this relationship. Thus, we speculate that a high UA level may induce ischemic stroke recurrence through inflammatory mechanisms.Numerous studies have shown that UA level is associated with cerebrovascular disease, but whether it is a protective factor or worsens the risk of developing cerebrovascular disease remains controversial. This study found that there is an association between UA and the recurrence of ischemic stroke, and the possible mechanisms are: (1) Oxidation and anti-oxidation and (2) Inflammation.Many studies have shown that UA can promote oxidative stress . UA may However, studies have also shown that UA has anti-oxidative stress and neuroprotective effects. UA is a powerful antioxidant and a scavenger of singlet oxygen and radicals. It has antioxidant activity, can scavenge ROS and protects cells from oxidative stress . In addiFrom the above studies, we surmise that the role of UA in cerebrovascular disease remains controversial. We speculate that the reasons may be as follows: (1) the research population, research objectives, and research methods are inconsistent; (2) UA stratification methods are inconsistent; and (3) the sample sizes of previous studies were not enough. Therefore, our research sought to further explore the role of UA in the recurrence of ischemic stroke. We found that UA is a risk factor for the recurrence of ischemic stroke, and patients with high levels of UA may have a 92.6% increased risk of recurrence.UA also can induce inflammation. A large population-based sample of elderly and normal UA participants showed that UA has a significant positive correlation with inflammatory markers, such as C-reactive protein and interleukin-6 . StudiesThe NLR is a novel biomarker that can single out individuals at risk for vascular events . NLR hasIn recent years, the relationship between UA and NLR has become a research hot-spot. Use of NLR as an indicator of inflammation is very valuable, and it can be evaluated by a simple blood count analysis. One study showed that UA is positively correlated with NLR, and NLR may be a determinant of inflammation and atherosclerosis in CKD patients . A ChineFrom the above discussion and analysis, we drew a conclusion that both UA and NLR are closely related to ischemic stroke, with a circular relationship existing among all 3 items. However, few studies to date have looked at UA, NLR, and stroke recurrence together.Therefore, this study explored the correlation between NLR and UA in the recurrence of ischemic stroke, and we found that NLR has a mediating effect in the association between UA and ischemic stroke recurrence. Although the effect is not strong, there was an additive interaction between NLR and UA.Many previous studies have revealed the relationship between UA and the outcomes of ischemic stroke , but theThus, our research provides evidence that high concentrations of UA promote ischemic stroke recurrence in older adults. This study confirms the non-linear relationship between UA and the recurrence of ischemic stroke and suggests that this relationship may be affected by NLR. This study reminds clinicians to pay attention to UA level and inflammation in ischemic stroke patients, which may help to prevent the recurrence of ischemic stroke.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Institutional Review Board and Research Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University. The patients/participants provided their written informed consent to participate in this study.H-YZ collected, analyzed the data, interpreted the data, and drafted the manuscript. S-ZZ prepared the figures and analyzed the data. M-LZ collected the data and prepared the tables. YW prepared the figures and collected the data. Z-MP collected the data. H-RC helped to draft the manuscript. KZ obtained funding and helped to revise the manuscript. ZW designed the study, obtained funding, and helped to draft the manuscript. All authors saw and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Development of early follicles, especially the activation of primordial follicles, is strictly modulated by a network of signaling pathways. Recent advance in ovarian physiology has been allowed the development of several therapies to improve reproductive outcomes by manipulating early folliculogenesis. Among these, in vitro activation (IVA) has been recently developed to extend the possibility of achieving genetically related offspring for patients with premature ovarian insufficiency and ovarian dysfunction. This method was established based on basic science studies of the intraovarian signaling pathways: the phosphoinositide 3-kinase (PI3K)/Akt and the Hippo signaling pathways. These two pathways were found to play crucial roles in folliculogenesis from the primordial follicle to the early antral follicle. Following the results of rodent experiments, IVA was implemented in clinical practice. There have been multiple recorded live births and ongoing pregnancies. Further investigations are essential to confirm the efficacy and safety of IVA before used widely in clinics. This review aimed to summarize the published literature on IVA and provide future perspectives for its improvement. The majority of achievements in medical science practice are based on basic scientific research. Improved understanding of reproductive physiology has also allowed remarkable advances in artificial reproductive technologies, giving a higher chance to achieve parenthood for millions of infertile couples .As a consequence of global modernization, a higher number of advanced aged infertile women with diminished ovarian reserve (DOR) have been diagnosed in the last decades ,3. Due tDuring last decades, oocyte donation which cannot fulfill wishes of patients to give birth to genetically related offspring is often the option for patients with DOR and POI. To expand reproductive possibilities to DOR and POI patients, considerable efforts to investigate molecular mechanisms underlying folliculogenesis, leading to a variety of new approaches including follicle regeneration, rejuvenation, and activation .Among these, the activation of early follicles (EFs) including primordial, primary, secondary, and early antral follicles has been recently achieved. For developing the efficient activation system, many basic studies using the animal-model were conducted to identify the signaling pathways governing the activation of early follicles.IVA has been recently introduced and gradually implemented in clinical practice. This innovation was established from numerous animal experiments, including genetic manipulation studies illustrating the molecular mechanism of two involving signaling pathways in folliculogenesis. The first one is the PI3K/Akt/forkhead box O3 (FOXO3) pathway, which has a crucial role in the activation of primordial follicles (PFs) ,7,8,9. TTo provide a full overview of basic science related to IVA to the scientific community, this review summarized published works from basic scientific studies to clinical reports illustrating the molecular mechanisms and recorded outcomes of this treatment.Folliculogenesis is a complicated process which is generally divided into two stages: the first one is early stage from the PFs to the early antral follicles stage, and the latter one is the early antral follicles to ovulatory stage  22]. In. In22]. To form the growing follicles, a small number of PFs are cyclically and periodically recruited in the growing pool. The most specific feature of PFs is their long-term dormancy and survival for several decades, ensuring the longevity for female reproductive possibility. Thus, they represent a finite source of ovarian follicles and their recruitment is an indispensable step as it determines reproductive lifespan ,24. The Although the number of PFs decreases gradually through apoptosis and recruitment, the pool of PFs is not completely exhausted even at the age of menopause . AdditioThe first in vitro experiment of PFs was conducted on mice by culturing the whole intact ovaries in serum-containing medium. The spontaneous activation of a small number of PFs in the medulla region, which is similar to the first wave of physiologic PFs activation, was noted. Additionally, the addition of epidermal growth factor (EGF) could enhance the follicle recovery . In boviThese findings suggested that the PFs could be suppressed under the physiological environment, or there are in vitro factors stimulating the PF activation . AlthougThe importance of PI3K/Akt pathway in primordial follicle activation has been well-established ,7,8,9. TThis pathway is the cascade of signaling pathway with several components . When thPTEN in oocytes in mice resulted in an extensive and precocious activation of PFs [PTEN led to increased granulosa cell proliferation, and ovulatory efficacy as well as to decreases in follicle atresia [Pdk1 experienced an early depletion at dormant state, leading to POI phenotype in early adulthood [Foxo3a, a higher rate of primordial follicle activation was noted, leading to early infertility [TSC1 or TSC2 increases protein translation in oocytes, resulting in follicular overactivation [PTEN and TSC1, an enhancement of oocyte growth was observed. Additionally, the loss of mTORC1 signaling in oocytes stimulates the PI3K signaling cascade to maintain normal follicular growth [PTEN, FOXO3 was constantly phosphorylated and suppressed [PTEN and FOXO3 resulted in genetic equivalence to specific deletion of PTEN, indicating the dependent role FOXO3 from PI3K pathway in PFs’ activation [Using genetically modified mouse models, a number of studies illustrated the crucial roles of the PI3K/Akt pathway in the activation of PFs. The deletion of n of PFs ,50. In a atresia . The majdulthood . Similarertility ,53. The tivation ,55. Of nr growth ,56. Thestivation .Several experiments were carried out in animal models to clarify the roles of PI3K/Akt pathway in PFs’ activation. In three-day-aged mice, ovaries treated with 100 μM bpV(HOpic) (a bisperoxovanadium inhibitor of protein phosphotyrosine phosphatases with selectivity for PTEN) plus 740YP (a PI3K activator) for 48 h were dramatically larger with higher number of follicles at antral stages than control ovaries. Mature oocytes could be retrieved and fertilized after in vitro fertilization (IVF), resulting in live birth of healthy pups . AnotherIn swine ovarian tissue incubated with bpV(HOpic) (1 μM) for 48 h, the number of growing follicles was significantly higher compared to the control ovaries with a higher percentage of growing follicles. The degeneration rate in the incubated follicle population was approximately 30%, which was not different between bpV(HOpic) and control groups . In ovinSome authors raised concerns about the impact of manipulation of PI3K/Akt on the follicular function and survival , especiaIn human ovary, after incubation with 1 µM bpV(HOpic) for 24 h, the number of growing follicles in the bpV(HOpic)-exposed ovarian tissues increased remarkably. However, the secondary follicles isolated from the exposed ovaries showed a lower survival rate when comparing to the control group . Of noteThe second major pathway in IVA is the Hippo signaling pathway, which has been described as a fundamental pathway regulating mammalian organ size ,70,71. TThis pathway controls cell proliferation and organ growth via the kinase complexes consisting of the mammalian sterile-20 like serine/threonine kinase 1/2 (MST 1/2) and Salvador (SAV) as well as the large tumor suppressor 1/2 (LATS 1/2). These components suppress the Yes-associated protein (YAP) and transcriptional co-activator PDZ-binding motif (TAZ) through phosphorylation and cytoplasmic retention. The disruption of Hippo signaling pathway decreases phosphorylation of YAP, leading to increased nuclear YAP levels . Upon biIn the mammalian ovary, the fragmentation of ovarian cortex into small cubes was found to disrupt the Hippo signaling pathway by increasing the polymerization of G-actin into F-actin. Consequently, increases in YAP nuclear translocation stimulated the expression of CCN growth factors and BIRC apoptosis inhibitors, resulting eventually in secondary follicle growth ,11 Figu.Several studies described the mechanism and importance of this pathway on folliculogenesis. In ten-day-aged mice, after one hour of ovarian fragmentation, the ratios of F-actin to G-actin increased transiently and decreased pYAP to total YAP ratios were observed, showing disruption of the Hippo pathway. The ovarian cubes were then grafted under hosts’ kidney capsules, which is the typical site for grafting tissues because of its high vascularity . After fMany other findings indicate the importance of Hippo signaling pathway in EFs’ development. MST1/2, LATS1/2, YAP, and TAZ are expressed in different development stages of follicles in mouse as well as human ovaries ,81,82. RA recent study revealed that culturing the intact neonatal mice ovaries without Hippo or PI3K/Akt modulators could also increase the CCN2 expression and the KL staining. However, sectioned ovaries resulted in a remarkable increase in follicular counts compared to the intact ovaries and fresh ovaries, but with a higher rate of cell death . The metLATS1 null mice presented with a POI phenotype because of an early extensive follicular activation [YAP-knockout mice had a smaller size as well as the number of growing follicles, accompanied by a higher number of atretic follicles compared to control [CCN exhibited a decrease in the number of preantral follicles in mice [Genetic evidence also illustrated the crucial role of Hippo signaling pathway in follicle growth. tivation . YAP-kno control . In the  control . Deletio in mice . In huma in mice ,89,90.YAP1 knockout mice had a considerable increase in the number PFs accompanied by the decline of primary follicles [Some studies indicated that the Hippo signaling pathway works synergistically with PI3K/Akt activators to accelerate primordial follicle recruitment ,66,91. Bollicles . Notablyollicles . In humaollicles ,93. Consollicles .The development of EFs is a complicated process governed by a number of signaling pathways and components ,95,96. RThere are several factors, generated from the oocyte, GCs, stromal cells, neighboring follicles, and local blood vessels, known to enhance the EFs’ development. Among these factors, transforming growth factor beta 1 (TGF-β1) family is considered the most prevalent in EFs’ development. Belonging to this family, growth and differentiation factor 9 (GDF-9) and bone morphogenetic proteins (BMPs) have been intensively investigated. GDF-9 derived from oocytes was described as necessary for EFs for follicular growth from the early PFs to the preantral stage, especially at the transition of primary to secondary follicle stage ,99. SimiAlso, neurotrophins (NTs) and glial cell line-derived neurotrophic factor (GDNF) also take part in the assembly and activation of PFs. In murine, culturing newborn ovaries with NTs showed an extensive activation of PFs . In consFSH is generally described as one key gonadotropin modulating the latter stage of folliculogenesis. However, several data suggested that FSH has specific roles in EFs’ development. In mice, gonadotropin-deficiency resulted in a higher number PFs . In anotAMH, a member of the TGF-β family, is secreted by growing EFs. Unlike other members, AMH has been proposed to inhibit the PFs’ activation and EF’s development ,64. In r3) to disrupt Hippo signaling. The cubes are incubated with a PTEN inhibitor and/or a PI3K stimulator for 2 days followed by autotransplantation beneath the serosa of the Fallopian tubes [Better understandings of the pathways and agents modulating the EFs’ growth could allow the development of therapeutic approaches to improve female fertility competence . Currentan tubes   and morAdditionally, IVA could be a beneficial option for oncological patients and other female populations attempting FP as the IVA approach could maximize the number of oocytes in infertile women . Indeed,In this approach, the activation of PFs is the initial and most important step as majority of PFs exist in the resting state. By combining with other steps, a recent report achieved the first human oocyte with mature structure derived from an in vitro–grown unilaminar follicles for the PFs . In addiYAP1, MOB1A, MOB1B, and WWTR1) and PCOS [In addition, several studies revealed the association of the expression of several Hippo signaling pathway genes (and PCOS ,20,128. and PCOS . Based oand PCOS . Hippo gand PCOS  (Figure However, like other innovations in medical science, further studies are needed to improve this procedure as a successful and safe approach. Because IVA is ineffective in patients without residual follicles, developing a novel method to identify the presence of residual follicles before the IVA surgery can improve the outcome of IVA. Some studies have suggested the optical coherence tomography as imaging techniques for live ovarian imaging to localize the residual follicles ,131,132.Numerous researches provided scientific evidence for pathways underlying the IVA approach. IVA offered encouraging outcomes to the poor prognostic infertile women in the clinic. Individualization in treatment is a crucial aspect of clinical practice. In patients with DOR or early stage of POI, drug-free IVA is more beneficial compared to conventional IVA as it decreases the invasiveness of surgical approach and avoids the unfavorable effects of tissue culture on follicles. In terms of FP, IVA should be applied to only patients with low ovarian reserve and the requirement for motherhood achievement is urgent.


In summary, the in silico analysis showed that the bronchial epithelium of severe asthmatic patients showed a deranged balance between Wnt enhancer and Wnt inhibitors. A Th2-high phenotype is associated with upregulated Wnt-negative regulators, while inflammatory and neutrophilic severe asthmatics showed higher canonical Wnt signaling member enrichment. Most of these genes are regulators of healthy lung development early in life and, if disturbed, can make people susceptible to developing asthma early in life and prone to developing a severe phenotype. Most of the Wnt members are secreted, and their effect can be in an autocrine fashion on the bronchial epithelium, paracrine on nearby adjacent structural cells like fibroblasts and smooth muscles, or systemic in blood. Our results showed that canonical Wnt signaling is needed for the proper response of cells to proliferative stimuli, which puts cells under stress. Cells in response to this proliferative stress will activate the senescence mechanism, which is also dependent on Wnt signaling. Inhibition of Wnt signaling using FH535 inhibits both proliferation and senescence markers in bronchial fibroblasts compared to DMSO-treated cells. In fibroblasts from asthmatic patients, inhibition of Wnt signaling did not show that effect as the Wnt signaling is deranged besides other pathways that might be non-functional.Both canonical and non-canonical Wnt signaling pathway alterations have been documented in pulmonary disease pathogenesis and progression; therefore, they can be an attractive target for pharmaceutical management of severe asthma. Wnt/β-catenin signaling was shown to link early embryonic lung development impairment to later in life asthmatic airway remodeling. Here we explored the changes in Wnt signaling associated with asthma initiation and progression in epithelial and fibroblasts using a comprehensive approach based on  The hybrid name “WNT” (for Wingless-related integration site) stands for a group of genes belonging to the INT1 (WNT1)/wingless family . In the Both canonical and non-canonical Wnt signaling pathway alterations have been documented in pulmonary disease pathogenesis and progression; therefore, they can be an attractive target for pharmaceutical management of severe asthma . Wnt sigProgenitor cells that give rise to lung epithelium use CTNNB1 to promote lung progenitor gene signature and employ Fgf (fibroblast growth factor)/Kras -mediated promotion of the progenitors . It is lin silico analysis followed by in vitro validation.Here we explored the changes in Wnt signaling associated with asthma initiation and progression in epithelial and fibroblasts using a comprehensive approach based on N = 17) and healthy volunteers (N = 23) as previously described  filter using their gcRMA-normalized expression. CV was calculated as the mean/standard deviation of each gene across all samples. Probes with a CV value of at least 10% across the two groups examined were considered to be variant and thus selected. Since many genes are different between males and females and should be identified, only genes that do not show a significant variance between males and female samples were passed.1 and gene-set libraries downloaded from the Enrichr site2. Gene sets where the identified genes showed significant enrichment with nominal p-value < 0.05 were selected for further analysis with classical GSEA. A signal-to-noise metric was used to generate a rank-ordered gene list based on the enrichment score of each gene in the dataset. The enriched genes were filtered based on a cutoff score ≥ 0.25 as upregulated genes in asthma; those ≤ 0.25 as downregulated genes in asthma. For each gene-set collection or library, the genes that were upregulated or downregulated in asthmatic patients compared to healthy controls in each of the significant pathways were identified and grouped. Subsequently, DEG in each dataset was intersected with the DEG from the other sets, and shared genes were identified. Consequently, genes that were enriched in more than the median value of the number of enriched pathways for each gene were selected to be the DEGs in severe asthmatic bronchial epithelium compared to healthy control. The flowchart of filtering is shown in After processing, normalization, and filtering of the CEL files for each dataset, the normalized probe expressions were uploaded to GSEA software using the AbsGSEA option first. Over 100,000 gene sets were downloaded from Molecular Signatures Database v7.1 of the GSEA tooln = 3), and fibroblasts from asthmatic patients (n = 3). Primary fibroblasts were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM)  with 10% fetal bovine serum (FBS)  supplemented with 100 units/mL penicillin/streptomycin . The original study was approved by the MUHC Research Ethics Board (2003–1879), and the subjects had provided written informed consent as previously described  as previously described  overnight at 4°C. On the next day, cells were incubated with Alexa-Fluor 488-anti rabbit secondary antibody  and visualized using IX53 inverted immunofluorescent microscope .Cells were seeded until they reached 80–90% confluency. Then, cells were washed with PBS and serum-starved for 1–2 h. Subsequently, cells were treated with 100 ng/ml Wnt agonist , a WNT agonist that mimics the effects of WNT ligand or 1 μM Wnt antagonist “FH535” , a β-catenin/Tcf inhibitor.Fibroblast cells  were collected and washed with PBS, after which the proteins were extracted using the laemmli or RIPA lysis buffer . All protein extracts were quantified using Bradford Protein Assay Kit, according to the manufacturer’s instructions . 10 μg (for fibroblasts) of protein was separated on SDS-PAGE and transferred to a nitrocellulose membrane. Expressions of β-catenin and β-actin were assessed using rabbit anti-human CTNNB1  and mouse anti-human β-actin , respectively. Anti-rabbit and anti-mouse IgG HRP-linked antibodies  were used along with Clarity Western ECL Substrate  for chemiluminescent detection of protein bands. Western blot analysis of cell fractions from asthmatic bronchial fibroblast using Cell Fractionation Antibody Sampler Kit #11843 showing cytoplasmic (C.F), organellular/membrane (M.F), and nuclear/cytoskeletal localization (N.F.). Whole-cell lysates (WCL) represent total protein. The fractionation was done under two conditions, the cultivation with high glucose medium and low glucose medium.Two hundred and thirty-four genes were identified to be differentially expressed between severe asthmatic and healthy bronchial epithelia. Those 234 identified genes were further filtered to shorten the list into those that participate in more than 97 gene sets (above the median number of enrichments by the identified genes), indicating their essential role in development, progression, and response to therapy in asthmatic bronchial epithelium. Thirty-five genes fulfilled these criteria, as shown in 3, was used. All genes showed a strong interaction at the protein level, and most of them were downstream of CTNNB1, as shown in To assess the protein–protein interaction between the products of the identified genes, the Protein–Protein Association Networks tool, STRINGTwelve genes out of the 35 were downregulated in severe asthma . Interestingly, 5 out of those 12 genes are members of Wnt signaling .Looking at other Wnt signaling pathways, DEGs in severe asthmatics’ bronchial epithelium compared to healthy controls showed that three more genes  showed a statistically significant difference between the two groups. All identified Wnt genes were downregulated in severe asthmatic bronchial epithelia except FOSL1, as shown in We investigated whether Wnt signaling members are differentially expressed in the bronchial epithelium of Th2 high, Th2 low versus healthy controls. CTNNBIP1, AXIN1, and TLE1 were upregulated in Th2-high bronchial epithelium compared to healthy controls, while WIF1 and CCND1 were significantly downregulated in Th2-high bronchial epithelium compared to healthy controls, as shown in Aberrant Wnt signaling contributed to diverse human conditions. At the same time, its dynamics in asthma development need more attention, so we decided to dissect this pathway with a focus on its role in fibrosis and fibroblast biology. We profiled the Wnt pathway gene expression in fibroblasts taken from the lungs of healthy and non-severe asthmatic patients using RT2 Profiler PCR Arrays that profile 84 related genes simultaneously, as shown in Interestingly, members of canonical Wnt signaling  were significantly upregulated in fibroblasts from asthmatic patients. On the other hand, members of the non-canonical and negative regulators of the canonical pathways were downregulated in fibroblasts from asthmatic patients like WIF1. Comprehensive mapping for the Wnt signaling members’ expression in asthmatic versus healthy fibroblasts is shown in Our initial analysis showed that members of the non-canonical and negative regulators of the canonical pathways were downregulated in fibroblasts from asthmatic patients. One of the essential non-canonical Wnt pathways is the Wnt/Ca2+ signaling pathway, which is a crucial mediator in development and involved in NFκB mediated inflammatory response . The calWnt cell signaling uses CTNNB1 protein as an essential part of relaying the signal to target genes inside the nucleus. The next step was to assess the CTNNB1 as a protein and explore its dynamics in fibroblasts from asthmatic patients. Immunoblot showed that fibroblasts from asthmatic patients express less CTNNB1 compared to healthy fibroblasts, as shown in Since CTNNB1 activity is based on shuttling between cell membrane, cytoplasm, and nucleus, we examined its intracellular localization using immunofluorescent and subcellular fractionation using immunoblot. Immunofluorescence detection showed that the nuclear fraction of CTNNB1 is more in fibroblasts from asthmatic patients compared to healthy controls, and the fractions are increased in later passages, as shown in The note of increasing CTNNB1 with increased passage necessitates further explanation to understand the mechanisms of CTNNB1 synthesis in fibroblasts. We compared high-glucose with low-glucose culture media to examine the effect of glucose concentration in media on CTNNB1 protein shuttling. Interestingly, a low-glucose medium increased the shuttling of CTNNB1 to the nucleus and membrane compartment, indicating its role in fibroblasts’ response to the change in its environment, as shown in Glucose restriction was shown to extend fibroblasts’ lifespan while high glucose induced their premature senescence at any passages . So we e® AQueous One Solution Cell Proliferation Assay. As shown in To evaluate the effect of Wnt signaling activation or inhibition on the fibroblast’s viability/proliferation, we treated healthy fibroblasts and fibroblasts from asthmatic patients with Wnt agonists and inhibitors then measured their proliferation using CellTiter 96in silico analysis showed that the members of Wnt signaling are part of the core genes differentially expressed in severe asthmatic tissues compared to healthy controls. Extensive in vitro experiments confirmed and explained the critical role of the Wnt signaling pathway in asthma development.Our Twelve genes out of the total 35 were downregulated in severe asthma, and 5 out of those 12 genes are members of Wnt signaling . Also, three more genes  showed statistically significant downregulation in severe asthmatic bronchial epithelium except for FOSL1. CREBBP is a known histone acetyltransferase that regulates gene expression and interacts with β-catenin to maintain cell proliferation rather than differentiation . It was ITPR1 encodes an intracellular receptor for inositol 1,4,5-trisphosphate that mediates calcium release from the endoplasmic reticulum and plays a significant role in airway smooth muscles. Decreasing the activity of ITPR1 can make lung smooth muscle cells less reactive to contractile agonists to control asthma . AnotherWnt5a, a prototype of a non-canonical Wnt signaling axis with known cross talk with TGFβ1 during repair and remodeling, was elevated in the airway epithelium of Th17 asthma patients and steroid-resistant asthma . In asthVANGL Planar Cell Polarity Protein 2 (VANGL2) is involved in the control of early morphogenesis and planar cell polarity and is required for fetal lung development, precisely in normal lung branching morphogenesis . VANGL2 In human asthmatic airways, multiple Wnt ligand genes showed differential expression in signature between Th2-high and Th2-low asthmatics . Our resCTNNBIP1 encodes a protein that binds CTNNB1 to prevent CTNNB1 and TCF and control the downstream signaling and is a selectively enriched cluster of alveolar epithelium cells  . It is oAXIN1, when binding CTNNB1, acts as a negative regulator of the Wnt signaling pathway. Since β-catenin can block the overproduction of inflammatory cytokines in LPS-induced inflammatory responses, disturbance of the AXIN1 augments LPS effect . LPS-chain silico analysis showed that the bronchial epithelium of severe asthmatic patients possess a deranged balance between Wnt enhancer and Wnt inhibitors. The Th2-high phenotype is associated with upregulated Wnt-negative regulators, while inflammatory and neutrophilic severe asthmatics showed higher canonical Wnt signaling member enrichment. Most of these genes are the regulator of healthy lung development early in life and, if disturbed, can make people susceptible to develop asthma early in life and prone to develop severe phenotype. Most of Wnt members are secreted, and their effect can be in an autocrine fashion on the bronchial epithelium, paracrine on nearby adjacent structural cells like fibroblasts and smooth muscles, or systemic in blood.In summary, the Wnt signaling is essential in T cell development, maturation, and hematopoiesis regulation . T cell in silico analysis, as members of canonical Wnt signaling  were significantly upregulated in fibroblasts from asthmatic patients. On the other hand, members of non-canonical and the negative regulators of the canonical pathways were downregulated in fibroblasts from asthmatic patients like WIF1.In asthmatic airways exposed to frequent injury and repair, fibrosis eventually will develop by the critical player “myofibroblasts,” which activate three integrated pathways: TGFβ, Wnt, and YAP/TAZ signaling . We deciNon-severe fibroblasts from asthmatic patients express less CTNNB1 compared to healthy fibroblasts in early passages, but in later passages, fibroblasts from asthmatic patients start to produce more CTNNB1. Fibroblasts with decreased Wnt activation can undergo regeneration, whereas β-catenin activation can reduce regeneration in wounds ; neverthAt the wound edge, cytosolic calcium oscillations are induced in the fibroblasts . In humaInhibiting Wnt signaling with FH535 inhibited the growth of healthy fibroblasts with no effect on fibroblasts from asthmatic patients, indicating the deranged signaling pathways in fibroblasts from asthmatic patients. FH535 anti-proliferative effect is mediated by inhibiting the recruitment of β-catenin coactivators . The knoOur results showed that canonical Wnt signaling is needed for the proper response of cells to proliferative stimuli, which put cells under stress. Cells in response to this proliferative stress will activate senescence mechanisms, which depend on Wnt signaling too. Inhibition of Wnt signaling using FH535 inhibits both proliferation and senescence markers in bronchial fibroblasts compared to DMSO-treated cells. In fibroblasts from asthmatic patients, inhibition of Wnt signaling did not show that effect as Wnt signaling is deranged besides other pathways that might be non-functional. Further understanding of the factors that made fibroblasts from asthmatic patients respond differently will need further exploration to elucudate the interactions between Wnt pathways and other genes that we found to be differentially expressed in asthma.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the MUHC Research Ethics Board (2003–1879) and the subjects had provided written informed consent to participate in this study.MH did the conceptualization, data curation, formal analysis, investigation, methodology, validation, software, visualization, and writing—original draft. NE did the data curation, formal analysis, investigation, methodology, validation, and writing—review. RR and KB did the investigation, methodology, and writing—review. IH and SA did the formal analysis, investigation, software, visualization, and writing—review. RO did the resources. QH did the conceptualization, funding acquisition, project administration, resources, supervision, and writing—review and editing. HB did the supervision and writing—review and editing. RH did the conceptualization, funding acquisition, methodology, resources, software, and writing—review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The retina is a light-sensing ocular tissue that sends information to the brain to enable vision. The blood–retinal barrier (BRB) contributes to maintaining homeostasis in the retinal microenvironment by selectively regulating flux of molecules between systemic circulation and the retina. Maintaining such physiological balance is fundamental to visual function by facilitating the delivery of nutrients and oxygen and for protection from blood-borne toxins. The inner BRB (iBRB), composed mostly of inner retinal vasculature, controls substance exchange mainly via transportation processes between (paracellular) and through (transcellular) the retinal microvascular endothelium. Disruption of iBRB, characterized by retinal edema, is observed in many eye diseases and disturbs the physiological quiescence in the retina’s extracellular space, resulting in vision loss. Consequently, understanding the mechanisms of iBRB formation, maintenance, and breakdown is pivotal to discovering potential targets to restore function to compromised physiological barriers. These unraveled targets can also inform potential drug delivery strategies across the BRB and the blood–brain barrier into retinas and brain tissues, respectively. This review summarizes mechanistic insights into the development and maintenance of iBRB in health and disease, with a specific focus on the Wnt signaling pathway and its regulatory role in both paracellular and transcellular transport across the retinal vascular endothelium. The retina, an extension of the brain and part of the central nervous system (CNS), plays an important function in converting photons into electrochemical signals to allow visual perception. Per tissue weight, the retina is one of the most metabolically active and demanding tissues in the body . This hiThe existence of BRB was first observed by Schnaudigel  in 1913 Dysfunction of the BRB is implicated in several debilitating, blinding retinal diseases wherein impaired BRB disturbs the stability of the retinal microenvironment with adverse effects on nutrient and oxygen supply, waste removal, and light absorption. For instance, loss of the iBRB is implicated in diabetic retinopathy (DR), retinopathy of prematurity (ROP), retinal vein occlusion, retinitis pigmentosa, and retinoblastoma ,9,10,11.This review focuses on the role of Wnt signaling in maintaining iBRB. The cellular and molecular composition of the iBRB together with its development is introduced first, followed by a summary of essential knowledge on the mechanistic foundations of Wnt signaling in iBRB function. Other crucial mechanisms of iBRB maintenance and breakdown in health and disease are also briefly discussed with their relevance to Wnt signaling. Finally, we postulate a possible future inquiry into the role of the Wnt signaling pathway in regulating ocular barriergenesis and the possibility of targeting this pathway as a therapeutic intervention to improve BRB function.Molecular flux across the iBRB is primarily regulated by a network of well-organized retinal vasculature and RMECs lining the lumen of these vessels . MicrovaWater and small, water-soluble compounds  can pas‘Zonula Occludens’ 1 (ZO-1) was the first protein discovered to be associated with tight junctions , followeThere are 27 family members of claudins, and each one can form complex structures with one another or with occludin ,33. In bThe formation of tight junctions between RMECs is preceded and stimulated by adherens junctions, which enhance intercellular contact during early embryonic development . For insThe assembly of adherens junctions and tight junctions is facilitated by gap junctions , suggestTo summarize, the junctional proteins of RMECs do not function in isolation but are structurally and functionally interlinked with one another to ensure the precise regulation of paracellular transport in maintaining iBRB integrity.+, K+/ATPase), sodium-potassium-two chloride (Na+/K+/2Cl−) cotransporter, sodium–hydrogen exchanger, chloride–bicarbonate exchanger, and sodium–calcium exchanger [Generally, the movement of solute or fluid ‘through’ RMECs in the inner retina is tightly regulated by energy-dependent membrane transporters and vesicular transport. The exception is a wide range of dissolved or gaseous lipid-soluble molecules, such as oxygen, where passive transport across RMECs occurs via diffusion following a concentration gradient. All other types of energy-mediated transcellular transport across RMECs can be grouped into five main categories: carrier-mediated transport, ion transport, active efflux transport, receptor-mediated transport, and caveolae-mediated transport D. For inxchanger ,47,48. Txchanger . The recxchanger ,54,55,56xchanger ,58. HereThe idea of endothelial transcytosis first emerged in the 1950s in studies by George Palade on vascular permeability, in which small vesicles near capillary plasma membrane were described to explain microvessel permeability . Later, Cav1−/− mice are resistant to drug-induced pulmonary vascular albumin hyperpermeability and edema [The core protein components of caveolae are caveolins (Cav) and cavins. There are three Cav (Cav13) and four cavin (Cavin14) proteins ,64. Amonnd edema , suggestnd edema , and alsnd edema . Whereasnd edema . The trand edema .Transcytosis may assist the migration of macromolecules, including albumin, transferrin, insulin, lipoproteins, and possibly immunoglobulins, from the circulation into the retina through their respective receptors located on vesicles. However, under normal physiological conditions, transcytosis is significantly limited in the retina compared with other tissues or organs in order to maintain its barrier property ,70. ThisIn the developing retina, functional iBRB integrity is acquired in both spatial and temporal manner following the developmental reduction in endothelial transcytosis , which hAnother molecule implicated in regulating transcytosis in the CNS is the major facilitator superfamily domain-containing 2a (MFSD2A). MFSD2A, selectively expressed in both brain and retinal microvessels, is a protein with dual roles in both lipid transport and transcytosis. MFSD2A assists brain uptake of docosahexaenoic acid (DHA)  in a form bound with lysophosphatidylcholine (LPC) ,79. In tTo summarize, under physiological conditions, transcellular-regulated transport appears to be the preferred route for the active transport of macromolecules facilitated by caveolae- and other receptor-mediated transport mechanisms across ECs in BBB, and similarly in RMECs that constitute the iBRB.As retinal vessel growth and barrier formation are closely associated , we willIn early human and rodent embryonic eye development, a transient hyaloid network of vasculature emerges from the central hyaloid artery in the optic nerve, providing vascular support for the developing lens and retina. Hyaloid vessels begin to regress around gestation week (GW) 14 in humans and after birth in rodents, concurrent with the development of new retinal vasculature ,86. AstrMuch of our understanding on iBRB formation comes from studies on mice B. The deTogether, not only is the maturation of cells, including RMECs, in the neurovascular unit important for physiological retinal vascularization, but also fundamental to iBRB development and maintenance. In the mouse, an intact iBRB is established early on while the retinal vasculature is still under development and before eye opening , suggestThe formation of blood vessels and barrier maturation in the eye are tightly coordinated, although the underlying mechanisms regulating angiogenesis vs. barriergenesis are different. For instance, VEGF is a well-known endothelial growth factor, yet it promotes the breakdown of vascular barriergenesis by inducing permeability; it was formerly named Vascular Permeability Factors when originally discovered . AccordiThe Wnt signaling pathway is crucial to developmental and pathological angiogenesis in many organs and tissues, including the eye and retina ,100,101.The Wnt signaling pathway consists of two main types: the canonical Wnt/β-catenin-dependent and the non-canonical Wnt/β-catenin-independent pathways. Although the latter has not been as well characterized as the former, examples of the non-canonical ligands include Wnt5a and Wnt11 , with tActivation of canonical Wnt signaling to the ‘on’ state starts with Wnt ligands binding to one of several Frizzled (Fzd) family receptors , typically in addition to a co-receptor, such as low-density lipoprotein receptor-related protein 5 (LRP5) or LRP6 A, to actConversely, in the absence of Wnt ligands (inactive ‘off’ state), β-catenin is recruited to a destruction complex consisting of Axin, adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and GSK3β. β-catenin is sequentially phosphorylated by CK1α and GSK3β to create a binding site for the ubiquitin E3-ligase βTrCP, which targets β-catenin for ubiquitination and proteasomal degradation in the cytoplasm , disabliNorrin-, Fzd4-, Lrp5-, and Tspan12-deficient mice, all of which exhibit delayed developmental angiogenesis and model these two rare human congenital eye diseases, are emerging as experimental models for studying not only retinal angiogenesis but also ocular barriergenesis due to their compromised BRB [Much of our understanding of Wnt signaling in retinal angiogenesis comes from experimental studies of two rare, congenital, potentially blinding eye diseases: X-linked Norrie disease and FEVR, which are linked with Wnt signaling mutations in humans in either receptors  or the ligand Norrin ,16, respised BRB ,126,127.ised BRB . These oNorrin-deficient retinas [The idea that Wnt/β-catenin signaling maintains iBRB, in part through restricting paracellular transport in vascular endothelium, has been supported by multiple studies. This is probably partly due to the fact that β-catenin binds VE-cadherin, which is a prerequisite for tight junction formation , suggest retinas . Similar retinas ,19 also  retinas .Alk5-deficient retina, it does reduce their vascular leakage in part through increasing claudin5, suggesting a potential interaction between TGFβ and Wnt signaling not in retinal angiogenesis but in barrier control [More recent studies have identified additional new players of Wnt signaling that can coordinate its effects on angiogenesis and barrier formation, some of those through claudin5. For example, the loss of Discs large homologue 1 (Dlg1), an intracellular scaffolding protein, in vascular endothelium reproduces retinal vascular defects and the breakdown of BRB and BBB, as seen in Wnt-deficient mice. This finding suggests a new role of Dlg1 in Wnt/β-catenin signaling that appears to be independent of Dlg1′s direct interaction with FZD4 . Similar control . Similar control  showed tTspan12−/− Wnt-deficient mice with the antibody (from P5 to P20) restored retinal angiogenesis and barrier function (claudin5) [Methods to target Wnt signaling have been explored as potential therapeutics to restore iBRB in both development and diseases. Recently, Chidiac and colleagues  developelaudin5) . Similarlaudin5) , suggestlaudin5)  showed tlaudin5) , indicatlaudin5)  demonstrTaken together, the canonical Wnt signaling pathway modulates junctional proteins of RMECs, particularly claudin5, in its interaction with other junctional components in paracellular transport across the iBRB in health and disease . Yet, meTspan12−/− Wnt-deficient mouse retinas [The Wnt signaling pathway maintains iBRB not only by tightly regulating the paracellular transport  between RMECs, but also by limiting transcellular transport (transmembrane transporters or vesicles) to ensure iBRB stability and integrity. A hint to its link to transcytosis derives from studies on PLVAP, an indicator and marker of EC transcytosis that has an increased expression in both Norrin KO and LRP5 KO mice deficient in Wnt signaling and exhibiting leaky retinal vessels ,128. Mul retinas , furtherDirect evidence of the Wnt signaling pathway mediated modulation of physiological transcytosis in retinal endothelial cells was provided by our recent study  where weWhereas the activation of the Wnt signaling pathway may be crucial to maintaining iBRB and BBB during development and under physiological conditions, aberrant or excessive activation of Wnt signaling, as observed in several blinding retinal diseases, may be detrimental to iBRB maintenance. For instance, Yan and colleagues  recentlyIn a nutshell, MFSD2A and caveolae are important mechanistic downstream effectors of the canonical Wnt/β-catenin signaling pathway for regulating transcytosis in RMECs under physiological conditions . WhetherThe mechanisms of iBRB maintenance and breakdown are clearly multifactorial and transcend the Wnt signaling pathway. For instance, VEGF is a well-known factor that can alter EC junctional proteins to disrupt iBRB integrity in retinal vascular diseases in part through protein kinase C (PKC), and both VEGF and PKC are common drug targets in diabetic retinopathy and diabetic macular edema (DME) . Yet, anDR is one of the most common microvascular complications of diabetes mellitus and a leading cause of vision loss among the working-age population worldwide ,145. It Among these growth factors, much work has been carried out on VEGF since the initial discovery that VEGF was upregulated in the ocular fluid of patients with DR and other vascular eye diseases . This fiIn addition to the vascular endothelium, aberrant signaling in other components of the neurovascular unit, such as pericytes , Mϋller Apart from pericytes, activated microglia can engender increased apoptosis, decreased autophagy, and the increased expression of VEGF in Müller glial cells to destabilize iBRB regulation in DR or under hypoxic conditions ,184. TheRecently, Weiner and colleagues  showed tTo recapitulate, several cellular and non-cellular mechanisms within the retinal neurovascular unit, including retinal ECs or retinal neurons, have been documented to underlie iBRB maintenance and breakdown in health and blinding retinal diseases, respectively. The insufficient efficacy and coverage of existing therapy for ameliorating retinal edema calls for alternative improved therapy or for combination therapy. The latter could be achieved by harnessing the drug development of newly discovered VEGF-independent targets underlying the molecular basis of retinal barriergenesis in health and disease. In this regard, the Wnt signaling pathway appears to be promising in terms of its probable therapeutic efficacy in ameliorating vision loss due to iBRB dysfunction and retinal edema. Research in the past two decades has provided substantial insights into the role of Wnt/β-catenin signaling in not only retinal and CNS angiogenesis but also the regulation of BBB and iBRB. Many genes essential to BBB and iBRB development and maintenance have been discovered as target genes downstream of the Wnt/β-catenin pathway . ClaudinRecent works identifying new components of the Wnt/β-catenin signaling pathway  or potential interactive partners (ILK and TGFβ) have largely expanded our knowledge of BBB and iBRB control related to and beyond Wnt signaling. Uncovering additional players of this pathway, its interaction with other signaling pathways, and its role in regulating the extracellular matrix  will potClaudin5 and Mfsd2a, and thereby loosening iBRB and BBB to promote drug delivery, will also represent a potential future direction of research [Targeting the Wnt signaling pathway to restore BBB and iBRB has been an active area of research. Recent work has demonstrated how Wnt ligand treatment or antibodies activating Wnt or Wnt ligands may restore iBRB breakdown in genetic or diabetic animal models with VEGF-induced hyperpermeability, yet aberrant overactivation of Wnt signaling may underlie iBRB breakdown in other studies. Given the pro-angiogenic effects of the Wnt pathway in pathological angiogenesis, titration of proper levels of Wnt signaling and selection of the optimal treatment window will need to be considered to avoid potential undesirable consequence of stimulating pathological angiogenesis in the proliferative stage of retinopathy. While iBRB and BBB are protective against pathogens and toxins from circulation, they also hinder the delivery of drugs into the CNS and the retina. Finding ways to transiently deactivate the Wnt pathway or suppress its target genes, including research ,189,190.


Hepatozoon and genera of the order Piroplasmorida) and Anaplasmataceae agents in Brazil. Thus, this study investigated the occurrence of Hepatozoon spp., Piroplasmorida, and Anaplasmataceae agents in small mammals in seven forest fragments in Brazil.Small mammals  have been poorly explored for the occurrence of apicomplexan  and one fragment in the State of Mato Grosso do Sul . Mammal blood, liver, spleen, and lung samples were tested molecularly for the presence of DNA of Hepatozoon spp. were detected in small mammals from different biomes. In São Paulo state, haplotype 1 was detected in rodents from Cerrado and a transition area of Cerrado and Atlantic Forest biomes, whereas haplotype 2 was detected in rodents from the Atlantic Forest biome. On the other hand, haplotypes 3 and 4 were restricted to rodents and marsupials, respectively, from the Pantanal biome of Mato Grosso do Sul. No host species shared more than one haplotype. Despite these distinct geographical and host associations, our phylogenetic analyses indicated that the four Hepatozoon haplotypes belonged to the same clade that contained nearly all haplotypes previously reported on rodents and marsupials, in addition to several reptile-associated haplotypes from different parts of the world. No mammal samples yielded detectable DNA of Piroplasmorida agents. On the other hand, the Anaplasmataceae-targeted polymerase chain reaction (PCR) assay amplified a sequence 100% identical to the Wolbachia pipientis endosymbiont of the rodent filarid Litomosoides galizai.A total of 524 mammals were captured, comprising seven species of marsupials, 14 rodents, two carnivores, and one Cingulata. Four novel haplotypes  of Hepatozoon haplotypes associated with small mammals in three Brazilian biomes: Cerrado, Atlantic Forest, and Pantanal. Through phylogenetic analyses, the Hepatozoon agents grouped in the rodent-marsupial-reptile large clade of Hepatozoon spp. from the world. The detection of a W. pipientis associated with the rodent filarid L. galizai indicates that the rodent was infected by filarial nematodes.We report a variety of  Hepatozoon and order Piroplasmorida  have been associated with infections and diseases of domestic and wild mammals in Brazil [Hepatozoon spp. are apicomplexan parasites characterized by a heteroxenous life-cycle, in which the intermediate hosts (vertebrates) become infected primarily through the ingestion of hematophagous arthropods (definite hosts such as ticks and mosquitoes) containing mature oocysts [Hepatozoon cysts in their tissues, have been described and seem to be an important infection route for carnivore hosts [Tick-borne protozoans of the genus n Brazil –5. Hepat oocysts . Alternare hosts , 8. The re hosts , 10.Anaplasmataceae family includes bacteria of the genera Anaplasma, Ehrlichia, Neorickettsia, and Wolbachia, from which the first two encompass tick-borne agents of veterinary and public health significance world widely [Wolbachia are known to infect vertebrate cells . Through the tick bite, the bacterium enters the bloodstream and infects specific host cell types, such as neutrophils, monocytes and macrophages, platelets, erythrocytes, or endothelial cells depending on the agent [The d widely . All genhe agent , 12.Hepatozoon spp.Small mammals such as wild rodents and marsupials are hosts of numerous species of ticks at some stage  of their life-cycle . In BrazAnaplasmataceae agents in wildlife in Brazil during this century [Hepatozoon spp., Piroplasmorida, and Anaplasmataceae agents in small mammals in seven forest fragments in Brazil.Various studies have reported the occurrence of apicomplexan and  century , 18–26, Seven forest fragments, six of them located in the State of São Paulo and one in the State of Mato Grosso do Sul, were prospected Fig. . Areas Acytb) was performed as described [Hepatozoon, protozoa of the order Piroplasmorida , and bacteria of the family Anaplasmataceae  gene of this protozoan genus. PCR assays were performed in a total volume of 25 μl, using DreamTaq Green PCR Master Mix . Primers, PCR conditions, and positive controls are provided in Table DNA extractions from blood, liver, lung, and spleen samples were carried out using the DNeasy Tissue & Blood Kit  according to the manufacturer’s instructions. In order to verify the success of extraction, an initial polymerase chain reaction (PCR) targeting the mammalian mitochondrial cytochrome b gene  of the 18S rRNA gene , and one for a larger fragment (≈1700 bp) of the 18S rRNA gene were constructed. Both trees were inferred by using maximum parsimony (MP), as implemented in PAUP version 4.0b10 [Partial 18S rDNA sequences of lustal X  and manulustal X  with corn 4.0b10  with 100n 4.0b10  with 1,0cytb gene were obtained in all 1355 samples, thus confirming successful DNA extractions.A total of 524 mammals were captured, comprising seven species of marsupials, 14 rodents, two carnivores, and one Cingulata , and six individual marsupials and four rodents from the state of Mato Grosso do Sul (A7) , liver only (3), spleen only (1), lung and spleen (4), and lung, liver, and spleen (5). Amplicons from the 21 PCR-positive small mammals were submitted to DNA sequencing, resulting in four 18S rRNA new haplotypes  of the genus Hepatozoon. Haplotypes 1 and 2 were restricted to rodents of the state of São Paulo, whereas the other two haplotypes were found only in the state of Mato Grosso do Sul, being haplotype 3 on rodents, and haplotype 4 on marsupials  yielded amplicons in the initial 7) Table . Only onver only , spleen Hepatozoon-haplotype 1, detected in the rodents Nectomys squamipes and Oligoryzomys nigripes, was 98.7% (532/539 bp) identical to Hepatozoon sp. detected in the tick Ixodes sp. from Chile (MH174345) and Hepatozoon sp. detected in the rodent Phyllotis darwini from Chile (MW881034). The Hepatozoon-haplotype 2, detected in the rodents Akodon sp. and Euryoryzomys russatus, was 99.8% (543/544 bp) identical to the same two Hepatozoon sp. sequences from Chile . Similarly, the Hepatozoon-haplotype 3, detected in the rodents Oecomys mamorae and Thrichomys pachyurus, was 98.2% (532/542 bp) identical to the same two Hepatozoon sp. sequences from Chile . Finally, the Hepatozoon-haplotype 4, detected in the marsupials Gracilinanus microtarsus and Monodelphis domestica, was 98.0% (532/543 bp) identical to Hepatozoon sp. detected in the reptile Caiman crocodilus from Brazil .Considering the closest identities under 100% query-cover through BLAST analysis, the Hepatozoon 18S rRNA gene from at least one small mammal of each haplotype. Although no larger fragment was generated from haplotype 1-representatives, we did generate it from four haplotype 2-representatives, from which their identical 18S rRNA sequences, here designated as Hepatozoon sp. HF005, HF009, HF014, and HF015, were by BLAST analysis 99.2% (1715/1729 bp) identical to Hepatozoon ayorgbor from the snake Python regius from Ghana (EF157822) (Table Hepatozoon sp. PS085) was 98.2% (1710/1741 bp) identical to the same H. ayorgbor sequence mentioned above (EF157822). Similarly, from a haplotype 4-representative (designated as Hepatozoon sp. PS001), its larger 18S rDNA fragment was 98.2% (1692/1723 bp) identical to H. ayorgbor (EF157822)  of the 2) Table . A large 1692/172 bp identHepatozoon spp., haplotypes 1, 2, 3, and 4 branched within a large clade composed by many Hepatozoon haplotypes associated with rodents and reptiles from different parts of the world. This large clade was sister to another large clade that contained Hepatozoon haplotypes associated with canids (Hepatozoon canis) and felids (Hepatozoon felis) under 66 (MP) and 0.8 (BA) bootstrap supports  and 1.0 (BA) bootstrap supports  of the 18S rRNA gene of Oligoryzomys sp. from Piracicaba (A1) yielded amplicons by the Anaplasmataceae-PCR protocol. These amplicons generated a DNA sequence 100% (306/306 bp) identical to the Wolbachia pipientis endosymbiont of the rodent filarid Litomosoides galizai (AJ548800).None of the 1355 samples yielded amplicons by the Piroplasmorida-PCR protocol. One blood sample from an Hepatozoon spp. were detected in small mammals from different biomes among two Brazilian states, São Paulo and Mato Grosso do Sul. In São Paulo state, haplotype 1 was detected in rodents from Cerrado and a transition area of Cerrado and Atlantic Forest biomes, whereas haplotype 2 was detected in rodents from the Atlantic Forest biome. On the other hand, haplotypes 3 and 4 were restricted to rodents and marsupials, respectively, from the Pantanal biome of Mato Grosso do Sul. No host species shared more than one haplotype. Despite these distinct geographical and host associations, our phylogenetic analyses indicated that the four Hepatozoon haplotypes belonged to the same clade that contained nearly all haplotypes previously reported on rodents and marsupials, in addition to several reptile-associated haplotypes from different parts of the world. This general topology was confirmed by our analysis inferred from nearly complete sequences (≈1700 bp) of the 18S rRNA gene of Hepatozoon spp.In the present study, four different haplotypes  of Hepatozoon spp. on small rodents and marsupials in Brazil are restricted to six studies [Hepatozoon sp. PCR165 on one Thylamys macrurus [H. canis on two Didelphis albiventris [. Except for this later record of H. canis, all previous records on small rodents and marsupials in Brazil demonstrated that the Hepatozoon spp. haplotypes belonged to the large clade composed of haplotypes associated with rodents, marsupials, and reptiles, in agreement with the results of the present study. Unfortunately, many of the sequences from previous studies were from regions of the 18S rRNA gene different from the present study  from previous Brazilian studies [Akodon sp. and E. russatus of the state of São Paulo) had internal regions that were 100% identical to 516–1007-bp haplotypes of Hepatozoon sp., previously reported on the rodents Akodon montensis, Akodon cursor, and Galea spixii from three Brazilian states, including São Paulo [O. mamorae from the Pantanal biome of Mato Grosso do Sul) had regions that were 100% identical to 573–625–bp haplotypes of Hepatozoon sp., previously reported on this same rodent species and the same Brazilian biome [O. mamorae in the Pantanal. On the other hand, no Hepatozoon sequence > 500 bp from GenBank matched 100% to haplotype PS001, which was detected in the marsupial M. domestica in the Pantanal biome. This result points PS001 as a novel Hepatozoon haplotype associated with marsupials in the Pantanal biome of Mato Grosso do Sul state, where other closely related haplotypes seem to be associated primarily with rodents and reptiles.We retrieved rodent-associated- studies , 33–36 fão Paulo , 33. Hapan biome , indicatHepatozoon infection through the ingestion of a hematophagous arthropod containing mature oocysts. Alternatively, infection can be acquired by intrauterine transmission or by predation of infected vertebrates containing merozoites (intermediate hosts) or cystozoites (paratenic host) [Hepatozoon meronts in the liver or lungs , as indicated by ours and previous phylogenetic analyses.Vertebrate hosts acquire ic host) . Previouiewed by ). Regardr et al.  detectedy stated , 36, 38,Hepatozoon spp. infection was detected only in areas A4, A5, A6, and A7. According to previous analyses [Hepatozoon by interspecies predation, a likely important transmission route of this protozoan [Hepatozoon transmission. Again, it is noteworthy that areas A4, A5, A6, and A7 presented higher diversity of ticks than the remaining areas [Hepatozoon spp. is under a complex interaction of vertebrate and invertebrate hosts.Among the seven sampled areas of the present study, analyses , these frotozoan . In adding areas , suggestRegarding the other PCR assays of the present study, none of the samples yielded detectable DNA to Piroplasmorida agents. This result contrasts with previous studies that reported different Piroplasmorida haplotypes among small mammals from other regions of Brazil, including studies that employed the same PCR protocol of the present study , 24, 39.Anaplasmataceae bacteria yielded amplicons from the blood of an Oligoryzomys sp.: a 16S rRNA partial sequence 100% identical to W. pipientis associated with the rodent filarid L. galizai. This is indirect evidence that this rodent was infected by filarial nematodes. In previous studies, similar PCR assays amplified Wolbachia fragments from wild mammals (monkeys and opossums) and domestic dogs, leading the authors to conclude that these animals were infected by filarial parasites [The PCR assay targeting arasites , 40.Hepatozoon haplotypes associated with small mammals in three Brazilian biomes: Cerrado, Atlantic Forest, and Pantanal. Through phylogenetic analyses, the Hepatozoon agents grouped in the rodent-marsupial-reptile large clade of Hepatozoon spp. from the world. Our phylogenetic analyses suggest that small mammals might play a role in the epidemiology cycle or reptile-associated Hepatozoon spp. rather than the Carnivora-associated Hepatozoon species. Since the Hepatozoon detections were restricted to areas with higher diversity of small mammals and ticks, this condition could facilitate Hepatozoon transmission. Finally, the detection of a W. pipientis associated with the rodent filarid L. galizai indicates that the rodent was infected by filarial nematodes.We report a variety of


HTLV-1 is a highly oncogenic yet neglected retrovirus, which primarily infects CD4+ T-cells in vivo and causes incurable diseases like HTLV-1-associated inflammatory conditions or Adult T-cell leukemia/lymphoma (ATLL) after lifelong viral persistence. Worldwide, at least 5–10 million people are HTLV-1-infected and most of them are unaware of their infection posing the risk of silent transmissions. HTLV-1 is transmitted via cell-containing body fluids such as blood products, semen, and breast milk, which constitutes the major route of mother-to-child transmission (MTCT). Risk of transmission increases with the duration of breastfeeding, however, abstinence from breastfeeding as it is recommended in some endemic countries is not an option in resource-limited settings or underrepresented areas and populations. Despite significant progress in understanding details of HTLV-1 cell-to-cell transmission, it is still not fully understood, which cells in which organs get infected via the oral route, how these cells get infected, how breast milk affects this route of infection and how to inhibit oral transmission despite breastfeeding, which is an urgent need especially in underrepresented areas of the world. Here, we review these questions and provide an outlook how future research could help to uncover prevention strategies that might ultimately allow infants to benefit from breastfeeding while reducing the risk of HTLV-1 transmission.Breastfeeding is recommended by the World Health Organization for at least 6 months up to 2 years of age, and breast milk protects against several diseases and infections. Intriguingly, few viruses are transmitted  Upon infection, HTLV-1 integrates into the host cell genome and PHTLV are life-long suffering from the burden of HTLV-1-infection and an increasingly recognized impairment of quality of life (via breastfeeding since the level of infection among babies that are exclusively formula fed is low (via breastfeeding are (1) high proviral loads (PVL) in milk and blood, (2) low income, (3) breastfeeding over a longer period, (4) previous HTLV-1-infected offspring, (5) HLA-concordance between mother and child, (6) coinfection with the nematode Strongyloides sp., or (7) being a HAM/TSP patient (Human T-cell leukemia virus type 1 (HTLV-1) is a neglected highly oncogenic retrovirus infecting at least 5–10 million people worldwide, which is probably an underestimation . A high  of life . Carrier of life . In ende of life , while t of life , 8, 9. A of life , the majd is low , 7. In bd is low , 8, 12. d child,  coinfect patient . Infecti patient .via breast milk including Human Cytomegalovirus (CMV), Human Immunodeficiency Virus (HIV), HTLV-1, and the related HTLV-2 , a major secondary lymphatic organ, arises as an ideal candidate for potential HTLV-1 entry. Beneath the protective mucus film containing a multitude of antimicrobial peptides like defensins and lysozymes, the intestinal epithelial cell layer followed by the lamina propria is located . These svia the oral route could occur in several ways, which has been nicely summarized in recent reviews  are transferred to target cells, so called viral biofilms (VB)  , 58. Addlms (VB) , 59–61. al route . Of specal route . Plasmac T-cells , 64. Thiemselves . Moreovein vitro . These fin vitro . Still, Breast milk constitutes of water, probiotic bacteria, breast milk cells, and macro- and micronutrients including lipids, fats, proteins and carbohydrates. The repertoire of breast milk cells is highly heterogeneous and contains (1) breast-derived cells including luminal epithelial cells , myoepithelial cells from the ducts, squamous epithelial cells (from skin and nipple of the breast), progenitor and stem cells, and (2) blood-derived cells including immune cells , and hematopoietic progenitor and stem cells –69. Morein vivo  neutralizing monoclonal antibody (mAb) termed LAT-27, which prevents in vivo transmission of HTLV-1 in a simple humanized mouse model of HTLV-1 infection  adherence” . In factnfection . Of notenfection . It remanfection . MoreoveOverall, there is a great need to develop novel prevention strategies of HTLV MTCT with the potential to protect the most vulnerable members of our society, newborn infants, from live-long, live-threatening, and incurable infections.SM and AT-K wrote the article. Both authors contributed to the article and approved the submitted version.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.


FGFR (fibroblast growth factor receptor) signaling controls fundamental processes in embryonic, fetal and adult human life. The magnitude, duration, and location of FGFR signaling must be strictly controlled in order to induce the correct biological response. Uncontrolled receptor signaling has been shown to lead to a variety of diseases, such as skeletal disorders and cancer. Here we review the numerous cellular mechanisms that regulate and turn off FGFR signaling, once the receptor is activated. These mechanisms include endocytosis and endocytic sorting, phosphatase activity, negative regulatory proteins and negative feedback phosphorylation events. The mechanisms act together simultaneously or sequentially, controlling the same or different steps in FGFR signaling. Although more work is needed to fully understand the regulation of FGFR signaling, it is clear that the cells in our body have evolved an extensive repertoire of mechanisms that together keep FGFR signaling tightly controlled and prevent excess FGFR signaling. The pleiotropic biological actions of FGFs (fibroblast growth factors) are exerted through binding to and activation of four conserved, transmembrane cell-surface FGFRs (fibroblast growth factor receptors) with tyrosine kinase activity, named FGFR1-4. In humans, 22 structurally related FGFs have been identified based on sequence homology . The betThe FGFs act as tissue growth factors  or metabolic hormones (endocrine FGFs)  and formIn vertebrates, FGFs/FGFRs form one of the largest protein signaling families. Already from this point of view, it is not surprising that aberrations in the FGF/FGFR axis signaling are often found in developmental and metabolic disorders as well as in highly malignant diseases like breast, lung and prostate cancers ,16,17. DThe binding of FGF ligands to their specific receptors results in receptor dimerization and conformational changes in the cytoplasmic part of the receptor. The conformational changes lead to trans-autophosphorylation of the tyrosine kinase domains of the receptors and subsequently the induction of several downstream signaling pathways. There are four major signaling pathways activated by FGF/FGFRs; the Ras-MAPK (mitogen-activated protein kinase) pathway, the PI3K -AKT pathway, the PLCγ (phospholipase Cγ)/PKC (protein kinase C) pathway and the STAT  pathway  Figure . There aAccurate signaling from receptor tyrosine kinases can be maintained through tight regulation by several mechanisms . HoweverEndocytosis is a cellular process involving membrane invagination and uptake of different cargos such as receptor-ligand complexes, antigens, viruses and protein toxins into intracellular vesicles . VesicleThe first step in receptor-mediated endocytosis is the recruitment of active receptor-ligand complexes into growing invaginations at the plasma membrane that eventually buds off to form intracellular vesicles. Receptor internalization is often divided into clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE). CIE pathways include caveolae-mediated, FEME (fast endophilin-mediated endocytosis) and CLIC/GEEC  . CME is In the case of another RTK (receptor tyrosine kinase), namely EGFR , the receptor is taken up via both CME and CIE depending on low (<2 ng/mL) or high (>2 ng/mL) ligand concentration, respectively . At evenIt is uncertain what might be the signal for recruitment of FGFRs to invaginations at the plasma membrane leading to their endocytosis. Most receptors internalized via CME are recruited to the clathrin-coated membrane invaginations via tyrosine-based sequences such as YXXФ (where Ф can be any hydrophobic residue) or [FY]XNPX[FY] motives . These sRecruitment of E3 ubiquitin ligases such as NEDD4-1  and c-CBL (Casitas B-lineage lymphoma) either directly to the receptor or via FRS2 (FGFR substrate 2)/Grb2 (growth factor receptor-bound 2), respectively, and subsequent ubiquitination of the receptor has also been implicated in FGFR endocytosis ,44,45. HOnce internalized, the number of receptors available for binding of ligands at the cell surface is reduced and the cells are less responsive to ligand. The receptor can continue to signal from endosomes and it has been suggested that different intracellular locations might give rise to different signaling simply because the subsets of downstream signaling molecules might vary from compartment to compartment . For exaFollowing endocytosis, receptors are transported to the lysosomes for degradation resulting in termination of signaling or for recycling back to the cell surface allowing additional rounds of signaling ,61 Figu. The newThe pathway that FGFRs follow after internalization depends on both the receptor-type but also the bound ligand  Figure . Upon stWhile the retrieval of receptors for recycling is poorly understood, sorting of receptors into intraluminal vesicles and the degradative pathway is well characterized. The key step for sorting of a receptor into intraluminal vesicles and subsequent degradation, is the attachment of ubiquitin to lysine residues in the intracellular part of the receptor by E3 ubiquitin ligases such as CBL and NEDD4 ,68. The To understand the different sorting of FGFR1 and FGFR4, a comparison of their intracellular sequences was performed and revealed that FGFR1 contains a higher number of lysines that are potential ubiquitination sites, than FGFR4 . Indeed,It is not completely clear which ubiquitin ligases are involved in FGFR ubiquitination but many reports have implicated c-CBL ,73,76,77Clearly, endocytosis and intracellular sorting influence receptor signaling in many ways. Receptor trafficking is not only altering the duration of signaling, but also the signaling pathways activated might vary with subcellular localization. This might give rise to a completely different signaling output. For instance, altered intracellular sorting of FGFR2b upon stimulation with FGF10 (recycling) reduced signaling duration and led to decreased breast cancer cell migration and inhibition of epithelial branching compared to stimulation with FGF7 (degradation) . SimilarMost studies on FGFR endocytosis and signaling are performed in cell lines. The understanding of the mechanisms and consequences of FGFR endocytosis in vivo is limited. It has been shown that expression of a mutant form of FGFR1, which is unable to bind to NEDD-4 leading to reduced endocytosis and sustained signaling, results in disrupted anterior neuronal patterning (head development) in zebrafish . AnotherProtein tyrosine phosphatases (PTPs) remove the phosphate group from tyrosine residues of phosphorylated proteins  and as sDusp6. MKP3 acts as a key modulator and controls MAPK deactivation by dephosphorylating ERK1 and ERK2 on phosphotyrosine and phosphothreonine residues [Dusp6 transcription is activated by FGF signaling and suggest a negative feedback role for MKP3 in FGF signaling [Dusp6 promoter [Phosphatases can also play a role in the positive regulation of FGFR signaling. The best example is the phosphatase SHP2 ), which binds to phosphorylated FRS2 upon FGF stimulation, but enhances FGFR signaling ,89. Onceresidues . Severalignaling ,93,94. Fpromoter . Downregpromoter . Overexp2 knockout rats had severe defects in lung morphogenesis [A class of well-studied molecules that regulate FGFR signaling are the SPRY proteins . Four meogenesis . Contrarogenesis ,106. SPRogenesis . Interesogenesis . Taken tSPRY share several features with the SPRED (Sprouty related with EVH1 (Ena/VASP homology 1)) protein family composed of SPRED-1, SPRED-2, and SPRED-3. Both protein families have a homologous cysteine-rich C terminus and function as inhibitors of the Ras-MAPK pathway downstream of various stimuli like growth factors and cytokines ,109. As Another molecule that acts as an antagonist of FGFR signaling is SEF (similar expression to FGF), a conserved inhibitor of the MAPK pathway . HoweverAnother protein that might modulate FGFR signaling is FGFR5 (FGFRL1). FGFR5 is the fifth member of the FGFR family but lacks tyrosine kinase activity and thus is not an active receptor. Instead, FGFR5 has been predicted to negatively regulate FGF signaling by competing with other FGFRs for ligand binding or by forming heterodimers with other members of the FGFR family and thereby preventing trans-autophosphorylation . HoweverGrb2 has also been suggested to play a role as a regulator of FGFR2 signaling . It was The adaptor protein Grb14 has been implicated in the regulation of PLCγ signaling downstream of FGFRs . Upon reClearly, multiple proteins can function at various steps as negative regulators in the FGFR signaling pathways to turn off FGFR signaling. In addition, some of these regulatory proteins might have numerous roles.In addition to several negative feedback mechanisms governing FGFR signaling such as FGF-induced CBL recruitment, MKP3 production and others discussed above, more direct negative feedback mechanism involving phosphorylation events also exists to prevent excess FGFR signaling . Nearly Lack of such negative feedback loops could give tumor cells a growth advantage and indeed, mutants of FGFR2 lacking S780 have been identified in human cancers ,136,137 Clearly, many mechanisms exist to control FGFR signaling. The tight regulation of signaling is necessary to maintain body homeostasis. These mechanisms can occur simultaneously or sequentially, and in different or, sometimes, the same steps of the signaling pathways. They might operate differently depending on cell type, the ligand or the receptor type. It is clear that our cells have evolved a whole menu of mechanisms reinforcing each other to control FGFR signaling. In case one of the mechanisms is not operational, other mechanisms and proteins involved in the regulation of cell signaling can take over and the cell might still be able to prevent excessive FGFR signaling. The development of all of these different mechanisms indicates the importance of keeping FGFR signaling under control. As mentioned in the introduction, several human conditions are caused by excessive FGFR signaling, among them dwarfism and cancer ,16,17,18


Dysregulation of the glutamatergic system underlies the pathophysiology of depression. Both negative and positive modulation of NMDARs exert rapid and sustained antidepressant effects by reversing the dysregulated glutamatergic system. Research in the past decades has identified key signaling pathways activated by these rapid acting antidepressants. Here, we review the converging signaling mechanisms shared by rapid acting antidepressants and discuss the recent progress on distinct actions of NMDAR antagonists and NMDAR positive modulators to trigger rapid antidepressant actions. Major depressive disorder (MDD) is a debilitating psychiatric disorder and one of the leading causes of disability worldwide. Although the monoaminergic hypothesis of depression dominated the field for several decades, research in the past two decades demonstrates that the glutamatergic system plays a central role in the pathophysiology of MDD . ConventHere, we review previous and current research on the mechanism underlying the actions of rapastinel and compare with ketamine, including both unique convergent mechanisms underlying the actions of these rapid antidepressants. We discuss novel NMDAR-PAMs and their therapeutic potential for treatment of depression and neuropsychiatric disorders.Rapastinel is a threonine-proline-proline-threonine (TPPT) tetrapeptide derived from a hypervariable region cloned and sequenced from the monoclonal antibody B6B216 ,9 ongoinA receptor agonist) was used to inhibit glutamatergic neuronal activation; the results demonstrated that muscimol completely blocked the increase in phospho-ERK and BDNF release induced by either ketamine or rapastinel. Together, these findings indicate that two different rapid acting antidepressants with opposing NMDAR effects share common molecular and cellular mechanisms.Recently, our studies focused on the converging cellular and molecular mechanisms in the antidepressant actions of rapastinel, an NMDAR-PAM as well as the NMDAR antagonist, ketamine. Our previous studies reported that activation of the mTORC1 signaling pathway , through ERK/AKT activation acts as a key underlying convergent mechanism for the fast-acting antidepressant properties of ketamine . In addiin vivo to reveal mechanistic differences between ketamine and rapastinel. This study demonstrated that like ketamine, a single dose of rapastinel rapidly activated mTORC1 pathway in medial prefrontal cortex (mPFC). Infusion of rapamycin, the selective mTORC1 inhibitor, blocked the antidepressant-like behavioral actions of rapastinel, demonstrating a requirement for the mTORC1 pathway. Further, rapastinel increased the number and function of spine synapses in the apical dendritic tuft of layer V pyramidal neurons in the mPFC, an effect also reported after ketamine treatment [in vivo, this study identified similar molecular and cellular mechanisms for ketamine and rapastinel. However, in this study, the authors also reported some differences. Rapastinel is a centrally active, intravenously administered  peptide, so Liu et al. [Liu et al.  further reatment . Therefou et al.  injectedIt is also likely that ketamine and rapastinel could act at NMDARs on different cell types or recruit unknown distinct signaling pathways that converge on ERK or mTOR signaling. Electrophysiological studies also demonstrated that rapastinel significantly increased the frequency of EPSCs to hypocretin, a measure of thalamocortical synapses. However, rapastinel did not increase the frequency of EPSCs to 5-HT, a measure of cortical-cortical response mediated by the 5-HT2A receptor. Previous studies revealed that ketamine increases both hypocretin- and 5-HT induced ESPC frequency ,12,17,18While both rapastinel and ketamine physically target NMDA receptors and share converging molecular signaling mechanisms, the two molecules have opposing actions on NMDARs. Ketamine is a non-competitive NMDAR antagonist while rapastinel acts as an NMDAR-PAM but how both these drugs exert similar rapid and sustained antidepressant effects and activate convergent downstream signaling pathways has been an unanswered question. One of the most accepted hypotheses that addresses this question is that ketamine and rapastinel could modulate NMDARs on different cell types in mPFC. Recent studies from our lab addressed this issue by using a Crerecombinase dependent viral shRNA approach to produce cell type specific knockdown of the GluN2B subunit of NMDARs in different Cre-lines  . GluN2B Ketamine and rapastinel produce rapid and sustained antidepressant effects. Both molecules increase mTORC1, pERK and pAKT, and BDNF release, resulting in increased synaptic number and function that are associated with antidepressant responses . However