N = 1883) or the control group (N = 1882). A significantly higher posttest mean score was observed for the intervention group compared to the control group (P = 0.0017). We also observed that posttest scores were significantly improved compared to pretest scores in both the intervention and control group (P < 0.001). When examining by specific test question, the proportion answering correctly increased at posttest compared to pretest for both intervention and control groups (P < 0.001). Furthermore, the intervention group showed a greater mean difference between posttest and pretest scores than the control group (P < 0.001). In conclusion, educational intervention using a smartphone application contributed to enhancing fertility treatment-related literacy.People of reproductive age have unmet needs related to deficiencies in fertility literacy. Here, we aimed to investigate whether providing fertility-related information via a smartphone application could improve fertility treatment-related literacy in participants. We performed a randomized control-group pretest posttest study and recruited participants between June 18 and 25, 2020. Participants’ fertility treatment-related literacy was assessed with a pretest that comprised of 28 questions and participants were allocated with stratified randomization to either intervention group or control group. The intervention comprised a one-week smartphone application-based provision of information on fertility-related information and the control group received general information about women’s healthcare. Effectiveness of intervention was assessed using a posttest. A total of 4137 participants were administered the questionnaire and pretest, among which 3765 participants (91.0 %) responded and were randomly allocated into either the intervention group ( With declining birth rates experienced by developed countries, infertility issues will add to economic hardship and is of significant public health importance. Internationally, it has been estimated that 56% of couples have sought medical care including fertility-related issues and treatment2, which is believed to be an underestimate of the overall population that experiences infertility challenges. In such contexts, Japan is recognized as one of the most active countries in reproductive medicine globally3, where nationwide surveys have shown that 1 in 5.5 couples of reproductive age had undergone fertility treatment (http://www.ipss.go.jp/site-ad/index_english/Survey-e.asp).Infertility is defined as a failure to conceive after 12 months of regular and unprotected sexual intercourse, and is estimated to affect 8–12% of couples of reproductive age worldwide4. According to the definition used by Centers for Disease Control and Prevention, assisted reproductive technology (ART) includes all fertility treatments in which either eggs or embryos are handled (https://www.cdc.gov/art/whatis.html). Artificial insemination is applied to achieve fertilization at the timing of ovulation, and this procedure is not included in ART. Alternatively, mature oocytes are retrieved directly from the ovary for fertilization in an IVF protocol. A typical IVF cycle includes gonadotropin stimulation, followed by oocyte retrieval. Oocytes can be fertilized in vitro either mixed with sperm or with intracytoplasmic sperm injection. Fertilized eggs (embryos) are cultured under optimal conditions, then transferred into the uterus (embryo transplantation). Reproductive endocrinologists suggest appropriate therapeutic strategies for the patients based on clinical examinations, such as hormonal level measurements, ultrasonographic images of the uterus and ovaries, radiological findings , and sperm analysis. However, for patients, the interpretation of examination results may be overly complicated and complex, and patients may have difficulty making sense of their own fertility problems. Indeed, people are unaware of their own fertility potential, the constraints on their fertility, the signs, symptoms or preventable causes of fertility problems7, or the available ART that can shape their reproductive lives7.Treatments available for couples experiencing infertility issues include ovulation induction with timed intercourse, artificial insemination with husband (AIH), in vitro fertilization (IVF) and intracytoplasmic sperm injection8. It has been reported that a median of 76% of the population among 18 advanced economies surveyed have smartphones9. In particular, considering the high rate of smartphone ownership in Japan (https://www.soumu.go.jp/johotsusintokei/whitepaper/ja/r01/html/nd232110.html), it is likely that couples of reproductive age would access information about fertility and increase their literacy using the Internet via smartphone. Indeed, in the field of cardiology, it has been demonstrated that digital patient education can increase patient literacy, and also improve the quality of life and lower feelings of depression and anxiety10. Online information is easily accessible; however, the information does not always reflect evidence-based recommendations11. In a previous study on the availability of Internet-based information for people trying to conceive, it was shown that inaccurate and non-evidence-based statements are frequently put forward in online websites which are readily accessible to people trying to conceive who may partake in online communities and distribute this information12. Although the evidence is still limited, considering the ease of access to low quality information, it is conceivable that misinformation and/or poor quality information may adversely affect patients seeking fertility treatment; thus, innovative approaches to providing both accessible and accurate information are desired.Recently, the Internet has become a common source for fertility treatment-related information, and social media is viewed as a potentially effective avenue for dissemination of fertility-related news and education15. Within the Japanese context, it is reasonable to hypothesize that providing high-quality information via a smartphone application can be an appealing strategy to help offset the damage incurred from vastly accessible low-quality information. To the best of our knowledge, the effectiveness of a smartphone application to improve literacy on fertility treatment has not been investigated. The smartphone application, Luna Luna, is the most popular female health care application in Japan and has been widely downloaded by about 16 million people. As one of its features, this application provides high-quality information about fertility treatment developed under the supervision of a reproductive endocrinology specialist.Accumulating evidence indicates that providing quality-assured information could improve patients’ fertility-related knowledge and help decision-makingLuna Luna platform to educate and collect large-scale data from users, we conducted a randomized control-group pretest posttest study to investigate whether providing high-quality fertility treatment information has the ability to enhance fertility treatment-related literacy among a large Japanese population. We also expected that the findings from this study may provide insight for other countries faced with similar challenges regarding reproductive health.In this study, using this Luna Luna users agreed to participate and were administered the questionnaire and pretest  or the control group (N = 1882). Characteristics of participants appeared similar between the groups reflecting that the randomization was successful in producing a balance in baseline characteristics (Table http://www.ipss.go.jp/ps-doukou/e/doukou15/Nfs15_gaiyoEng.html).A total of 4137 In the pretest, the overall mean test score was 57.8%, and scores were similar between the intervention and control groups Table . FocusinLuna Luna and Taion note, 207 participants in the intervention group and 222 participants in the control group were available for pretest-posttest analysis  and after excluding duplicate users of 7.5% and Examination of the overall posttest performance showed that the number of questions for which less than 50% of participants answered correctly decreased to 4 questions in the posttest  among the intervention group Table . These fP = 0.0017)  (Table P < 0.001) and control (P < 0.001) groups (Table P < 0.001)  Fig. . Interes1) Table . When exps Table b and c. 01) Fig. . These fWe further examined the quality of the smartphone application based on information provided in the follow-up questionnaire Fig. . We aske16. However, CFKS-J does not cover fertility treatment-related literacy; thus, we conducted this study with an emphasis on the effectiveness of an educational intervention on fertility treatment-related literacy. This is the largest randomized control trial to examine fertility treatment-related literacy using a smartphone application in a general population, including those who have already received fertility treatment. Questions included in this current study are those which we expect can be answered by people who have already received fertility treatment. In other words, if a couple has a chance to receive treatment due to infertility, it is hopeful that this level of understanding is acquired. Even if not currently undergoing fertility treatment, it would be worthwhile for the general public to know this information in case infertility becomes an issue for them in the future. Furthermore, it is also valuable for medical professionals to know the extent to which these medical issues are understandable for the general public. Patient-centered care, good communication and a strong patient–healthcare provider relationship are essential for effective management of patients with infertility17. These measures may also improve patient adherence to treatment schedules, reduce the physical and emotional burdens associated with treatment, and decrease the rates of treatment discontinuation19. Finally, by improving fertility treatment literacy in general, including the degree of effectiveness for procedures like ART, it might help to enhance self-determination regarding pregnancy issues17.As a primary finding of this study, we found that fertility- and treatment-related information provided via a smartphone application contributed to enhancing fertility literacy. In this study, we developed a fertility literacy assessment tool based on the CFKS-J, which has been used in international comparative studies across 79 countries, including Japan, and its validity and reliability is well-recognized21, it is possible that the control group experienced something similar to a placebo effect. Although the content provided to the controls did not specifically cover infertility issues, it is possible that the act of studying women’s health issues may have helped them to recall topics they have previously heard about or studied. We also cannot rule out the possibility that the controls may have accessed other education materials prior to the posttest as described in the limitations. In addition to assessing the effectiveness of the education intervention, important insights were gained from observing the pattern of incorrect answers and the characteristics associated with them. First, there appeared to be high expectations for fertility treatment. Participants’ perception of pregnancy success rates for AIH and ART was higher than the actual observed rates. A previous study also showed that large proportions of men and women from the general population have limited knowledge of the factors influencing fertility, and tended to overestimate the ability of medically assisted reproduction to overcome age-related infertility22. Second, most participants misunderstood the procedures for AIH as IVF, suggesting that Japanese people do not have sufficient knowledge about types of fertility treatment. Third, participants believe that if they are examined with a certain fertility test, everything can be uncovered, i.e., they do not understand what type of information each test reveals. In decision-making, accurate understanding of what people desire and what people understand should be critical.Interestingly, significant improvement of literacy in the control group was observed in this study. Similar to the researches using smartphone applicationhttps://www.soumu.go.jp/johotsusintokei/whitepaper/ja/h26/html/nc143120.html).With growing fertility issues experienced by many countries, the findings from this Japanese context may help to inform strategies for addressing similar circumstances in other regions of the world as well. We assumed that people with concerns on credibility would be influenced by the source of the information; however, we observed that participants seem to obtain information without particularly caring about the source of information, even though they may have doubts about its reliability. This discrepancy may reflect information literacy circumstance of the Japanese population (https://www.mhlw.go.jp/shingi/2006/10/s1018-7h01.html). Our study showed that people of childbearing age have a reasonable understanding of the public insurance system. In light of the Japanese government’s recent consideration of expanding public insurance to cover medically-assisted reproduction, the findings from our study that targeted the general population of Japan, provides important insight into the current population circumstance associated with fertility treatment (https://www8.cao.go.jp/shoushi/shoushika/law/pdf/r020529/shoushika_taikou_b1.pdf). In particular, in the context of understanding that fertility declines with age, we found that the participants are generally familiar with the age-related fertility decline, and this type of educational provision could enhance knowledge on the clinical significance of AMH which is recognized as a biomarker for ovarian reserve23. The Japanese government is considering to limit the application of public insurance support and subsidies based on the women’s age with reference to the success rate of fertility treatment (https://www.tokyo-np.co.jp/article/122691). In order to determine whether this policy would be generally accepted by the public, it would be worthwhile to gauge the current level of public understanding regarding age and fertility treatment success rates. These findings can contribute to evidence-based policy making when setting the age limit in applying for public insurance.In Japan, care offered by reproductive endocrinologists for fertility management, including timed intercourse with ovarian stimulation, is covered by public insurance; however, medically-assisted reproduction, including AIH and ART, is not covered in principle and financial support is often needed . Furthermore, long-term public health strategies are essential. In the UK, a group of stakeholders have set up the Fertility Education Initiative to increase opportunities for fertility education and to help ensure all women and men are able to make informed choicesLuna Luna, which equates to signing the informed consent form before inclusion. This randomized controlled trial was registered on 15 June 2020 with UMIN Clinical Trials Registry number UMIN000040721 (https://upload.umin.ac.jp/cgi-bin/ctr/ctr_view_reg.cgi?recptno=R000046393).Ethical approval for the implementation of the present study was obtained from the Institutional Review Board of the National Center for Child Health and Development of Japan . All participants in the study agreed to be enrolled and selected the agreement button in http://www.mti.co.jp/eng/) Luna Luna. Luna Luna is a smartphone application that assists users in predicting their menstrual cycle and ovulation under normal physical conditions. The basic functions of this application are freely available on the Android and iOS platforms. This application is used for both pregnancy and contraception planning and for general health management, thus targeting the Japanese female population in general36.We conducted a randomized control-group pretest posttest study among current application users of MTI Ltd.’s  in order to allocate them into two groups with similar distributions of high to low performance. Participants within each strata were assigned a unique randomly generated number, and were sequentially allocated to intervention or control group in an alternating fashion. Immediately after random allocation, participants were sent a notification, through the On the last day of providing information, the posttest (same contents as the pretest) was administered under the same conditions as the pretest to measure the effectiveness of the intervention. The participants were given a week to answer the posttest. The pretest and posttest were scored separately by summing the number of correct answers divided by the total number of questions and multiplied by 100 (expressed as %), and the difference in the scores were also calculated.Luna Luna operators at MTI Ltd. and investigators at the National Center for Child Health and Development Research Institute.Through administration of a follow-up questionnaire during the week subsequent to the posttest, we also assessed the participants’ satisfaction with the application and information provided, and their usual information-seeking behaviors about fertility treatment. We asked the participants the following questions regarding the application: “How much did you know about the contents (Recognition)?”, “How beneficial did you find it (Benefits)?”, “How satisfied were you with the content (Satisfaction)?”, “Was the content easy to understand (Understanding)?”, “How was the amount of content (Amount of content)?”, and “Were there any points to be improved (Points for improvement)?”. Regarding information-seeking behaviors for fertility-related information, we asked about “Source of information”, “Bothersome issues”, and “Influential factors”. Finally, at the end of the study, all participants were provided with the answers to the test questions, and further explanation was provided about the pretest and posttest as a way to provide them knowledge about fertility treatment. All study implementation procedures were pursued with close coordination between Luna Luna application.Sample size calculations were performed based on detecting a difference of 20% in score between two groups and setting the type I and II error rate at 0.05 and 0.1, respectively. Mean score of in the control group was expected to be about 50%, resulting in a total number of 250 individuals (at a 1:1 scheme resulting in 125 per group). Considering an expectedly high withdrawal rate at follow-up for this application-based study, we empirically assumed that a baseline population of nearly 4000 participants allocated to each group would result in sufficient numbers based on the response and withdrawal rates experienced with previous surveys conducted through the p value of less than 0.05 was considered to be statistically significant for all analyses.Characteristics between the intervention group and the control group were compared using the Pearson’s chi-square test and Fisher’s exact test for categorical variables. Age and test score  were examined using the Mann–Whitney U test. Changes in test score between the pretest and posttest were compared using the Wilcoxon matched-pairs signed rank test. Effectiveness of intervention was analyzed by comparing the changes in test score and proportion answering correctly between the pretest and posttest within the intervention group and control group using the McNemar test. SPSS (IBM) and Prism 8.01 software  were used for the statistical analyses. A two-sided Further information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationReporting Summary


Circular RNAs (circRNAs) are single-stranded RNA molecules that form a covalently closed loop structure. They are characterized by distinct features and multifariously implicated in the regulation of both physiological and pathological states. The aberrant expression of circRNAs has been evidenced in various malignancies. Circular transcripts can effectively modulate gene expression. The most prevalent manner through which circRNAs promote cancer development and progression is their interaction with key components of major signaling pathways. In particular, abnormally expressed circRNAs can dictate the crosstalk between signaling cascades. In recent years, there has been great progress regarding circRNA research in the context of cancer progression, and various regulatory axes have been described. As our knowledge of signaling regulation by circRNAs continuously expands, novel therapeutic approaches can be assessed and established, seeking to overcome clinical challenges, such as the treatment of cancer patients with distant metastasis and those who relapse.Signal transduction is an essential process that regulates and coordinates fundamental cellular processes, such as development, immunity, energy metabolism, and apoptosis. Through signaling, cells are capable of perceiving their environment and adjusting to changes, and most signaling cascades ultimately lead to alterations in gene expression. Circular RNAs (circRNAs) constitute an emerging type of endogenous transcripts with regulatory roles and unique properties. They are stable and expressed in a tissue-, cell-, and developmental stage-specific manner, while they are involved in the pathogenesis of several diseases, including cancer. Aberrantly expressed circRNAs can mediate cancer progression through regulation of the activity of major signaling cascades, such as the VEGF, WNT/β-catenin, MAPK, PI3K/AKT, and Notch signaling pathways, as well as by interfering with signaling crosstalk. Deregulated signaling can then function to induce angiogenesis, promote invasion, migration, and metastasis, and, generally, modulate the hallmarks of cancer. In this review article, we summarize the most recently described and intriguing cases of circRNA-mediated signaling regulation that are involved in cancer progression, and discuss the biomarker potential of circRNAs, as well as future therapeutic applications. Cell signaling can be defined as the fundamental process of extra- and intercellular communication, which controls basic cellular activities that are essential for survival. The ability of cells to receive, process, and respond to signals from their environment is the foundation for their growth, division, differentiation, genetic stability, and cellular homeostasis ,2. ThereGiven that signal transduction is vital for maintaining normal cellular characteristics, the deregulation of signaling pathways is intricately involved in the development of pathological states, such as autoimmunity and cancer ,6. In paThe critical role of signaling pathways is irrefutable in both cancer initiation and progression. In the last decades, the 5-year survival rate of patients with solid tumors and many hematological malignancies has increased, as a result of the clinical advances that allowed earlier detection and local tumor control ,10. HowecircRNAs constitute a type of evolutionarily conserved, single-stranded RNA molecules that form a covalently closed loop, and their continuous structure denotes the lack of exposed 5′ and 3′ ends and polyadenylation tails . The resNumerous evidence supports that circRNAs are correlated with the progression of human diseases through their interaction with signaling pathways, and their biomarker potential has been proved by multiple studies ,17. In pcircRNAs are formed through the back-splicing of a primary transcript, when the 3′ end of an exon (donor site) is directly joined to the 5′ end of an upstream exon (acceptor site)  21,22].,22.21,22In the first case, which is also referred to as “exon-skipping”, circularization takes place during splicing, where circRNAs are formed from lariat intermediate structures . Three dcircRNA formation can also be mediated by RNA-binding proteins (RBPs), through their binding to flanking intronic sites which leads to hybridization. The most common examples include quaking (QKI) , musclebThe main biological functions of circRNAs have been elucidated in both normal and pathological states . The mosMBNL1) contains binding sites for the MBNL1 protein, evidencing that protein production and the circularization of transcripts from the same gene are closely related. Furthermore, circRNAs can decoy and stabilize proteins to a specific cellular compartment, affecting downstream targets. This process includes the binding and retaining of an RBP, which leads to higher expression rates of the protein target genes in this particular compartment , integrins, and ion channels. This binding activates the transmembrane receptors and, subsequently, the signal transduction cascade through cytosolic intermediates, and finally leads to the regulation of transcription/translation of effector genes . Four MADeregulated MAPK signaling is implicated in the progression of a wide range of cancers and occurs via multiple mechanisms, including abnormal expression of RTKs and/or genetic mutations that trigger the activation of RTKs and, thus, of downstream signaling molecules in the absence of appropriate stimuli . NumerouNF1. The well-known tumor suppressor NF1 then promotes the hydrolysis of GTP by RAS GTPase, inactivates RAS and negatively regulates the activity of RAS downstream targets, including the RAF/MEK/ERK signaling pathway. The suppressed RAS signaling fails to activate RREB1, a transcription factor that has been associated with tumor development. This leads to elevated expression of the linear transcript of ITGA7, which further suppresses tumor growth and metastasis, as shown in RIN1 axis. RIN1 can positively regulate RAS signaling at different levels. Therefore, its upregulation induces the survival and proliferation of cancer cells [circRNA-driven MAPK signaling regulation is a hot topic of research, and there are several examples of such interactions in a variety of human malignancies. One such interesting example is the circ-ITGA7-mediated MAPK signaling regulation. This circRNA is downregulated in colorectal cancer, while its upregulation is associated with reduced tumor growth. circ-ITGA7 hinders tumor progression in a dual way, in which the expression of its linear transcript is implicated. Specifically, it sponges miR-370-3p, which leads to an increase in the levels of its target, er cells .In general, circRNAs can affect the MAPK signaling activity at multiple levels, affecting the binding of the ligand to the receptor, the MAPKs themselves, and the regulators of MAPKs as well. For instance, circRNAs can regulate MAPK signaling by affecting the levels of the IGFBP family. These proteins bind to the insulin-like growth factors (IGFs), prolong their half-life, and regulate their tissue distribution. Additionally, they play a key role in normal cell function and are rarely mutated in cancer. However, studies support that IGFBPs can exert either an oncogenic or an onco-suppressive role, via the regulation of the IGFR/MAPK and IGFR/PI3K/AKT signaling pathways . It has NRAS [RASA1 axis; RASA1 is an inhibitor of MAPK signaling. In contrast, NEK2 is an activator that interacts with MAPK1, promotes cell cycle progression, and hence cell proliferation. NEK2 expression was found to be upregulated by circ-PDSS1 in gastric cancer, which stimulated cancer progression [Moreover, circ-AGFG1 upregulates the RAF1 kinase levels, through the sponging of miR-370-3p, thereby initiating the MAPK signaling cascade and enhancing cervical cancer progression . circ-DLNRAS . Regardigression .FAM83A by acting as a ceRNA for miR-330-5p. The high levels of FAM83A, which led to reduced activation of the MAPK signaling pathway and increased cancer cell proliferation [Intriguingly, circRNAs can promote cancer progression by inactivating MAPK signaling as well. A recent study in non-small cell lung cancer revealed that circ-ZKSCAN1 assisted in the upregulation of feration . TherefoAll the aforementioned circRNA interactions are the most intriguing cases out of the several so far investigated, and firmly support the key regulatory role of these novel molecules in the regulation of the MAPK signaling pathway with regard to cancer progression. Although this field is still in its infancy, the complex regulatory networks discussed above provide a new perspective in the matter of metastatic burden. A more detailed list of circRNAs with a regulatory role in MAPK signaling can be found in The PI3K/AKT is an important signaling pathway that is responsible for a variety of cellular activities, such as cell growth, proliferation, differentiation, and migration; this pathway is activated by both EGFR and IGFR signaling. PI3Ks are a family of related intracellular signal transducer enzymes that are activated by the stimulation of RTKs and/or activation of RAS family proteins, while AKT1, a Ser/Thr protein kinase, mediates the PI3K effects on tumor growth and progression. The phosphorylation of AKT1 is associated with cell proliferation and inhibition of apoptosis in several malignancies, since it regulates downstream targets, which promote angiogenesis and cancer progression. The PTEN protein, a known tumor-suppressor that is frequently mutated in cancer, attenuates the activation of the PI3K/AKT pathway. Overall, the PI3K/AKT signaling pathway serves an oncogenic role in the initiation and progression of cancer, and several reports are evidencing that targeted inhibition of this pathway obstructs tumor development .PRKCI. Subsequently, the kinase PRKCI phosphorylates AKT1, which in turn activates the signaling cascade that decreases autophagy and increases the proliferation rate and migration of cancer cells [Various studies, prompted by the key role of this pathway, highlight the implication of circRNAs in PI3K/AKT signaling regulation , while rer cells . ConsideAKT1 [ATL1) in ovarian cancer and osteosarcoma, respectively [MMP2) in papillary thyroid carcinoma [In general, there are circRNAs that influence the PI3K/AKT signaling pathway directly, by affecting the expression of key components of the pathway, such as by sequestering a miRNA that normally binds to AKT1 , or in aAKT1  and circectively . The regarcinoma  and MET arcinoma .Besides the well-known regulators of the PI3K/AKT signaling pathway, HEMGN has been shown to accelerate thyroid cancer cell proliferation rate via the activation of this pathway. HEMGN can facilitate the proliferation and differentiation of hematopoietic cells via the NF-kB, a downstream target of AKT1. Of note, a recent study revealed an alternative way of HEMGN regulation in papillary thyroid carcinoma, where circ-PSD3 sponge miR-637 increases the levels of HEMGN, and activates PI3K/AKT signaling .IGF1R and IGF1, respectively, leading to enhanced PI3K/AKT signaling activity, elevated cancer cell proliferation rate, and EMT [PTEN. Therefore, circ-VRK1 restores the expression levels of PTEN. Consequently, the activity of the PI3K/AKT signaling pathway is obstructed, and tumor progression is hampered as well. Additionally, it was shown that the overexpression of circ-VRK1 could reverse the patients’ radio-resistance, indicating that the targeting of this circRNA can serve as a potential therapeutic approach [It is important to mention that there are circRNAs—for instance, circ_0002577 and circ-RUNX1—which increase the expression levels of  and EMT ,123. Howapproach .SEC61A1) is upregulated in plasma samples of patients with multiple myeloma and exerts an oncogenic role via the miR-338-3p/BRD4 axis [MYC and BCL2. In the same study, it was shown that activation of the PI3K/AKT signaling pathway could be regulated through the aforementioned cascade. Additionally, it was uncovered that exosomes generated from mesenchymal stromal cells (MSCs) could accelerate the progression of multiple myeloma via circ_0007841, but the exact mechanism of action remains unknown [Besides solid tumors, the regulatory role of circRNAs in the PI3K/AKT signaling pathway has been investigated in hematopoietic malignancies as well. More specifically, circ_0007841  was found to have an opposite effect on hepatocellular cancer [TEX2) has an opposite effect on hepatocellular cancer [HAMP, therefore restoring the suppressing role of HAMP on JAK2/STAT3 signaling.The effect of circRNAs on the JAK/STAT signaling pathway has been investigated in several instances so far, in the context of tumor progression . Firstlyr cancer . It imper cancer . It impeVEGFA as well, further supporting the intricate crosstalk between these pathways [BCL2 and FOS.Another feedback interaction was also reported in hepatoblastoma . In partpathways ,47. Morepathways . circ-ZNThe biomarker potential of circRNAs was further supported in gastric cancer, where circ-CUL3 was found to accelerate the Warburg effect progression and impact the overall survival of patients. The Warburg effect, one of the hallmarks of cancer, is an alternative form of cellular metabolism where glucose is mainly converted to lactate . ConsideTGFB1, TGFB2, and TGFB3. In particular, they encode three structurally and functionally similar isoforms ; most frequently, each isoform is simply mentioned as TGF-β. TGF-β ligands bind to type II receptors, which act as Ser/Thr kinases, and recruit and phosphorylate type I receptors, thus forming heteromeric complexes. TGF-β stimulates signaling cascades that are responsible for vital cellular procedures, such as morphogenesis, immune regulation, and differentiation. The main mediators of TGF-β signaling are SMAD transcription factors, which regulate the expression of several genes. The role of this pathway in cancer is rather ambiguous, as its function in early tumor development is suppressive, but later on, the secretion of TGF-β facilitates tumor invasion and metastasis [The transforming growth factor-beta family, mainly known as TGF-β, consists of proteins encoded by three genes, namely tastasis ,144. Alttastasis ,145,146.TRIM33 constitutes a direct target of both miR-429 and miR-200b-3p. In non-small cell lung cancer, high expression levels of circ_0008305 (gene of origin: PTK2) have been reported; this circRNA sponges both these miRNAs and leads to TRIM33 upregulation and subsequent repression of the TGF-β-dependent EMT [ANKS1B) promotes EMT and metastasis of triple-negative breast cancer cells by enhancing TGF-β expression. Mechanistically, this circRNA acts as a ceRNA, similarly to circ_0008305. Specifically, it sponges miR-148a-3p and miR-152-3p [TGF-β promoter and stimulates its transcription [TGFB2 transcription by interacting with elements on its promoter as well [HOXA10 and, subsequently, promotes nasopharyngeal carcinoma progression through the upregulation of TGFB2 [Most circRNAs affecting the TGF-β/SMAD signaling pathway have been shown to regulate EMT. To begin with, an important regulator of the TGF-β/SMAD signaling pathway is the transcriptional repressor TRIM33 (T1F1γ), which acts suppressively on this pathway. Specifically, it represses the TGF-β-dependent EMT in mammary epithelial cells, as well as in non-small cell lung cancer cells. dent EMT . In contR-152-3p . Both thcription . HOXA10, as well . HOXA10 of TGFB2 .ZFR). Its binding to miR-377-3p in bladder cancer cells leads to elevated expression of ZEB2. ZEB2 is implicated in the repression of E-cadherin (CDH1) and thus promotes TGF-β-dependent EMT [ZFR has been reported to promote hepatocellular cancer progression, through the upregulation of β-catenin [Besides directly affecting TGF-β, some circRNAs have been witnessed to affect downstream molecules of this pathway to facilitate EMT. A relevant example is that of circ_0072088  phosphorylate other kinases, namely LATS1 and LATS2, which in turn activate the transcriptional factors YAP1 and TAZ (TAFAZZIN). Normally, this pathway, commonly known as the Hippo/YAP signaling pathway, inhibits cell proliferation and promotes apoptosis [The Ser/Thr kinases STK3 and STK4  increases the expression levels of LATS2, through binding miR-31-5p, as this kinase is downregulated by miR-31-5p. In this way, circ_0000140 promotes YAP1 phosphorylation and inhibits its translocation to the nucleus, leading to suppression of cell growth and metastasis of oral squamous cell carcinoma cells [Most of the investigated circRNAs that are indirectly involved in this pathway contribute to cancer initiation; however, few circRNAs affecting Hippo/YAP signaling are also associated with tumor progression. Firstly, circ_0000140  are found in mammals. Upon binding to the respective ligands, the active intracellular domain of NOTCH receptors is translocated to the nucleus, in order to interact with transcription factors. The role of Notch signaling in cancer is ambivalent, as it has been reported to act either onco-suppressively or onco-promotively .NOTCH1 expression is inhibited by this miRNA, high levels of circ-NFIX in glioma cells triggers the upregulation of NOTCH1 and, in this case, the progression of the disease [CBFA2T2) affects bladder cancer progression by increasing the levels of the mRNA encoded by its parental gene, through miR-155-5p and miR-330-5p sponging. CBFA2T2 (MTGR1) acts as a Notch signaling suppressor and, in this case, Notch-mediated signaling repression leads to the reinforcement of the migratory and invasive capabilities of bladder cancer cells [Regarding the role of circRNAs in Notch signaling, they either directly affect the receptor and/or its ligands, or alternatively other regulators of the pathway. A characteristic example is that of circ-NFIX, which acts as a sponge for miR-34a-5p, a miRNA known for its tumor-suppressive role. As  disease . Within  disease . Lastly,er cells . These dEach signaling pathway does not act independently of the others; there is significant crosstalk between them, leading to the formation of a complex signaling network. The aberrant interplay between pathways is an issue that, when it is better explored and understood, can lead to breakthroughs regarding cancer progression. A representative part of this broad network initiates from RTKs; typical RTK members are EGFR, VEGFR, MET, and DDR2. RTKs activate FAK and SRC tyrosine kinases, which are mediators for many signaling pathways, including the PI3K/AKT and MAPK, while FAK also interacts with the WNT/β-catenin pathway ,155.DDR2, leading to DDR2 upregulation. In this way, the activity of vital downstream signaling pathways is deregulated, including that of MAPK and PI3K/AKT, resulting in disease progression [MET, results in increased phosphorylation of its downstream targets, namely AKT1 and MAP2K7, facilitating invasion and migration of esophageal cancer cells [Regarding the role of circRNAs in the context of the intricate signaling network, some of them have been reported to directly affect the function of an RTK ,134. Thegression . In thiser cells .G3BP2, enhancing its translation. circ-FNDC3B acts as a sponge for miR-1178-3p, leading to the downregulation of G3BP2 and, consequently, to the inhibition of the FAK/SRC-mediated signaling [STEAP4. The respective protein binds to FAK and prevents its auto-phosphorylation at tyrosine 937 (Y937), affecting multiple downstream signaling pathways [FAK gene was shown to affect the TGF-β/SMAD pathway, as described above [In other cases, circRNAs have been evidenced to affect the kinase complex FAK/SRC, which has been characterized as signaling mediator. More specifically, in bladder cancer, two circRNAs have been reported to disturb FAK/SRC-mediated signaling and result in reduced tumor invasion and metastasis. G3BP2 constitutes a member of the RAS GTPase-activating proteins and is involved in tumor invasion by promoting SRC and FAK phosphorylation. Interestingly, miR-1178-3p binds to the 5′ UTR of ignaling . Similarpathways . These red above . This fiCDC5L; the respective protein subsequently upregulates the non-receptor tyrosine kinase PEAK1. In turn, PEAK1 phosphorylates MAPK1 and MAPK3, as well as JAK2, leading to ovarian cancer progression [Finally, some circRNAs affect key components of multiple signaling pathways, which, interestingly, interact with each other. For instance, extensive crosstalk has been evidenced between the JAK/STAT and other pathways, such as MAPK and PI3K/AKT, as previously discussed , and it gression . Similargression . These cConsidering the multiple biological functions of circRNAs, it is not surprising that numerous researchers have focused on elucidating their distinct properties. For this reason, their implication in the regulation of aberrant signaling, cancer onset, progression, and therapy resistance has repeatedly been demonstrated ,35, and The research for novel molecules that can serve as effective cancer therapeutic agents is ongoing, and various approaches have designated circRNAs as promising therapeutic targets. It has been demonstrated that when the activity of oncogenic circRNAs is obstructed, tumor progression is halted and cancer cell apoptosis rate is increased . FurtherMoreover, an increasing number of studies emphasize the participation of circRNAs in the regulation of signaling pathways linked to cancer stem cells (CSCs) . CSCs arAnother promising field of circRNA research includes viral circRNAs. Even though circular transcripts were firstly reported in RNA viruses, viral-derived circRNAs have only recently caught the attention of researchers. Several circRNAs are encoded by highly tumorigenic viruses, such as the Epstein–Barr virus, the Kaposi’s sarcoma-associated herpesvirus, the human papillomavirus, and the hepatitis B virus . MoreoveDespite the great progress in research efforts, several limitations remain unsolved. For instance, most studies focus on identifying the downstream targets of a circRNA/miRNA axis, and neglect to assess the availability of the circRNA or the number of binding sites that it possesses; many miRNA sponges are expressed in low copy numbers and are insufficient in exerting a regulatory role. Furthermore, circRNAs are still far from being incorporated into standard clinical practice, and the complicated experimental procedures required for recommending circRNAs with biomarker potential is a major reason for this. Advances in RNA sequencing have allowed breakthroughs in accurate circRNA detection in cancer patients’ samples; however, it is an expensive and time-consuming method, and more studies should explore easier and user-friendlier methods. More large-scale studies are required as well, prior to affirming the utility of tumor-specific circRNAs in cancer diagnosis, prognosis, and treatment. Nonetheless, the research interest for circRNAs is rapidly growing, and our understanding of these molecules and their intricate functions will be more thorough as more studies are conducted and better analysis pipelines are described.Overall, circRNAs interact multifariously with major signaling pathways and affect the progression of cancer. They can regulate angiogenesis through the VEGF signaling pathway, cancer cell proliferation through the WNT/β-catenin signaling pathway, tumor growth and invasion through the MAPK signaling pathway, and apoptosis through the Hippo/YAP signaling pathway, to name a few. Evidently, signal transduction is not limited to the function of a single pathway; several pathways intertwine and work in conjunction to drive cancer progression, and circRNAs reinforce or hinder this crosstalk in various ways. Future studies are expected to further establish circRNAs as effective biomarkers to monitor cancer progression, create more opportunities on targeted therapies, and shed light on intriguing circRNA aspects that remain elusive.


Despite the known importance of this neuron-immune axis in CNS function and pathology, until recently it was not clear whether this signaling axis played a role in macrophage chemotaxis and SGN survival following cochlear injury. In this review, we will explore the importance of innate immunity in neurodegenerative disease development, specifically focusing on the regulation of the CX3CL1-CX3CR1 axis, and present evidence for a role of FKN signaling in cochlear neuroprotection.Immune system dysregulation is increasingly being attributed to the development of a multitude of neurodegenerative diseases. This, in large part, is due to the delicate relationship that exists between neurons in the central nervous system (CNS) and peripheral nervous system (PNS), and the resident immune cells that aid in homeostasis and immune surveillance within a tissue. Classically, the inner ear was thought to be immune privileged due to the presence of a blood-labyrinth barrier. However, it is now well-established that both vestibular and auditory end organs in the inner ear contain a resident  population of macrophages which are the phagocytic cells of the innate-immune system. Upon cochlear sterile injury or infection, there is robust activation of these resident macrophages and a predominant increase in the numbers of macrophages as well as other types of leukocytes. Despite this, the source, nature, fate, and functions of these immune cells during cochlear physiology and pathology remains unclear. Migration of local macrophages and infiltration of bone-marrow-derived peripheral blood macrophages into the damaged cochlea occur through various signaling cascades, mediated by the release of specific chemical signals from damaged sensory and non-sensory cells of the cochlea. One such signaling pathway is CX The immune system is the body’s defense mechanism against pathogens and tissue injury. The immune system consists of an intricate network of cells and molecules that work together to protect the body from damage. These cells include lymphoid cells, such as T-cells, B-cells, and NK cells, and myeloid cells, such as monocytes, dendritic cells, macrophages, and granulocytes. Most immune cells, including T/B-cells, monocytes, and macrophages can be found under physiological conditions circulating in the blood. These cells have surface receptors that act in surveillance of the body searching for chemical signals that are released from damaged cells and tissues Chaplin, . These “Classically, the study of neurodegenerative diseases has focused on the pathological progression of the disease, specifically targeting toxic protein aggregation leading to neuronal dysfunction and death, rather than the underlying disease mechanism  and its cognate receptor (CX3CR1). FKN is a unique transmembrane protein that is constitutively expressed by neurons and endothelial cells . Finally, we will postulate further work that needs to be conducted to determine the mechanisms by which macrophages and FKN signaling mediate neuroprotection during cochlear damage, to harness these protective capabilities clinically.First discovered by Elie Metchnikoff in the late 19th century, macrophages are the main effector cells of innate immunity that are derived from precursor monocytes Tauber, . The maiCsf1) which is a cytokine required for differentiation of hematopoietic stem cells into macrophages and labels yolk sac-derived macrophages. Alternatively, fetal liver-derived macrophages differentiate from the cluster of differentiation molecule 11b (CD11b) expressing progenitor monocytes. Yolk sac-derived macrophages originate around mouse embryonic day 7.5 (E7.5) and travel to the otocyst around mouse E10.5. Fetal liver-derived macrophages travel to the developing cochlea around mouse E14.5  mice revealed drastically reduced density of macrophages in the spiral ligament and spiral ganglion, suggesting that these macrophages predominantly originate from the yolk sac  content of cochlear endolymph, creating an environment that is likely toxic for macrophage survival. Under normal conditions, resident macrophages in the cochlea display site-specific morphology. Apical macrophages are characterized by dendritic morphology with long and thin processes (pseudopodia). This dendritic morphology is characteristic of macrophages in surveillance mode , stria vascularis (SVA), spiral ligament, spiral limbus, basilar membrane, and spiral ganglion  on POU class 4 transcription factor 3 (Pou4f3) expressing inner hair cells (IHCs) and outer hair cells (OHCs) to selectively ablate hair cells upon diphtheria toxin (DT) administration , with the X representing the presence of an amino acid  is unique in that it only contains one analog, FKN  signaling, where ADAM10 cleavage is constitutive in unstimulated cells  high expressing monocytes have low levels of CX3CR1, and CCR2 low expressing monocytes express high levels of CX3CR1 , CD14highCD16−) which express low levels of CX3CR1, and non-classical monocytes (CD14lowCD16+) which express high levels of CX3CR1 , also known as CX3CR1GFP/GFP or CX3CR1 KO  on astrocytes  in the substantia nigra pars compacts (SNpc) of the midbrain, resulting in severe motor, balance, and coordination deficits. In PD, loss of dopaminergic neurons is precipitated by intraneuronal aggregation of α-synuclein, commonly referred to as Lewy bodies  death and increased levels of proinflammatory interleukin 1 beta (IL-1β). These results suggest that in a DR mouse model, intact FKN signaling is neuroprotective towards RGCs by reducing microglia activity and diminishing proinflammatory mediator production. Furthermore, in a 2016 study by Mendiola et al. (Diabetic retinopathy (DR) is a common microvascular disorder associated with diabetes and a leading cause of blindness worldwide  is a genetic mutation disease characterized by the progressive degeneration of photoreceptors and is a main cause of blindness worldwide, specifically in children  and MS. CX3CR1 has two single nucleotide polymorphisms (SNPs) in humans: V249I and T280M , Stojkovic et al.  on all cells as a model to disrupt FKN signaling to understand its function in macrophage chemotaxis in the injured cochlea  and CX3CR1 heterozygous  mice with aminoglycoside kanamycin to induce ototoxicity. The number of macrophages increased after kanamycin-induced hair cell death however, the degree of increase was not significantly different between CX3CR1 heterozygous and KO mice, and rather was found to be higher in the spiral ligament in certain cochlear frequency locations of the CX3CR1 global KO mice , resulted in a significant loss of OHCs in the 5–16 kHz range of the cochlea. This loss of OHCs correlated to elevated ABR thresholds in CX3CR1 global KO animals in the same tonotopic cochlear regions  potentials which is necessary for the release of excitatory glutamate from IHC. This, in turn, inhibits action potential generation in afferent cochlear SGN fibers, and therefore hearing is lost  when compared to animals with intact FKN signaling. Additionally, in this study, they also used a model of Kanamycin/Furosemide-induced ototoxicity  and distortion product otoacoustic emissions (DPOAE) thresholds) without causing sensory hair cell death. Interestingly, despite the complete recovery of ABR and DPOAE thresholds, they found that suprathreshold ABR Wave 1 amplitudes never fully recovered. This was supported by histological analysis showing permanent loss of IHC ribbon synapses, and progressive SGN death. Clinically, the loss of IHC ribbon synapses causing SGN death has profound effects on sound localization and understanding speech in noisy environments . Such noise trauma resulted in a rapid degeneration of ~50% of IHC-ribbon synapses in the mid-basal and basal regions of the cochlea and an increase in macrophage numbers in the IHC synaptic region despite any evident hair cell death. The noise-damaged synapses gradually repaired in mice with intact FKN signaling, however the absence of CX3CR1 impaired such spontaneous synaptic repair that correlated with reduced ABR Wave 1 input-output function (amplitudes). Altogether, this work suggests that FKN signaling also plays a vital role in the spontaneous repair of damaged ribbon synapses. However, further work needs to be done to determine the exact roles of macrophages and FKN signaling in ribbon synapse degeneration and repair in order to develop novel immuno-therapies for hidden-hearing loss. In the final section, we will look at some of the work being done examining the therapeutic benefits of FKN overexpression on a variety of neurodegenerative diseases.The idea of “hidden hearing loss” was first described by Kujawa and Liberman  in 2009,3CR1, this signaling axis has the potential to be an intriguing therapeutic target for any disease involving chronic neuroinflammation. In this section, we will investigate some of the work being done on targeting the FKN signaling to treat neurodegenerative diseases.The previous sections outlined the multitude of studies surrounding the role FKN signaling has in modulating the immune response during neurodegenerative disease development and progression. Due to the role of FKN signaling in microglia/macrophage activation, as well as FKN’s exclusivity for its receptor CXvia AAV serotype 2 (AAV2) delivery led to a rescue in cognitive function, as seen through novel recognition tasks and radial arm water maze behavior, in the same mouse model of tauopathy. This suggests exogenous FKN expression can rescue pathology associated with tauopathy. Furthermore, a 2011 study by Pabon et al. (3CR1 receptor agonists, to target the FKN signaling axis could prove to be a viable treatment option for a variety of neurodegenerative diseases.In 2013, Nash et al. , they fon et al. , where An et al. . Finallyn et al.  showed t3CR1 SNPs, there is diminished recruitment of peripheral macrophages and dysregulated production of proinflammatory mediators, leading to unregulated phagocytosis and SGN death. If accurate, these hypotheses would support the viability of exogenous FKN as a therapeutic mechanism to treat inner ear pathology. However, to fully determine the role of FKN signaling in cochlear physiology and pathology, the following questions need to be carefully addressed:The study of immune dysfunction continues to accumulate evidence for a primary role in the development and progression of neurodegenerative diseases. Specifically, interest in immune signaling pathways has grown, due to their involvement in tissue development, tissue homeostasis, and immune surveillance. Dysfunction in these communication pathways can result in neurotoxicity, resulting from unregulated immune activation. Evidence in several neurodegenerative diseases, including AD, PD, MS, and diseases of the retina suggests FKN signaling is responsible for dampening microglia activation, resulting in neuroprotection. Emerging research also suggests FKN signaling plays an important role in inner ear neuroprotection. However, the question remains, what is the specific role of macrophages and FKN signaling in cochlear pathology as well as other neurological conditions? We hypothesize that FKN signaling plays dual roles during cochlear pathology, as a chemokine and a checkpoint molecule. As a chemokine, FKN signaling mediates cochlear sensory and neuron protection by recruitment of anti-inflammatory and pro-healing macrophages from the circulation to the site of damage, which may be context or injury dependent. As a check point molecule, FKN signaling modulates the cochlear inflammatory state by regulating the inflammatory and phagocytic phenotype of resident and circulating macrophages. These functions may be inclusive or exclusive . However3CR1. Therefore, determining the effects of presence and absence of FKN ligand in the normal and damaged cochlea and in oto- and neuroprotection and the factors that regulate the endogenous levels of soluble and membrane-bound FKN in the injured cochlea are equally vital.What is the role of the FKN ligand in normal and damaged cochlea? To this point, FKN’s role in modulating cochlear pathology has only been investigated in terms of the presence or absence of its cognate receptor CX3CR1 expressing resident and recruited macrophages in SGN survival? CX3CR1 is expressed on both cochlear resident macrophages and on macrophages that migrate to the damaged cochlea from blood circulation. However, to what extent CX3CR1-expressing resident and/or recruited macrophages contribute towards the long-term survival of SGNs in the damaged cochlea remains to be determined.What is the precise role of CXIs FKN a chemotactic signal for resident or recruited macrophages in the injured cochlea? The FKN signaling has been explored elsewhere in the body for its role in chemotactic signaling to recruit additional immune cells to sites of tissue damage. Following cochlear trauma, there is an infiltration of circulating monocyte-derived macrophages to the site of injury. However, it is unclear if the FKN signaling axis is responsible for this migration of macrophages to the cochlea. Additionally, it is unclear if within the cochlea, FKN signaling acts as a chemotactic signal to draw additional resident macrophages from other regions of the cochlea to the site of cellular damage.3CR1 regulate cochlear inflammation and immune response to promote SGN survival following injury? It is not known if FKN signaling is directly responsible for protecting SGNs following trauma, and furthermore, if this neuroprotection is related to its regulation of proinflammatory mediator production and macrophage phagocytosis in the injured cochlea.Does CX3CR1 SNPs in acquired SNHL? Studies have shown a causal link between human CX3CR1 SNPs and increased susceptibility to various neurodegenerative diseases. However, it remains unknown if these SNPs are also associated with the development, progression, or severity of cochlear pathology and hearing loss in humans.What is the association of human CX3CR1 activity be used as a viable therapeutic target for SGN survival and ribbon synapse regeneration? Based on the number of studies indicated above on the therapeutic viability of using AAV or peptides to overexpress soluble FKN for the treatment of neurodegenerative diseases, it will be of great interest to examine the effects of overexpression of FKN as a viable treatment option for the survival of SGNs and regeneration of damaged ribbon synapses following cochlear trauma. Such putative therapeutics options may be a viable treatment for hidden-hearing loss.Can FKN overexpression or increase in CXAddressing the above questions, we will better understand the role FKN signaling has on immune regulation in the inner ear during cochlear trauma. By doing so, we can begin to develop therapeutic targets for not only prophylactic protection but post-trauma recovery. Together, understanding the role of FKN signaling in inner ear pathology has great promise for unraveling the mysteries tied to the treatment of hearing loss.AS reviewed the literature and wrote the manuscript. TK wrote and edited the manuscript. 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.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.


Phytochromes are targets of diverse PTMs; among them, phosphorylation was identified decades ago  Although the consequences of phosphorylation at certain amino acids on the function of phytochrome proteins were examined, the detailed phytochrome “phospho-map” is still missing. Furthermore, at the moment we do not know how the interplay of different PTMs fine-tune phytochrome signaling. (ii) We also do not know to what extent PHY autophosphorylation, and the activity of different kinases and phosphatases are responsible for the actual phospho-state of the available phytochrome pool and the performance of phytochrome signaling. Further targets of phytochrome kinase activity and its significance on the signaling process also need to be identified in the future. (iii) Two studies in this Research Topic focused on the molecular structure of phytochromes and their partners indicating the continued interest in the subject. Despite recent advances, we still need to achieve high resolution structural models of full length plant phytochromes in Pr and Pfr to understand the overall structural rearrangements during photoconversion. (iv) Hopefully, this structural information will allow designing structure-function models for phytochrome signaling that could be tested also experimentally. (v) Long-lasting efforts from the scientific community resulted in the identification of phytochrome interacting partners. We expect that examining the phytochrome containing photobodies will help us to understand the interaction dynamics of phytochromes with their signaling partners and to map these signaling networks in details. (vi) Furthermore, it is crucial to examine the role of phytochromes not only in light signaling but also in signal interactions of interconnecting pathways in order to understand how plants respond to complex environmental stimuli.We, the Guest Editors of this article collection, believe that the set of papers published here stimulates further discussions and initiates studies answering the pending questions.AV wrote the manuscript. CK, AH, and FN corrected and contributed to the text. All authors agreed in the final version.This work was supported by grants from the Hungarian Scientific Research Fund  and the German Research Foundation (DFG) under Germany's Excellence Strategy .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.


Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, and Aspergillusspecies. Since antibiotic treatment alone failed to resolve the infection, percutaneous drainage of the infected bulla was performed. Although a prolonged treatment period was necessary, the infected lung bulla and the lung abscess were eventually resolved. During her illness, the patient also developed arthritis, possibly related to the HTLV-1 infection. Thus, persons infected with HTLV-1 can develop refractory infections, myelopathy, and arthritis. Percutaneous drainage is an option to treat refractory infected lung bullae.We present a case of a 54-year-old Japanese woman with established human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy who developed a refractory infected lung bulla and lung abscess caused by  Human T-lymphotropic virus type 1 (HTLV-1), a human retrovirus discovered in 1980, is a causative pathogen of adult T-cell leukemia/lymphoma (ATL) and several HTLV-1-associated organ dysfunctions, including myelopathy, pulmonary diseases, and arthritis. HTLV-I is primarily transmitted by breastfeeding, although can spread via blood transfusion, sharing of needles, and sexual intercourse. HTLV-1 is distributed globally and is estimated to infect five to 10 million people worldwide . In thesHTLV-1-associated myelopathy (HAM) is characterized by progressive spastic weakness of the lower limbs, neurogenic bladder, and lower back pain, thus imposing a substantial health burden . The lifA 54-year-old Japanese woman was transferred to Hamanomachi Hospital, Fukuoka, Japan, for persistent fever with chest imaging abnormalities. She had a smoking history of 30 pack years. She had been diagnosed with oropharyngeal cancer and had been treated with chemoradiotherapy five years earlier. Two years prior to this clinical presentation, the patient had an established diagnosis of HTLV-1-associated myelopathy (HAM), with neurological findings of neurogenic bladder, orthostatic hypotension, bilateral lower limb spasticity, increased deep tendon reflexes, and positive bilateral Babinski reflexes, as well as positive serum and cerebrospinal fluid tests for anti-HTLV-1 antibodies.Post diagnosis of HAM, the patient experienced recurrent episodes of aspiration pneumonia and had a gastrostomy placed a year and a half earlier. A year earlier, she developed acute progressive HTLV-1-related myelopathy of bilateral lower limbs and was treated with methylprednisolone 1000mg pulse therapy, followed by 5mg of prednisone maintenance therapy at Fukuoka Central Hospital, Fukuoka, Japan. In the outpatient clinic at Fukuoka Central Hospital, she presented a fever with mild sputum. Her chest computed tomography (CT) showed infiltration in the upper right lobe and she was diagnosed with pneumonia. Despite treatment with levofloxacin for five days, a high fever persisted, and she was admitted to Fukuoka Central Hospital.At Fukuoka Central Hospital, she was placed on total parenteral nutrition, suspected of aspiration pneumonia, and was treated with tazobactam/piperacillin (TAZ/PIPC), followed by meropenem (MEPM) and vancomycin (VCM). She was also suspected to have vasculitis from the findings of purpura on her both lower limbs, and the corticosteroid dose was temporarily increased. Despite this effort of examinations and treatment for one month, her fever, high levels of serum C-reactive protein (CRP), and chest imaging abnormalities persisted. She was then transferred to Hamanomachi Hospital for further investigation and treatment on hospital day 28 Figure .2 98% on room air. Crackles were not detected in her chest. Persistent, increased levels of CRP (7.7 mg/dL) and a white blood cell count of 6400 /μL were noted , which were detected by sputum culture at Fukuoka Central Hospital. The differential diagnoses were opportunistic infections such as aspergillosis, mycobacteriosis, vasculitis, HTLV-1-associated bronchioalveolitis, and adult T-cell leukemia infiltration.On admission to Hamanomachi Hospital, she was sarcopenic with a body mass index of 13.5. She had muscle weakness of manual muscle testing (MMT) of 2-3/5, mainly in her lower limbs, which were the same levels of her baseline. Her respiratory condition was normal, with a respiratory rate of 15/min and SpOPseudomonas aeruginosa and MRSA, consistent with the sputum culture at Fukuoka Central Hospital and on admission to Hamanomachi Hospital. Neither Aspergillus species nor Mycobacterium species was detected. Hematoxylin and eosin staining of TBLB showed bronchial tissues with mild to moderate chronic inflammation and lymphocyte-predominant infiltration  and galactomannan (0.1 index) were negative.Based on these results, the patient was initiated with antibiotics . She was also treated with 5 g/day of intravenous immunoglobulin for three days, considering her immunosuppressive condition. However, she remained febrile with the upward trend of CRP. Considering the refractory state of the infected lung bulla, we performed percutaneous ultrasound-guided drainage of the bulla on day 38, following daily washing using sterile saline  and the side effects of the antibiotics. Amikacin was used for the combination treatment of drug-resistant Pseudomonas aeruginosa from day 60. Complications arising from anti-MRSA drugs included serum AST/ALT elevation caused by VCM, pancytopenia triggered by linezolid, and serum creatine kinase (CK) elevation (>1000 U/L) in response to daptomycin , but no elevation of anti-CCP antibodies (0.8 U/mL). Joint ultrasound showed active synovitis in both hand joints. The differential diagnosis of her poly-arthritis was antibiotic use-related arthritis, sepsis-related arthritis, pseudogout crystal arthropathy, rheumatoid arthritis, and HTLV-1-associated arthritis. She was treated with non-steroidal anti-inflammatory drugs (NSAIDs). Corticosteroids or other immunomodulating drugs could not be used until the infected bulla and lung abscess were completely healed. Her poly-arthritis persisted several months after the transfer to Fukuoka Central Hospital. From these results and clinical course, she was considered to have rheumatoid arthritis or HTLV-1-associated arthritis, which had been suppressed by the corticosteroid treatment.Thereafter, the fever gradually resolved, and the inflammatory response trended downwards. Chest imaging showed improvement of infiltration and reduction of the size of the infected bulla Figure , panel CWe describe an HTLV-1-affected patient who developed several complications. The patient needed to be transferred to Hamanomachi Hospital for further investigation of uncertain lung abnormalities with inflammation, since there were several other differential diagnoses, such as mycobacteriosis, ANCA-associated vasculitis, HTLV-1-associated bronchioalveolitis, and adult T-cell leukemia infiltration . We wereThe patient had numerous lung bulla. Her smoking history might be the main reason for the bullous change. Further, HTLV-1-infected status might also have caused developing such a giant bulla, since HTLV-1 associated bronchioloalveolar disorder often indicates progression of air trapping .Regarding lung pathology, we observed lymphocyte-predominant inflammation from lung tissues in the right upper lobe. Typically, biopsies of lung abscesses show neutrophilic granulocytes with dilated blood vessels and inflammatory edema in the acute phase . In the We performed percutaneous catheter drainage of the infected bulla. There were concerns regarding potential complications, e.g., pneumothorax or bleeding, of percutaneous drainage for lung parenchyma, which differs from drainage for pyothorax. However, 10 days after transfer to Hamanomachi Hospital, we determined that the infection could not be improved without drainage. Several reports have documented the utility of percutaneous drainage of infected bullae -14. FurtDuring admission, the patient developed arthritis of multiple joints, which was later considered to be rheumatoid arthritis or HTLV-1-associated arthritis. The prevalence of HTLV-1 infection is higher in patients with rheumatoid arthritis than in healthy controls . ChronicHTLV-1 is a causative pathogen of ATL and several HTLV-1-associated organ dysfunctions, including HAM, bronchioalveolitis, and arthritis. The case highlights that these HTLV-1-associated organ dysfunctions can be complicated in one patient, although no case reports have been published. The case also highlights the importance of the infectious disease principle of "source control," that is, percutaneous drainage for the treatment of refractory infected lung bullae.


Erk1/2 in the cranial mesenchyme results in a diminished domain of osteoblast marker expression and increased expression of cartilage fate markers and ectopic cartilage formation in the frontal bone primordia. Second, we show that mesenchyme Wnt/β-catenin signaling and Wntless are required for ERK activation in calvarial osteoblasts. Third, we demonstrate that Wnt and ERK signaling pathways function together to repress SOX9 expression in mouse cranial mesenchyme. Our results demonstrate an interaction between the Wnt and ERK signaling pathways in regulating lineage selection in a subset of calvarial cells and provide new insights into Wnt-dependent cell fate decisions.Wnt signaling regulates cell fate decisions in diverse contexts during development, and loss of Wnt signaling in the cranial mesenchyme results in a robust and binary cell fate switch from cranial bone to ectopic cartilage. The Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) and Wnt signaling pathways are activated during calvarial osteoblast cell fate selection. Here, we test the hypothesis that ERK signaling is a mediator of Wnt-dependent cell fate decisions in the cranial mesenchyme. First, we show that loss of  The development of the roof of the skull, the calvaria, involves several signaling pathways, differentiation program factors, and cellular movements in the supraorbital arch (SOA) mesenchyme (SOM) ,2. The fRunx2 by calvarial osteoblast progenitors starting at E11.5 in the mouse. Ossification and differentiation of the bone progenitors into committed osteoblasts is specified by Osx downstream of Runx2 by E14.0. These osteoblasts secrete bone-specific extracellular matrix proteins which results in mineralized and ossified bone formation [2+ signaling pathways. Importantly, canonical Wnt signaling is necessary in providing the osteo-inductive cue that initiates the calvarial differentiation program and inhibits cartilage fate [Calvarial differentiation is regulated by a sequence of transcription factors and signaling pathways. The bone transcription program begins with expression of ormation ,6,7. Sigormation . Wnt-depage fate . Canonicage fate ,9,10. Asage fate ,13,14,15age fate ,17,18. Sage fate , and actThe ERK  signaling pathway cross-talks with Wnt/β-catenin signaling during endochondral osteoblast differentiation in vivo and in vitro, and may have roles in chondrogenic repression ,20. ERK1Prx1-cre; βfl/fl-catenin embryos exhibit ectopic cartilage formation in the periosteum of long bones, ectopic cartilage and loss of bone mineralization in the skull and calvarial mesenchyme in vitro and in vivo [Similar to Wnt signaling , Wls flox  to activate CreER and conditionally delete Wls or Erk2 in the cranial mesenchyme. Embryos were harvested and processed for frozen sections as previously described [Mouse lines used in the conditional functional studies include  Joyner, ), PDGFRαrd Lang, ), β-Cate deleted , β-Catenl floxed . Erk1−/−[Erk1−/− , and Erkrk2fl/fl  were graescribed . For eacEmbryos were fixed in 4% paraformaldehyde at 4 °C, sucrose dehydrated, and embedded in O.C.T. Compound  and sectioned at 14 μm as previously described ,40. H&E Immunofluorescence (IF) on cryosections was performed as previously described , and secFor the p-ERK1/2 IF data, tyramide signal amplification using TSA plus Cyanine3.5 kit  was used to amplify the signal. Cryosections were dried at room temperature, washed in PBS then underwent antigen retrieval at 60 °C for 20 min in a citrate buffer. Slides were washed in 1X TBS-T (TBS + 0.5%Tween-20) then blocked in 3% peroxide for 10 min, blocked with goat serum, and incubated in primary antibody overnight. The slides were then washed in 1X TBS-T and incubated in HRP-conjugated goat-anti-rabbit secondary  for 45 min. The slides were then incubated with the TSA Plus Working solution as per the manufacturer’s recommendations. Following amplification, the slides were washed with DAPI (0.5 µg/mL) and mounted with Fluoroshield . Images were captured on confocal microscopy as described above.Fiji/ImageJ was used to generate z-stacks for confocal images max-projected to the inner 5 microns. Each image was set to 8-bit and background removed with a 10-pixel “rolling ball”. The signal threshold was set to . “Watershed” was utilized to separate individual nuclei, which were then counted using the “Analyze Particles” function . For nuclear protein expression patterns , data were analyzed as a ratio of targeted nuclei to DAPI stained nuclei per region of the SOA (hand traced). For the non-nuclear signal (p-ERK1/2), total corrected fluorescence was measured using Fiji/ImageJ . Total chttps://www.molecularinstruments.com/products, Los Angeles, CA, USA) following the manufacturer’s protocol. The Col2a1 probe utilized in the assay was designed by Molecular Instruments.HCR–in situ hybridization was performed on cryosections using HCR V3.0 bundle set designed by Molecular Instruments (Erk1 (Mm01278702_gH), Erk2 (Mm01171077_m1), Lef1 (Mm00550265_m1), Tcf1 (mm00493445_m1), Axin2 (Mm00443610_m1), Sox9 (Mm00448840_m1), OSX/Sp7 (Mm04209856_m1), Runx2 (Mm00501584_m1), Col2a1, Aggrecan (Mm00545794_m1), β-actin , Pdgfrα (Mm00440701_m1), and Wls (Mm00509695_m1).The relative expression of mRNA was analyzed using RT-qPCR as previously described . EmbryosDDPCR was used to quantify copy number of target transcripts. cDNA isolated as described above was used for all samples/targets. Reaction mixtures included ddPCR Supermix for Probes (no dUTP) , target probe (1 µL), and sample cDNA  to a final volume of 20 µL. Each reaction was loaded into a sample well of an eight-well disposable DG8 cartridge  along with 70 µL Droplet Generation Oil for Probes  then covered with a gasket and loaded into a QX200 Droplet Generator  to form droplets. Droplets were then transferred to a 96-well ddPCR plate , heat-sealed with foil, and amplified using a thermocycler using the following parameters: 95 °C for 10 min, 40 cycles of 94 °C for 30 s then 60 °C for 1 min, then 98 °C for 10 min. Completed PCR samples were loaded into the QX200 Droplet Reader  and droplets were analyzed per the manufacturer’s instructions. Data were analyzed using QuantaSoft software .Enriched cranial mesenchyme was isolated as described above at E12.5 and protein was isolated as previously described . Proteinp-values for statistical tests in all figures are represented as * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.All graphs and statistical analysis were generated using GraphPad Prism version 9.0.2 for Windows . Data are presented as mean ± SEM in all graphs. All pairwise sample comparisons were performed using a Student’s t-test or Mann–Whitney test where indicated. The PdgfrαCreER is active broadly across the cranial mesenchyme as early as E9.0 as previously described [CreER recombination in the mouse cranial mesenchyme prior to calvarial osteoblast lineage decisions by administering tamoxifen at E8.5 and E9.5 (Erk2 in an Erk1 null background (CM-Erk1/2) (Runx2 and Osx (Sp7)  B. We fir-Erk1/2) C,D. We fsx (Sp7) . Though eoblasts E,F, we ocontrols . Despite mutants G,H. Thisradation .Erk1/2 mice. At E16.5, the CM-Erk1/2 mutant exhibits gross morphological defects including rounded skull, cleft lip/palate, stunted jaw and snout, polysyndactyly, and overall smaller stature   A,B. In cErk1/2KD E,F, democontrols G,H,M. Ecrimordia I,J. Exprrimordia K,L. We dEn1Cre as previously described [En1Cre; βΔ/+-catenin control, phosphorylation of ERK1/2 proteins was diminished by 92% in En1Cre; βΔ/fl-catenin (CM-β-catΔ/fl) cranial mesenchyme  is a transmembrane protein that is specifically required for secretion of all Wnt ligands [Crect, a surface ectoderm restricted Cre line [Wls in the surface ectoderm which results in complete loss of Wnt signaling in the underlying cranial mesenchyme while leaving β-catenin intact   A,B. At Eat E12.5 C–E. Nextcontrols C–E. TotaWls resulted in discriminate changes in the level of canonical Wnt signaling before cell fate decisions in the cranial mesenchyme. Wnt ligands, inhibitors, and target genes are patterned in distinct domains in the cranial mesenchyme. Graded expression of nuclear β-catenin protein, Axin2 mRNA, and Lef1 mRNA in the cranial mesenchyme between E11.5–12.5 suggest that ectoderm Wnts contribute to a gradient of Wnt signaling response in the mesenchyme [Tcf1 and Lef1, require different levels of Wnt signaling for transcription [Axin2, Tcf1, and Lef1 mRNA. We saw a significant decrease in copy number of Tcf1 (−33%) and Axin2 (−25%) in our mutants compared to the controls, while there was no effect in Lef1 copy number  in two different Cre+ triple compound heterozygotes (Erk1/2KD and CM-Wls mutants, indicating that heterozygosity of the β-catenin and Erk1/2 alleles is sufficient to phenocopy loss of ERK and loss of mesenchyme Wnts at the level of SOX9 repression (Finally, to test if β-catenin and the ERK signaling pathway genetically interact in the cranial mesenchyme, we generated a novel compound heterozygous conditional mouse model for the β alleles A using belopment A,F [13,5ozygotes B–H’’’. Tpression I,J. Thespression P.Wnt-dependent repression of cartilage fate during calvarial development is poorly understood. Using a suite of conditional genetic mouse models, our data suggest a new working model that identifies ERK signaling as a key downstream mediator for Wnt-dependent chondrocyte repression in a subset of calvarial osteoblast precursors during cell fate selection. ERK signaling activation is dependent on β-catenin and Wnt ligands in the cranial mesenchyme. Our in vivo genetic mutants also demonstrate that β-catenin and ERK1/2 interact genetically to repress SOX9 expression and chondrocyte fate in the cranial mesenchyme in vivo.Twist genes. Our lab has demonstrated that expression of the transcription factors Twist1/Twist2 is regulated by Wnt/β-catenin signaling. Although Twist1 represses Sox9 mRNA expression [Twist1 in the cranial mesenchyme only results in a partial phenocopy of cartilage conversion of the occipital bone [Our in vivo analysis shows that Wnt signaling is required for ERK pathway activation in the cranial mesenchyme, but how is unclear. β-catenin is a transcriptional regulator which binds to TCF/LEF to initiate transcription during canonical Wnt signaling. Downstream of canonical Wnt signaling, and a putative candidate for Wnt/β-catenin ERK activation, are the pression ,59, condtal bone . Twist ctal bone . HoweverWls mutants, we were able to further dissect the role of Wnt signaling beyond canonical Wnt/β-catenin signaling. In support of a linear pathway, we previously showed that canonical Wnt signaling is required to transcriptionally regulate non-canonical Wnt ligands Wnt5a and Wnt11 in the cranial mesenchyme, suggesting that non-canonical Wnt signaling is downstream of canonical Wnt signaling [PDGFRαCreER deletion of Wntless support a linear pathway because there is a temporal delay in the observed cell fate switch to chondrocyte fate in the CM-Wls mutants (Wls embryos (Wls embryos show significant but modest decreases in two canonical Wnt signaling target genes, Axin2 and Tcf1. These decreases may indicate that canonical Wnt signaling is just beginning to decrease by E12.5 as a result of spatial differences as the mesenchyme grows away from the ectoderm, or as a result of changes in a gradient of Wnt signaling present in the cranial mesenchyme following loss of mesenchyme Wnts. Further studies will be necessary to understand how mesenchyme Wnt ligands and non-canonical Wnt signaling change throughout the cranial mesenchyme to activate the ERK signaling pathway, and how they intersect with canonical Wnt/β-catenin dependent activation of ERK. Future studies using specific mouse models targeting the functions of the Wnt/PCP and Wnt/Ca2+ signaling are necessary to test the roles of the non-canonical Wnt signaling pathway on ERK activation in the cranial mesenchyme in vivo.Our results suggest that mesenchyme Wnts mediating β-catenin-independent Wnt signaling may function in either a parallel or linear pathway to canonical Wnt/β-catenin-dependent signaling to activate ERK signaling . Using Cignaling . Our res mutants . Alterna embryos . These dErk1/2 mutant is more subtle, suggesting that there may be variability in the survival of the calvarial osteoblasts. Our data suggest a conserved role in the function of mesenchyme Wnts and ERK signaling on cell fate regulation within this subpopulation of calvarial osteoblasts. The phenotype of the CM-Wls mutants resembles CM-Erk mutants in the localization of ectopic SOX9 and cartilage nodule formation. Spatial segregation of Wnt ligands, Wnt inhibitors combined with graded expression of Wnt signaling target genes, suggests there can be regional variation in the level of Wnt signaling experienced by individual calvarial osteoblasts [Erk and CM-Wls mutants may experience less canonical Wnt signaling due to the high levels of the Wnt inhibitor DKK2, and the increased distance from the ectoderm Wnts [The role of Wnt signaling on the ERK signaling pathway is well studied in individual cell types in vitro. However, there are several limitations in understanding how these two signaling pathways guide calvarial osteoblast fate decisions from in vitro studies which do not capture the complexity of the environment in which the calvaria develops in vivo. Unlike long bones, calvarial osteoblasts develop between the brain and meningeal mesenchyme medially, and dermal fibroblasts and surface ectoderm laterally. The close proximity of each cell type results in cross-talk of cell signaling and communication between cell populations. Furthermore, there are mechanical cues and regional differences in the composition and signaling of the extracellular matrix in the cranial mesenchyme as the cranial bones expand apically . These reoblasts ,56. The erm Wnts . RegionaIn summary, our data reveal that Wnt/β-catenin signaling can activate ERK signaling to ensure a subset of calvarial osteoblasts are specified towards the intramembranous differentiation and repress the cartilage fate. We show a distinct requirement of ERK signaling in cell fate specification during early calvarial development, and demonstrate cross-talk between Wnt signaling and ERK signaling on cartilage repression that is required for normal calvarial development. Our results provide evidence towards identifying a specialized subset of periosteal cells that are highly sensitive to signaling dynamics of the Wnt and ERK signaling pathways. Our conclusions provide broad insights into Wnt-dependent cell fate specification that will be informative for intramembranous bone repair and regeneration.