Background: Orthostatic hypotension (OH) caused by autonomic dysfunction is a common symptom in older people and patients with idiopathic rapid eye movement sleep behavior disorder (iRBD). The orthostatic challenge test is a standard autonomic function test that measures a decrease of blood pressure during a postural change from supine to standing positions. Although previous studies have reported that changes in heart rate variability (HRV) are associated with autonomic dysfunction, no study has investigated the relationship between HRV before standing and the occurrence of OH in an orthostatic challenge test. This study aims to examine the connection between HRV in the supine position and the occurrence of OH in an orthostatic challenge test.Methods: We measured the electrocardiograms of patients with iRBD and healthy older people during an orthostatic challenge test, in which the supine and standing positions were held for 15 min, respectively. The subjects were divided into three groups: healthy controls (HC), OH-negative iRBD [OH (–) iRBD], and OH-positive iRBD [OH (+) iRBD]. HRV measured in the supine position during the test were calculated by time-domain analysis and Poincaré plots to evaluate the autonomic dysfunction.Results: Forty-two HC, 12 OH (–) iRBD, and nine OH (+) iRBD subjects were included. HRV indices in the OH (–) and the OH (+) iRBD groups were significantly smaller than those in the HC group. The multivariate logistic regression analysis for OH identification for the iRBD groups showed the model whose inputs were the HRV indices, i.e., standard deviation 2 (SD2) and the percentage of adjacent intervals that varied by more than 50 ms (pNN50), had a receiver operating characteristic curve with area under the curve of 0.840, the sensitivity to OH (+) of 1.000, and the specificity to OH (–) of 0.583 (p = 0.023).Conclusions: This study showed the possibility that short-term HRV indices in the supine position would predict subsequent OH in iRBD patients. Our results are of clinical importance in terms of showing the possibility that OH can be predicted using only HRV in the supine position without an orthostatic challenge test, which would improve the efficiency and safety of testing. Orthostatic hypotension (OH) is a major health concern in older people, affecting approximately one-third to two-thirds of them –3. A recOH is defined as a persistent decrease of systolic blood pressure (BP) ≥20 mm Hg, a persistent decrease of diastolic BP ≥10 mm Hg, or a decrease in systolic BP to <90 mm Hg within 3 min of standing or head-up tilt, or the manifestation of any OH clinical symptoms , 7. It hRapid eye movement (REM) sleep behavior disorder (RBD) is REM parasomnia characterized by dream-enacting behaviors . AlthougIn order to prevent falls of iRBD patients caused by OH, the severity of OH needs to be evaluated. One standard test for evaluating the severity of OH is the orthostatic challenge test, which usually takes place in clinics or hospitals . In the Autonomic dysfunction and OH are common among older people, particularly patients with α-synucleinopathies, such as PD, DLB, and MSA , 22. It 123I-metaiodobenzylguanidine (123I-MIBG) in the iRBD patients tends to decrease, which indicates cardiac sympathetic denervation   may overIn this work, we focus on heart rate variability (HRV) which is spontaneous beat-to-beat oscillation in the R-R interval (RRI) of an electrocardiogram (ECG) and reflects the autonomic nervous function . AccordiWe assumed that signs of autonomic dysfunction associated with OH appear even when in the supine position before standing in the orthostatic challenge test. Therefore, we conducted the orthostatic challenge test with ECG measurement for patients with iRBD and healthy older people in order to investigate the relationship between HRV in the supine position and the occurrence of OH. In addition, we discuss the possibility of the use of HRV analysis as an alternative method to the orthostatic challenge test for evaluating OH severity.Participants in this study consisted of healthy subjects and patients with iRBD. The details of this study were explained to them and written informed consent was obtained from each subject. In addition, the protocol was approved by the ethical committee at the Shiga University of Medical Science (R2017-199). Healthy subjects and patients with iRBD were recruited between December 2017 and August 2019.We recruited healthy subjects over 60 years of age and without history of autonomic dysfunction.In this study, patients who satisfied diagnostic criteria of RBD according to the 3rd edition of the International Classification of Sleep Disorders (ICSD-3) were recruited from among patients who visited the Shiga University of Medical Science. PSG was performed on all of the patients; who were then diagnosed with RBD. In order to focus on iRBD , patientA total of 60 healthy subjects and 29 iRBD patients were enrolled. One healthy subject was excluded because of a history of autonomic failure. Since a comorbidity of arrhythmia could affect the results of HRV analysis, 9 healthy participants and 3 iRBD patients with the comorbidity of arrhythmia were excluded .The orthostatic challenge test  was conducted on the remaining 50 healthy subjects and 26 iRBD patients. One healthy subject who did not maintain the standing position during the standing phase was excluded, and 3 healthy subjects who satisfied the OH criteria were also excluded . One iRBD patient to whom the examiner failed to attach devices was also excluded. The adaptation of the Hampel and the quotient filters , 39 resuFinally, 42 healthy controls (HC) and 21 patients with iRBD [12 OH (–) iRBD and 9 OH (+) iRBD] were included in this study. The definition of the subject grouping are described in “Orthostatic Hypotension Criteria and Grouping” in the “Orthostatic Challenge Test” section.The study consisted of a clinical interview and an orthostatic challenge test. The participants were instructed not to ingest alcohol, caffeine, or to smoke the night before the experiment because these substances could affect the autonomic nervous system . The expThe subjects were interviewed regarding their past histories including arterial hypertension, coronary artery disease, myocardial infarction, and diabetes mellitus, as well as their smoking habits, which could affect BP dynamics and autonomic nervous functions . InterviIn addition, patients with iRBD were asked about the duration of their RBD disease, the onset of which was confirmed by family members.In order to evaluate behavior in daily life, an active standing test called the orthostatic challenge test was performed instead of passive standing tests such as the tilt test . The ortA wearable RRI sensor  was used for RRI measurement  54). Th. Th54). In addition, BP and pulse rate (PR) were measured with a digital brachial BP monitor HEM-7500 . The BP monitor was attached to the left upper arm of the participant so that the cuff of the sphygmomanometer was at heart level. When the examiner pressed a button on the sphygmomanometer, the cuff was automatically pressurized and depressurized to record systolic BP (sBP), diastolic BP (dBP), and PR.The orthostatic challenge test was composed of two phases: supine- and standing-position phases . Before After checking the device operation in the supine position (>2 min), the measurements of BP and PR began, they were performed four times with 3-min intervals. After being in the supine position for 15 min, the participants were instructed to quickly stand up and to keep their standing position for 15 min. After standing for 1 min, BP and PR measurement was performed once per min for 15 min. The examiner carefully monitored the safety of the participant during the test. After the orthostatic challenge test, the examiner, by asking the participants, confirmed the presence/absence of the following symptoms: dizziness, titubation, blurry vision, syncope, or nausea.The average of five BP and PR measurements of each participant in the supine position was defined as the baseline. The orthostatic challenge test was determined to be positive when the participant satisfied one of the following conditions: (1) decrease of sBP ≥20 mm Hg, or decrease of dBP ≥10 mm Hg in comparison with the baseline BP at 1 or 3 min after standing, (2) a decrease in systolic BP to <90 mm Hg, or (3) manifestation of any clinical symptom including falling, dizziness, blurry vision, syncope, and nausea , 7, 9. HIn patients with iRBD, those who met the orthostatic challenge test criteria were defined as “OH (+) iRBD,” and those who did not meet the criteria were defined as “OH (–) iRBD.” Among healthy subjects, those who did not meet the OH criteria were defined as “HC ;” on the other hand, those who met the OH criteria were excluded because they might have potential autonomic nervous dysfunction .The HRV data extracted from the RRI data measured when in the supine position were analyzed . Data frIncidental arrhythmias such as premature atrial contractions (PAC) or premature ventricular contractions (PVC) are often observed in continuous ECG measurements of elderly people, although arrhythmias are not identified in short-term ECG examination. A high proportion of PAC and PVC in the elderly has been reported , 58; thunth RRI and its corresponding y value is the (n + 1)th RRI. A typical Poincaré plot forms an ellipse with about a 45 tilt, and the diffusion ranges around the major axis and the minor axis of the ellipse are defined as the standard deviation 2 and 1 (SD2 and SD1), respectively. In addition, their ratio is defined as SD1/SD2 , the root mean square of successive differences (RMSSD), and the percentage of adjacent intervals that varied by more than 50 ms (pNN50) was performed.Various indices of HRV have been reported to be associated with the sympathetic and the parasympathetic nervous system . For theThe demographic data of the HC, OH (–) iRBD, and OH (+) iRBD groups were examined. In addition, the experimental data of these three groups—BP and PR of the baseline and the delta values at 1 and 3 min after standing, and the ratio of clinical symptoms manifested after standing—were compared. The HRV features when in the supine position were compared among the three groups.2-test. The normality of the distribution of the variable was checked by the Shapiro-Wilk test . Parametric variables were presented as mean ± SD; non-parametric variables were presented as the median and interquartile range (IQR). For the three groups, parametric variables were compared using one-way analyses of variance (ANOVAs) with Tukey's post-hoc analysis followed by Bonferroni correction; non-parametric variables were compared using the Kruskal-Wallis test and post-hoc Mann-Whitney test followed by Bonferroni correction. For comparison with the two groups [OH (–) iRBD and OH (+) iRBD], parametric variables were compared using Student's t-test.For comparison with the three groups, nominal data were compared using the χ2-test. The disease duration of RBD was analyzed using the Student's t-test. In the orthostatic challenge test, BP and PR of the baseline and the delta values at 1 and 3 min after standing were compared using ANOVAs at each time point. For the correction for the multiple comparisons, Tukey's post-hoc analysis was used for the comparison of multiple groups, and Bonferroni adjustment was performed for multiple time points (p < 0.05/3 ≈ 0.017). The ratio of the clinical symptoms that appeared after standing was assessed using the χ2-test. In addition, HRV indices when in the supine position were compared using the Kruskal-Wallis test and post-hoc Mann-Whitney test followed by Bonferroni correction.Age and BMI were compared using ANOVAs. Years of education, a score of MMSE, and a score of RBDSQ-J were compared using the Kruskal-Wallis test. Gender differences, rate of comorbidities, and rate of medication usage were compared using the χp < 0.05 was considered significant in this study. For parametric variables, the Cohen's d effect size index was used to calculate the pairwise differences. Cohen classified the effect sizes into small (d = 0.2), medium (d = 0.5), and large (d = 0.8) , medium (r = 0.3), and large (r = 0.5)  . For nonr = 0.5) .Next, among the OH (–) iRBD and OH (+) iRBD patients, a multivariate logistic regression analysis was conducted to focus on the relationship between the OH (+/–) and HRV indices, with OH (–) or OH (+) as the dependent variable and HRV indices  as the independent variables. The Bayesian information criterion (BIC) was used to select the appropriate independent variables. In a model that minimized the BIC, we calculated the area under the curve (AUC) of the receiver operating characteristic (ROC) curve, the sensitivity of OH (+), and the specificity of OH (–). Besides, the value of HRV indices that maximizes Youden's index was calculated to determine the optimal cutoff values .These statistical tests were performed using IBM SPSS Statistics for Macintosh, Version 22.0 . HRV were evaluated with an HRV Analysis software .The demographic data of the included participants are shown in Of the comorbidities that could affect BP or HRV, there were no significant differences in the promotions of arterial hypertension, coronary artery disease, and diabetes mellitus. However, the proportion of myocardial infarction was significantly higher in the OH (+) iRBD group.For medications that could affect BP or HRV, calcium channel blockers, ACE and AT II inhibitors, and organic nitrates were used by the participants. There was a significant difference in the proportion of ACE and AT II inhibitor use. Of the calcium channel blockers, diltiazem may decrease the low-frequency component of HRV and nifedipine may not . ACE andAs to smoking habit and the use of dopaminergic agonists, there were few participants (zero or one) in each group of HC, OH (–), and OH (+) iRBD.The results of the orthostatic challenge test are shown in p = 0.003). PR in the HC group increased greatly after standing and then decreased rapidly. Similar changes were not observed in the OH (+) or OH (–) iRBD groups.PR increased after standing in all three groups . Althougp < 0.001) and at 3 min after standing (p < 0.001), and ΔdBP at 1 min (p < 0.001) and at 3 min after standing (p < 0.001). Subsequent post-hoc analyses revealed that ΔsBP at 1 min after standing in the OH (+) iRBD group was significantly lower than that in the HC group and OH (–) iRBD group with large effect sizes . Similarly, ΔsBP at 3 min after standing in the OH (+) iRBD group was significantly lower than the HC group with a large effect size (Cohen's d = 1.727). ΔdBP at 1 min after standing in the OH (–) iRBD and OH (+) iRBD groups were significantly lower than that in the HC group with large effect sizes .Delta values of BP and PR  and the occurrence of subjective symptoms related to the OH +/– criteria were compared among the three groups . sBP, dBn = 2; 22.2%), and the incidence of dizziness was significantly different in the three groups (p = 0.002).Only dizziness was reported in this test as a subjective symptom in the OH (+) iRBD group . Subsequent post-hoc analyses revealed that SDNN in the OH (–) iRBD and the OH (+) iRBD groups were significantly smaller than that in the HC group with medium and large effect sizes ; RMSSD in the OH (–) iRBD and the OH (+) iRBD groups were significantly smaller than that in the HC group with medium effect sizes ; and pNN50 in the OH (–) iRBD group was significantly smaller than that in the HC group with a medium effect size (r = 0.370).First, as for the time-domain analysis during segment 2, significant differences in SDNN, RMSSD, and pNN50 were confirmed among the three groups . Subsequent post-hoc analyses revealed that SD1 in the OH (–) iRBD and the OH (+) iRBD groups were significantly smaller than that in the HC group with medium effect sizes ; SD2 in the OH (–) iRBD and the OH (+) iRBD groups were significantly smaller than that in the HC group with medium and large effect sizes .Second, in terms of the Poincaré plots, there were no significant differences among the three groups in SD1/SD2; however, significant differences were confirmed in SD1 and SD2  iRBD patients, had smaller values of HRV indices than the HC group.p = 0.012). In this model, the AUC of the ROC curve was 0.840, and the sensitivity of OH (+) was 1.000, and the specificity of OH (–) was 0.583  was performed using HRV indices during segment 2. We identified SD2 and pNN50 associated with OH (+/–) with the minimal value of BIC . In the segment 1 model  with the minimal values of BIC in segment 1 and the whole period  and OH (+) iRBD patients using HRV indices presented a good model and ROC curve . HRV indOur results are of clinical importance in terms of showing the possibility that OH of older people can be predicted using only HRV data in the supine position without an orthostatic challenge test, which could improve the efficiency and safety of testing. However, several steps are required for the clinical application of OH detection by HRV indices. Since the number of OH patients were limited in this study, we performed a multivariate logistic regression analysis for only the construction of models. In the future, it is needed to validate the current model. Besides there are issues to be addressed to meet the clinical needs, such as whether using only the HRV indices is enough for OH detection or whether a combination of the OH questionnaire and the HRV indices is useful. A new device that can predict the occurrence of a fall can thereby be realized, since a wearable device for measuring RRI and calculating HRV in real-time has already been developed, which would contribute to preventing the falls of older people.There were two benefits of recruiting iRBD patients in this study. The first was that the evaluation of autonomic dysfunction in the whole population of iRBD patients [OH (–) and OH (+) iRBD] was carried out, and the second was that we were able to perform an analysis to detect severe cases of autonomic dysfunction [OH (+) iRBD group]. iRBD patients have a high rate of autonomic dysfunction, and the degree of autonomic dysfunction was considered to be graded from mild to severe. Since PD, DLB, and MSA are associated with severe autonomic dysfunction, and most of the iRBD patients phenoconvert to PD, DLB, and MSA over a long period of time , 15, 20,Demographics of the patients with iRBD included in our experiments were not much different from previous studies , althougOn the other hand, the RBDSQ-J score of our population was lower than in the previous report , and theThe time of the experiment, the last meal, and the temperature and humidity in the laboratory were controlled to reduce their effects on the autonomic activities. The participants were instructed to stop consuming alcohol and tobacco the night before. However, autonomic activities in daily life are affected by various factors such as circadian rhythms and diet as well as alcohol, tobacco, and temperature , 78–80. Because Xie et al. suggested that cognitive impairment-related gait disorders occur while walking in daily life among patients with amnestic mild cognitive impairment , comprehThe limitations of this research include medication and comorbidities; although older people regularly use blood pressure-related medications due to hypertension or complications of angina, our study did not control for these medications and comorbidities. Some patients took calcium channel blockers, ACE and AT II inhibitors, and organic nitrates. There were significant differences in the proportion of myocardial infarction comorbidity and the use of ACE and AT II inhibitors . To addrAnother limitation was the ratio of male to female in the HC group. In this study, there were more females than males in HC , although the number of males was more significant than that of females in the patient group reflecting the characteristics of RBD prevalence. There was the possibility that this gender difference might affect HRV and decreases in BP. To address this issue, we prepared randomized HC samples with the number of HC adjusted for the ratio of males to females in the iRBD groups  because the sample size of iRBD was smaller than that of HC. Namely, of the 26 HC females, we randomly selected 4 females 10 times, then created a sample of HC  10 times. The mean or median of variables of the 10 randomized samples of the 20 HC, whose male-to-female ratio was matched to the iRBD, are shown in In this study, iRBD patients showed significant decreases in BP after standing and significant decreases in HRV indices during a supine position compared to HC. The combination of HRV indices would predict subsequent OH in iRBD patients. Our results are clinically useful in terms of showing the possibility that OH can be predicted without an orthostatic challenge test using only HRV data in the supine position, which could improve the efficiency and the safety of testing. Future studies are required to develop a system that can predict OH before standing up by analyzing HRV in real-time.The datasets analyzed in the current study are available from the corresponding author upon reasonable request.The studies involving human participants were reviewed and approved by the ethical committee at Shiga University of Medical Science. The patients/participants provided their written informed consent to participate in this study.YS, MM, HK, KF, and CN conceived the idea. YS, CN, and TK performed the experiments and analyzed the data. KF and TY contributed to the reagents, materials, and analysis tools. YS, KF, and HK wrote the draft. MM, YO, HK, KF, CN, YG, MK, TY, MH-O, and KO interpreted the data and revised the manuscript. All authors contributed to the article and approved the submitted version.HK's laboratory is supported by a donation from Fukuda Lifetech Co., Ltd., Fukuda Life Tech Keiji Co., Ltd., Tanaka Sleep Clinic, Akita Sleep Clinic, and Ai Care Co., Ltd. to the Shiga University of Medical Science. HK received a grant from Merck Sharp & Dohme Corp/MSD K.K. (the Investigator-Initiated Studies Program). The opinions expressed in this paper are those of the authors and do not necessarily represent those of Merck Sharp & Dohme Corp/MSD K.K. KF, TY, and MK participate in Quadlytics Inc. The remaining 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.


Nonleukemic variant of HTLV‐1‐associated adult T‐cell leukemia lymphoma (ATLL) is a rare variant, and herein, we describe a case with strong and diffuse positivity of neoplastic cells for CD30. Even though ATLL is aggressive entity with poor prognosis, in our case, there was very good clinical response achieved with brentuximab‐containing regimen. Therefore, HTLV‐1‐associated ATLL can be included in the differential diagnostic approach of CD30‐positive lymphoproliferative disorders. Nonleukemic variant of HTLV‐1‐associated adult T‐cell leukemia lymphoma (ATLL) is a rare variant, and herein, we describe a case with strong and diffuse positivity of neoplastic cells for CD30. Even though ATLL is aggressive entity with poor prognosis, in our case, there was very good clinical response achieved with brentuximab‐containing regimen. Therefore, HTLV‐1‐associated ATLL can be included in the differential diagnostic approach of CD30‐positive lymphoproliferative disorders. A 38‐year‐old previously healthy woman of Haitian origin presented with splenomegaly and extensive lymphadenopathy involving cervical, supraclavicular, axillary, mediastinal, hilar, para‐esophageal, para‐aortic, mesenteric, and inguinal lymph nodes. The largest lymph node measured 2.9 cm in size, and PET‐CT scan showed maximum SUV of approximately 32 mSv. No skin lesions were identified. Serum calcium was normal. Peripheral blood counts were within normal range, and no circulating abnormal lymphoid cells were seen.The patient underwent a biopsy of a cervical lymph node. The lymph node architecture was effaced by an atypical infiltrate of large pleomorphic lymphoid cells with moderate eosinophilic cytoplasm, irregular nuclei, coarse to vesicular chromatin, and occasional prominent nucleoli Figure  amid a bBy immunohistochemistry, neoplastic cells were positive for CD3 Figure , CD4 Fi, CD25 F, and negSerologic tests showed anti‐human T‐cell leukemia virus (HTLV)‐1/2 antibodies with subsequent Western blot confirmation of HTLV‐1 infection. In this context, the histopathologic and immunophenotypic findings were consistent with adult T‐cell leukemia/lymphoma (ATLL), HTLV‐1 associated.Staging bone marrow evaluation did not demonstrate involvement by lymphoma. Evaluation of cerebrospinal fluid showed no abnormal cells by cytology or flow cytometry. The patient began treatment with brentuximab, cyclophosphamide, adriamycin, vincristine, etoposide, and prednisone (BvCHEP) with intrathecal methotrexate with significant improvement in adenopathy after one cycle. Treatment is planned for a total of four cycles followed by allogenic stem cell transplant.HTLV‐1 retrovirus has been present since ancient times.The major difference between HIV and HTLV‐1 is the fact that the latter stimulates mitosis of target CD4 T cells rather than viral replication.All HTLV‐1‐related disorders are broadly classified into neoplastic, inflammatory, and infectious. Neoplastic HTLV‐1 associated disorder is represented by ATLL, and examples of infectious and inflammatory HTLV‐1 related entities include, but are not limited to, tropical spastic paraparesis , dermatitis, arthritis, uveitis, and myositis.The 2017 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues separates four clinical variants of ATLL: smoldering, chronic, acute, and lymphomatous.Strong and diffuse expression of CD30, as seen in our case, is an unusual finding in ATLL. However, Takeshita et al described expression of CD30 in 21 of 91 Japanese patients with ATLL.Authors have no conflict of interest to declare.Both authors (MP and AS): equally contributed to manuscript preparation and submission as follows: MP: contributed to composing text and submission of the manuscript. AS: contributed to reviewing main text body and preparation of the corresponding images.The study was approved by the Institutional Review Board of Partners Healthcare/Mass General Brigham.


Dendrobium cariniferum is a valuable ornamental and medicinal plant rich with polysaccharides, alkaloid, and other bioactive compounds, which are potential raw materials for pharmacological utilization. In this study, an efficient protocol for the rapid propagation of D. cariniferum was developed. By using the tissue culture protocol, the effects of pH, hormone combinations, temperatures, light intensity, culture time protocorm proliferation, seedlings rooting, and accumulation of biomass with bioactive compounds were investigated. The experiments showed that the medium [1/2 MS + activated carbon1.0 g/L+ agar strip 7.5 g/L + sucrose 25 g/L] effectively promoted the germination of D. cariniferum seeds. The optimal culture conditions were found at pH 5.7, temperature 23 ± 2°C, and light intensity of 1000 Lx in the protocorm proliferation stage. Adding 1.5 g/L peptone in the medium effectively promoted the seedling rooting. The optimal culture conditions for accumulation of bioactive compounds  of seedlings were found at temperature of 25 ± 2°C, light intensity of 1500–2000 Lx after the 60-day (d). Our study constructed a rapid propagation system in vitro for D. cariniferum, as well as the methods for efficient accumulation of active substances in seedling culture, which will serve as guidance for industrial production of D. cariniferum seedlings for both medicinal raw materials and ornamental plants. In addition, our study provided a new idea that we can directly use the high bioactive compound seedlings to extract medicinal components in industry conditions without transferring to the field.  Adding 1.5 g/L peptone to the medium effectively promoted the seedlings rooting. At the accumulation stage of bioactive compounds, the optimal culture conditions were found at a temperature range of 25 ± 2°C and a light intensity of 1500–2000 Lx. After 60 d of tissue culture, the seedlings need to be transferred in time. This culture system effectively promoted the rapid propagation in vitro and accumulation of bioactive compounds of 4.4.1.D. cariniferum seed germination, the culture medium of  effectively promoted the germination of seeds, which was obviously better than the MS medium. The results indicated that the germination of D. cariniferum seeds required a lower salt concentration; too high or too low inorganic salt concentration are not beneficial for seed germination. The MS medium is rich in inorganic salts, especially ammonium nitrate, potassium ion, and ammonium ions, which is not suitable for seed germination. In addition, from the perspective of economy, the cost of one-half MS basic medium was relatively low. Therefore, one-half MS medium was selected as the basic medium for D. carniferum seed germination, which was found to be more economical and efficient. Chen et al. [In the stage of n et al.  proposed4.2.D. cariniferum protocorm proliferation stage. The experiments show that D. cariniferum seeds were more suitable to the acidic medium condition. Moreover, the result shows that 23 ± 2°C was the optimum temperature range for protocorm proliferation. Too low or too high temperature will have disadvantages on the quality of culture seedlings for D. cariniferum [Dendrobium and Oncidium [Cymbidium japonicus [D. cariniferum was set to 1000 Lx. In addition, the combination of medium (0.1 mg/L NAA+1.0 mg/L 6-BA) was found to be the optimal hormone combination for protocorm proliferation. Similarly, the study on Cymbidium hybridum by Zhang et al. [At a medium pH value of 5.7, the protocorm proliferated and differentiated faster and loosely, which should be regarded as the optimum medium pH value for iniferum . We founOncidium . We discaponicus . Therefog et al.  believed4.3.Cymbidum rhizomes. Our experiment concluded that the addition of 1.5 g/L peptone in the medium effectively promoted the rooting of D. cariniferum seedlings, which can be widely used for D. cariniferum seedling production.Peptone is a light-yellow powder hydrolyzed with acid or protease which is rich in various nutrients, such as nitrogen and carbon source . Adding 4.4.D. cariniferum was determined by the biomass and the content of active substances. With the extension of time, the biomass was gradually increased; however, the active substances decreased, which is also found in a previous study [D. cariniferum has strict requirements for the light intensity. Therefore, special attention should be paid to regulate the light intensity at the seedling stage [The yield of us study . In ordeng stage –23.D. cariniferum were sensitive to the temperature; if the cultivation temperature is not suitable, it could lead to the change of enzyme activity of seedlings. The experimental result showed that the biomass accumulated at the highest amount under 23 ± 2°C culture temperature. However, the nutrition of seedlings was consumed for growth, which resulted in few bioactive compounds accumulated. While under 25 ± 2°C culture temperature, the seedlings grew slower, but bioactive compound accumulation increased with the highest level compared with other cultivation temperature.Finally, the cultivation temperature could lead to the change of enzyme activity in seedlings. Our research concluded that temperature (23 ± 2°C) for cultivating seedlings will consume a large amount of material in the growth, which affected the accumulation of active material. Therefore, in order to ensure the normal accumulation of active substance, the temperature could set between range from 23-25 ± 2°C. Our results showed the seedlings of 5.D. cariniferum. In addition, the optimum conditions for the accumulation of bioactive compounds in seedling culture were analysed through the development of a rapid in vitro propagation system. Our study provided a new idea that cultivated seedlings that containhigh active substances can be directly used to extract medicinal raw materials to save the process of transferring to the field growth.Our study used the tissue culture process for developing a rapid propagation system in vitro and the methods for efficient accumulation of bioactive compounds in seedling culture for the endangered


R206H, a mutation associated with Fibrodysplasia Ossificans Progressiva (FOP). How activin A inhibits ALK2WT-mediated BMP signaling but activates ALK2R206H-mediated BMP signaling is not well understood, and here we offer some insights into its molecular mechanism. We first demonstrated that among four BMP type I receptors, ALK2 is the only subtype able to mediate the activin A-induced BMP signaling upon the dissociation of FKBP12. We further showed that BMP4 does not cross-signal TGF-β pathway upon FKBP12 inhibition. In addition, although the roles of type II receptors in the ligand-independent BMP signaling activated by FOP-associated mutant ALK2 have been reported, their roles in activin A-induced BMP signaling remains unclear. We demonstrated in this study that the known type II BMP receptors contribute to activin A-induced BMP signaling through their kinase activity. Together, the current study provided important mechanistic insights at the molecular level into further understanding physiological and pathophysiological BMP signaling.Activins transduce the TGF-β pathway through a heteromeric signaling complex consisting of type I and type II receptors, and activins also inhibit bone morphogenetic protein (BMP) signaling mediated by type I receptor ALK2. Recent studies indicated that activin A cross-activates the BMP pathway through ALK2 Bone morphogenetic proteins (BMPs) and activins belong to the Transforming Growth Factor β (TGF-β) superfamily . Their bR206H produces basal leaky BMP signaling in the absence of BMP ligands and elicits abnormally elevated BMP signaling in the presence of BMP ligands [R206H in FOP [WT) [WT-mediated BMP signaling, but activates ALK2R206H-mediated BMP signaling is not well known.Abnormal BMP signaling activation can lead to pathological ectopic ossification such as Fibrodysplasia Ossificans Progressiva (FOP) and the dominant mutation R206H in ALK2 is responsible for over 95% patients with FOP ,8,9,10.  ligands ,15,16,17H in FOP ,19,20,21R206H, ALK2Q207E and ALK2Q207D) located at the binding interface of ALK2 and FKBP12 share the same loss of interaction between FKBP12 and ALK2 (most significantly between G90 on FKBP12 and F246 on ALK2), which allosterically shift the kinase towards catalytic competent configuration [WT for mediating the activin A-induced BMP signaling [A possible mechanism may involve the ubiquitous repressor protein FKBP12 in TGF-β superfamily signaling. In the absence of ligands, FKBP12 is a negative signaling regulator which binds to a glycine-serine rich (GS) domain of type I receptors and locks the kinase catalytic domain of type I receptors in an inactivate conformation . Previouguration ,24. Seveguration ,19. For guration . In addiignaling ,25. HoweWT to mediate activin A-induced BMP signaling. In brief, we transiently transfected C2C12BRA cells (C2C12 cells stably transfected with the Id1 promoter-firefly luciferase reporter) with ALK2WT plasmid and renilla luciferase reporter pRL-TK plasmid as internal control, followed by the treatment with FKBP12 inhibitor FK506. The luciferase assay showed that 1 µM FK506 dramatically induced BMP signaling in the ALK2WT- transfected C2C12BRA cells treated with activin A, and modestly activated BMP signaling in the absence of activin A, which could be attributed to the trace amount of BMPs or activins in the culture media  to mediate the activin A-induced BMP signaling [WT for the activin A-induced BMP signaling [−17 to 10−10 M was used and such low concentrations may not be sufficient for FKBP12 dissociation [−10 M) is unable to activate ALK2WT for activin A-induced BMP signaling . We next knocked down the ALK2 gene with siRNA in HepG2BRA cells and transfected them with ALK1, ALK3 or ALK6, respectively. Transfected cells were then treated with activin A, FK506 or their combination. Our RT-PCR assay confirmed that ALK2 expression was effectively knocked down by siRNA treatment (Since FKBP12 acts as a ubiquitous repressor for the four subtypes of the BMP type I receptor (ALK1/2/3/6), our results raised the possibility that activin A could also mediate BMP signaling via ALK1/3/6. We addressed this question by using HepG2BRA cells  as these cells were previously reported to be extremely sensitive for BMP signaling and express only ALK2 as BMP type I receptor . To confreatment B. In addreatment C. The sareatment D, suggesreatment .Since FKBP12 acts as an ubiquitous repressor for both BMP and TGF-β kinases and our results demonstrate a cross-talk between a TGF-β ligand (activin A) and a BMP receptor (ALK2) upon FKBP12 dissociation, we investigated whether a BMP ligand could, in turn, induce TGF-β signaling upon FKBP12 dissociation. To test this hypothesis, HEK293 cells were treated with BMP4, FK506 and their combination and the cell lysates were subjected to Western blotting. As expected, our control assays showed that TGF-β signaling was induced by activin A but not by BMP4 A. The FKR206H). Since ActR-IIA and ActR-IIB are the only known type II receptors for activin A signaling, we first generated the two kinase-deficient mutants ActR-IIAK219R and ActR-IIBK217R. Then, the following combinations of plasmids, in combination with renilla luciferase reporter pRL-TK plasmid as an internal control, were transiently transfected into HepG2BRA cells: (1) ActR-IIAWT/ALK2R206H, (2) ActR-IIAK219R/ALK2R206H, (3) ActR-IIBWT/ALK2R206H, (4) ActR-IIBK217R/ALK2R206H, (5) ActR-IIAWT/ActR-IIBWT/ALK2R206H and (6) ActR-IIAK219R/ActR-IIBK217R/ALK2R206H. After overnight incubation with activin A, a dual luciferase assay was conducted. Our data showed that the over-expression of either kinase-deficient ActR-IIAK219R or ActR-IIBK217R as well as their combination significantly downregulated activin A-induced BMP signaling via ALK2R206H, suggesting that the kinase activity of the type II receptor is required for activin A-induced BMP signaling in FOP disease  BMPR-2WT/ALK2WT, (2) BMPR-2WT/ALK2R206H and (3) the kinase-deficient BMPR-2K230R/ALK2R206H. As was the case with ActR-IIB and ActR-IIA type II receptors, co-expressing BMPR-2WT and ALK2R206H produces a significantly higher BMP signal as compared to co-expressing BMPR-2K230R and ALK2R206H. This shows that the BMPR-2 receptor kinase’s activity contributes to activin A-induced BMP signaling . No cross-signaling for BMPs and activins were recognized until a recent discovery that activin A can induce BMP signaling via FOP ALK2 mutants such as ALK2ignaling . On the ignaling . By utilMoreover, we proved that only ALK2, but not any other three BMP type I receptor subtypes (ALK1/3/6), can mediate the activin A cross-talking BMP signaling upon FKBP12 dissociation. In addition, as FKBP12 is a common negative inhibitory factor in regulating both BMP and TGF-β signaling pathways, we investigated whether BMP ligands can also cross-signal TGF-β signaling upon FKBP12 dissociation in a similar way to the activin A activation for BMP signaling. Our data indicate that BMP ligands failed to cross-signal TGF-β signaling in the FKBP12 dissociation, suggesting other factors additional to FKBP12 dissociation may govern the ligand-specific TGF-β signaling.R206H for BMP signaling activation [In the previous ligand-independent BMP signaling of the FOP model, two groups showed conflicting results about the role of the type II receptor’s roles ,25. Fujitivation . However2.C2C12-BRA cells (Mouse myoblast cells stably transfected with BRE-Luc construct) and HepG2-BRA (human hepatoma cell lines stably transfected with BRE-Luc construct) were obtained from Dr. Rifkin’s lab at New York University , HEK293 cells  were cultured in DMEM supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin–streptomycin . Cultures were maintained in a humidified incubator at 37 °C in 5% COK230R were kindly provided by Dr. Petra Knaus at Freie University Berlin . The following plasmids were used in the current study: pCDNA3 plasmids harboring ALK2WT, ALK3WT and ALK6WT were purchased from Addgene . pCMV3 plasmids harboring activin A receptor type 2A (ActR2A) and human activin A receptor type 2B (ActR2B) were obtained from Sino Biological Inc. The point mutations in human ALK2 (ALK2R206H), human ActR2A (ActR2AK219R) and ActR2B (ActR2BK217R) were made in-house using the QuikChange II site directed mutagenesis method. The sequences for mutagenic oligonucleotides are shown in The pcDNA3.1 plasmids harboring the long form BMPR-2 and its kinase dead mutant BMPR-2Transient transfection for overexpression was performed using Fugene HD transfection reagent  according to the manufacturer’s instructions. Briefly, C2C12-BRA cells or HepG2-BRA cells or HEK293 cells were seeded in 96-well plates to culture in the growth medium DMEM/10% FBS without antibiotics. After overnight culture, the cells were transfected with plasmids. For the knockdown experiments, the C2C12-BRA or HepG2-BRA cells were transfected with Fugene HD transfection reagent (Promega) using pools of FKBP12 Mouse siRNA oligo Duplex  or ALK2 Human siRNA oligo Duplex  at a concentration of 20 nM, respectively. When the siRNA pools were combined, 20 nM of each 27 siRNA duplexes was used. Samples for the dual-luciferase assay or Western blot were taken 30 h post-transfection after the treatment with drugs.The cells were transfected with different kinds of plasmids along with renilla luciferase reporter pRL-TK plasmid. Five hours after transfection, the cells were starved in DMEM containing 0.5% FBS for 4 h. Then, the cells were untreated or treated with 1 μM FK506 (Cayman) or 100 ng/mL activin A (R&D) or 1 μM FK506/100 ng/mL activin A or 50 ng/mL BMP4 (Euzlab) or 50 ng/mL BMP4/1μM FK506 overnight before lysis. Luciferase activities were determined according to the dual-luciferase1reporter assay system (Promega) using the Renilla for normalization of transfection efficiency.RNA was extracted by resuspending the cells in lysis buffer and purified by filtration following the manufacturer’s protocol . The first-strand cDNAs were synthesized using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instructions. Using cDNA as template, real-time (RT)-PCR reactions were carried out using the Fast Syber Green (2×) Master Mix Applied Biosystems). The reactions were performed in triplicate on a Bio-Rad CFX connected Real-Time PCR system. Human or mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. The primer sets used in this study are shown in p-Smad1/5/9 , rabbit anti-Smad 1, mouse anti-beta actin , rabbit anti-p-Smad 2  and rabbit anti-Smad 2 . The proteins were detected by Odyssey system (Li-Cor bioscience) followed by the secondary antibodies including IRDye 680-conjugated goat anti-rabbit IgG (Li-Cor Bioscience) and IRDye 800CWconjugated goat anti-mouse IgG .Cells were lysed with RIPA buffer (Sigma) containing protein inhibitors  and phosphatase inhibitors . Samples were denatured by incubating at 95 °C for 5 min in sample buffer and separated by using SDS-PAGE . Then, the samples were transferred to a PDVF membrane (Millipore). The membrane was blocked with Odyssey Blocking solution  for 1 h at room temperature, followed by primary antibody incubation at 4 °C overnight. The primary antibodies used in the present study included rabbit anti-g. The supernatant was collected and the aliquots (30 µL) were subjected to an ALP activity assay at 405 nm absorbance by using the ALP activity assay kit .The C2C12 cells were plated at 8000 cells/well in 96-well plates and grown overnight in DMEM containing 10% FBS. siRNA ALK2 or siRNA control were transfected for some treated groups. Then, the culture medium was replaced with DMEM containing 2% FBS and the cells were untreated or treated with 100 ng/mL activin A (R&D) or 1 μM FK506/100 ng/mL activin A. After 72 h treatments, the cells were washed with PBS buffer and lysed with 50 µL lysis buffer (0.5% Triton-I00/H2O) and frozen/thawed three times to disrupt the cell membranes. The cell lysates were centrifuged for 5 min at 13,000× t test, and the results were considered statistically significant if the p-value was < 0.05.All values are expressed as the means ± SEM (standard error). The comparison of the means was conducted using the Student’s


The formation of the neocortex relies on intracellular and extracellular signaling molecules that are involved in the sequential steps of corticogenesis, ranging from the proliferation and differentiation of neural progenitor cells to the migration and dendrite formation of neocortical neurons. Abnormalities in these steps lead to disruption of the cortical structure and circuit, and underly various neurodevelopmental diseases, including dyslexia and autism spectrum disorder (ASD). In this review, we focus on the axon guidance signaling Slit-Robo, and address the multifaceted roles of Slit-Robo signaling in neocortical development. Recent studies have clarified the roles of Slit-Robo signaling not only in axon guidance but also in progenitor cell proliferation and migration, and the maturation of neocortical neurons. We further discuss the etiology of neurodevelopmental diseases, which are caused by defects in Slit-Robo signaling during neocortical formation. The neocortex is the six-layered outermost structure of the cerebrum, and is considered to be an evolutionarily new region of the brain that appeared soon after the emergence of mammals. Humans have the largest neocortex relative to their body size, which is thought to underlie their higher brain functions, such as cognition and emotion .The neocortex consists of two main types of neurons, i.e., excitatory projection neurons and inhibitory interneurons, which are generated from distinct germinal zones in the developing cerebrum, corresponding to the dorsal and ventral telencephalon, respectively. In both regions, the germinal zones are divided into two territories. The first is the ventricular zone (VZ), which lines the ventricles and occupies the apical-most region of the cerebral cortex. The second is the subventricular zone (SVZ), which is located adjacent to the VZ and basally toward the surface of the neocortex. The VZ comprises apical radial glial cells (aRGCs), which integrate into the apical junctional belt and extend long basal processes toward the pial surface . After tIn mice, after the onset of neurogenesis, neurons generated from aRGCs migrate radially toward the marginal zone (MZ) through direct somal translocation by processes that extend from the soma to the pial surface . SubsequProper leading and trailing process formation and the subsequent migration of neurons are crucial for the establishment of neural networks. It has been shown that such neuronal morphogenesis and migration are regulated by environmental cues, including axon guidance molecules and cell adhesion molecules . AbnormaIn this review, we focus on Slit and Robo, which were originally identified as axon guidance molecules, and discuss the novel roles of Slit-Robo signaling in neocortical development. We highlight the pleiotropic functions of Slit-Robo signaling beyond axon guidance, by focusing on their new roles in the proliferation, migration, and maturation of cortical neurons during development, and further discuss the involvement of Slit-Robo signaling in human neurodevelopmental disorders.Drosophila mutants demonstrating abnormal projections of commissural axons in the central nervous system   and four mammals , several mammals . These fTo date, several downstream signals of the Robo receptor have been identified . Here, wsrGAP2 (srGAP2A), the human genome has three human-specific paralogs of srGAP2, namely, srGAP2B, srGAP2C, and srGAP2D, which arose by gene duplications  domain, GTPase-activating protein (GAP) domain, and Src homology 3 (SH3) domain. Each srGAP has specific binding characteristics to the Rho family small GTPases; i.e., srGAP1 interacts with Cdc42 and RhoA upon Slit stimulation 2/3 complex and neuronal Wiskott-Aldrich syndrome protein , resulting in actin depolymerization. This leads to the axon repulsion and the inhibition of cell migration .In addition to srGAPs, the crosstalk between Slit-Robo signaling and cell adhesion signals is mediated by cadherins . CadheriThe binding of Slit to the Robo receptor induces an interaction between the Robo receptor and N-cadherin-Cable complex via Abelson (Abl) kinase, which binds to the intracellular domain (CC3) of Robo. This Robo and N-cadherin interaction leads to the phosphorylation of β-catenin by Abl, and thereby phosphorylated β-catenin is detached from N-cadherin. This, in turn, weakens N-cadherin-mediated cell adhesion. The phosphorylated β-catenin translocates to the nucleus and activates transcription , 2007. TRobo mutant mouse lines have implicated the roles of Slit-Robo signaling in controlling the balance between cell proliferation and differentiation.During the early stages of cortical development, multiple signaling pathways regulate the proliferation and division modes of cortical progenitor cells. In this context, studies using Robo1/Robo2 expression is detected in the VZ. Loss of Robo1/2 signaling leads to a decrease in the number of aRGCs and a concomitant increase in the number of basally located cells expressing the bIP marker Tbr2. However, these Tbr2-expressing cells retain apical processes and are integrated into the ventricular  surface, suggesting that Robo signaling regulates two events, i.e., the production of intermediate progenitors (IPs) from aRGCs and their delamination from the apical junction belt.Borrell’s group reported that Slit-Robo signaling is involved in the proliferation-differentiation balance of neural progenitor cells . Robo1/RRobo1/Robo2-deficient mice is largely consistent with another report using distinct Robo1/Robo2 mutant mice  . ImmaturRobo1-knockdown neurons migrate through the CP and reach the MZ-CP border; however, these neurons accumulate there , through the analysis of a female patient with 3p deletion syndrome who had hypotonia and severe intellectual disability . srGAP3 Recently, the association between Robo and spine formation has been reported. Robo2 is localized at the postsynaptic membrane of hippocampal CA1 pyramidal neurons, and directly binds to presynaptic neurexin irrespective of Slit . This biIn summary, srGAPs play a role in spine formation through its Rho GAP domain and/or F-BAR domain . HoweverAbnormal development of the neocortex affects neural circuit formation and causes neuropsychiatric disorders. Here, we discuss two etiologies known to be caused by abnormalities in Slit-Robo signaling, i.e., dyslexia and autism spectrum disorder (ASD).ROBO1 and ROBO2 genes are mapped at the dyslexia susceptibility loci DYX5, which is located on chromosome 3 (3p12-q13). Silent and 3′UTR SNPs of ROBO1 and a translocation t  that causes reduced ROBO1 transcription were found in individuals with dyslexia (ROBO1 and ROBO2 have been associated with dyslexia . ROBO1 adyslexia . Furtherdyslexia , which aHowever, recently, a magnetic resonance imaging study of children with dyslexia demonstrated the abnormal morphology of neurites in the language-associated regions of the neocortex . In lineROBO1 mutations may be caused by the abnormal formation of dendrites and terminal positioning of neurons. As dendrite formation and terminal positioning of neurons are potentially regulated by signals from the MZ and occur during the neonatal period, an interaction between ROBO1 and Slit or unknown molecules that reside in the MZ during the neonatal period might be important. Altogether, ROBO plays a crucial role in human neocortical development by regulating dendrite formation and neuron positioning, and such abnormalities occurring in language-associated regions can lead to dyslexia.Taken all together, the dyslexic phenotype in patients with In addition to dyslexia, the downregulation of ROBO expression has also been associated with ASD, presumably through the modulation of serotonin levels in the neocortex.Drosophila (Serotonin reuptake by serotonin transporters is crucial for maintaining normal levels of serotonin in the neocortex. Dysfunctions of serotonin transporters and resultant high serotonin levels are observed in ASD patients . As Roboosophila , and theosophila , decreasosophila , 2011, dSRGAP2 gene is localized, causes Van der Woude syndrome accompanied with intellectual disabilities (SRGAP2C, a human-specific paralog of srGAP2, was identified in patients with ASD and intellectual disability (In addition, mutations in srGAPs are associated with intellectual and cognitive disabilities . The disbilities . In addisability . srGAP3-sability . srGAPs Whereas the roles of Slit-Robo signaling in the developing brain have been well studied regarding axon guidance, during the previous decade, new roles of Slit-Robo signaling in progenitor cell proliferation and dendritic formation have emerged. These studies have shed light on the fundamental roles of Slit-Robo signaling in multiple events of neocortical development, from the proliferation of progenitor cells to circuit formation .Although the significance of Slit-Robo signaling in cortical development has been highlighted in this review, the detailed molecular mechanisms underlying Slit-Robo-mediated corticogenesis merits further investigation. As Slit molecules and Robo receptors have multiple binding partners in addition to their conventional ones, a comprehensive understanding of the Slit and Robo interactome in different cell types at different developmental stages is essential to understand the upstream and downstream signaling networks of Slit and Robo. This in turn will help us to understand the etiology of human diseases caused by abnormalities in Robo signaling.The recent implication of Robo signaling in brain evolution  has provTaken together, now is the time to revise our classical view of Slit-Robo signaling as a regulator of axon guidance, and build a new perspective on these key molecules in orchestrating multiple steps of neocortical circuit assembly and function.YG wrote and edited the manuscript. TN and CH edited the manuscript. All authors reviewed, discussed, and commented on 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.


Adipose tissue inflammation plays an important role in the regulation of glucose and lipids metabolism. It is unknown whether Ursolic acid (UA) could regulate adipose tissue inflammation, though it can regulate inflammation in many other tissues. In this study, 3T3-L1 adipocytes, DIO mice and lean mice were treated with UA or vehicle. Gene expression of inflammatory factors, chemokines and immune markers in adipocytes and adipose tissue, cytokines in cell culture medium and serum, and inflammation regulatory pathways in adipocytes were detected. Results showed that UA increased the expression of interleukins and chemokines, but not TNFα, in both adipocytes and adipose tissue. IL6 and MCP1 levels in the cell culture medium and mouse serum were induced by UA treatment. Cd14 expression level and number of CD14+ monocytes were higher in UA treated adipose tissue than those in the control group. Glucose tolerance test was impaired by UA treatment in DIO mice. Mechanistically, UA induced the expression of Tlr4 and the phosphorylation levels of ERK and NFκB in adipocytes. In conclusion, our study indicated that short-term UA administration could induce CD14+ monocytes infiltration by increasing the production of interleukins and chemokines in mouse adipose tissue, which might further impair glucose tolerance test. Adipose tissue plays an important role in metabolic health, for it serves as not only a storage organ for excess energy in the form of triacylglycerol but also an endocrine organ for many cytokines, such as interleukin 6 (IL6), tumour necrosis factor α (TNFα), monocyte chemokine protein 1 (MCP1), macrophage inflammatory protein 2 (MIP2), adiponectin and leptin, which regulate systemic insulin sensitivity and energy homoeostasis . Lipids Ursolic acid (UA), a pentacyclic triterpenic acid distributed in a variety of traditional medicine herbs and edible plants, has multiple biological functions, including regulation of glucose and lipids metabolism, anti-cancer, anti-oxidation and anti-inflammation . UA has Nuclear factor kappa B (NFκB) signalling pathway is a key regulator for the secretion of IL6, TNFα, interleukin 1β (IL1β) and other pro-inflammatory cytokines under the treatment of UA ,20. FurtTo clarify the effect of UA on adipose tissue inflammation, we treated both 3T3-L1 adipocytes and mice with UA, and found that short-term UA administration could induce the expression of interleukins, chemokines and inflammation regulatory genes in both cultured adipocytes and adipose tissue. Moreover, UA treatment stimulated the adipose tissue to recruit CD14 positive monocytes.Animal study protocol (SICAU-2017-028) was reviewed and approved by the Animal Care and Use Committee of Sichuan Agricultural University. All animal procedures were performed according to the guide for the care and use of laboratory animals of the National Institute of Health. C57BL/6 J male and female mice were obtained from Laboratory Animal Research Centre of Sichuan University , and were housed under the temperature of 22 ± 2°C and humidity of 60 ± 5% with 12 h light/dark cycle.Fifteen 5-week-old female mice with similar body weight were fed with high fat diet . At 19-week of age, mice were randomized into 3 groups with similar body weight (BW). Mice in group 1  were intraperitoneally (i.p.) injected with the vehicle , mice in group 2  were injected with UA  at 10 mg/kg BW and mice in group 3  were injected with UA at 20 mg/kg BW.Thirteen 12-week-old male lean mice with the similar weight were randomly divided into two groups. One group (n = 7) was injected with DMSO at 0.35 mL/kg BW, and the other group (n = 6) was injected with UA at 20 mg/kg BW. All mice were fed with normal chow diet .All mice were injected once daily for 3 d. In the evening of the third day, mice were starved for 12 h. The next morning, mice were sacrificed 2 h after UA or vehicle injection. Bodyweight was recorded, tail vein blood glucose levels were measured with glucose strips . Blood, gonadal adipose tissues, perirenal adipose tissues and subcutaneous adipose tissues were collected, weighed and frozen in liquid nitrogen, followed by storing at −80°C for further analysis. One piece of gonadal fat tissue was fixed in 4% formalin for haematoxylin and eosin stain and immunohistology stain. The weights of gonadal adipose tissues, perirenal adipose tissues and subcutaneous adipose tissues were summed as weight of total white adipose tissue (WAT).Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed as previously reported . BrieflyITT was performed on day 7 of UA treatment after 6 h-fasting. Mice were intraperitoneally injected with 1.2 U/kg BW of insulin. Blood glucose levels were measured at 0, 15, 30, 45, 60 and 90 min post insulin injection.2. Cells were subcultured into a 12-well plate at the density of 5 × 105 per well. Two days after 100% confluence, cells were incubated in 10% FBS containing DMEM, plus 1 µg/mL insulin (Sigma), 1 µM dexamethasone (Sigma) and 0.5 mM isobutyl methylxanthine (Sigma) for 4 d. Cells were then continually maintained in 10% FBS containing DMEM with 1 µg/mL insulin for another 6 d. Medium was freshly changed every other day. Cells were then treated with UA (Sigma) or vehicle (DMSO) after 12 h-incubation in serum-free DMEM.The culture and differentiation of 3T3-L1 preadipocytes (ATCC) were performed as previously reported . BrieflySerum levels of IL6, TNFα and MCP1 were measured with the respective ELISA kits  according to the instructions of manufacturer, as previously reported .For the detection of IL6 and MCP1 levels in the cell culture medium, fully differentiated 3T3-L1 adipocytes were treated with 25 µM UA or vehicle for 12 h in serum-free DMEM. Levels of IL6 and MCP1 in the medium were quantified with the respective ELISA kits (BioLegend).The formalin fixed gonadal adipose tissues were embedded in paraffin and were sliced into 4 µm sections. H&E staining was performed as previously reported . BrieflyImmunohistochemistry straining was performed by Servicebio  using the DAB-stain method. Anti-CD14 antibody  was used at the dilution of 1:200.Images were captured on a microscope  with a CCD  using imaging software .Bactin as the reference gene. Sequences of the primers were given in Supplementary Table S1.Total RNA was isolated from gonadal adipose tissues and adipocytes with TRI Reagent  under the manufacturer’s instruction. The quality of total RNA was assessed by agarose gel and the concentration was measured with a spectrophotometer . cDNA was synthesized with 1 µg total RNA using a reverse-transcription PCR kit . Real-time quantitative PCR was performed with Power SYBR Green RT-PCR reagents  using the QuantStudioTM 6 Flex System . The PCR conditions were 1 cycle of 95°C for 30 s and 40 cycles of 95°C for 15 s followed by 60°C for 1 min. Data were analysed by 2-delta CT method with Western blot analysis was performed as previously reported . BrieflyP value <0.05 was considered statistically significant.Data were analysed using the software of SAS 9.4. One-way ANOVA with post hoc analysis was applied for multiple comparisons; independent t-test was utilized to compare the difference between two groups; repeated-measures ANOVA was used to analyse the statistical difference of GTT and ITT study. All data were presented as mean ± SEM and Il6, but not Tnfa, could be induced by UA in adipocytes as compared with control group . Meant manner ,b). Conct manner ).Figure Il6 and chemokine genes Mcp1, Mcp3 and Mip2 in adipocytes. To investigate the in vivo effect of UA on adipose inflammatory and chemokine gene expression, diet-induced obese (DIO) mice were treated with UA for 3 d. Results showed that UA treatment did not change the body weight, blood glucose level or adipose tissue weight . The expression of Il6, Il1b, Mcp1, Mcp3 and Mip2 in gonadal adipose tissue (gWAT) were remarkably induced by UA, as compared to those in the control group . Howevethe gWAT ). Furthethe gWAT ). No difthe gWAT ).Figure Il6, Il1b, Mcp1, Mcp3 and Mip2, but not Tnfa, were upregulated by UA in the gWAT of lean mice, as compared with those of the control mice . Besiderol mice ). Howeverol mice ).Figure Acc1, Fasn and Sterol regulatory element-binding protein 1 c (Srebp1c) in adipocytes at the doses of 25 and 50 µM, as compared to the control group . Besideol group ). As forol cells ). These F4/80 and Cd11 c, markers of macrophage, were not changed by UA treatment in the gWAT of lean mice, as compared to those in the control mice .Increased expression and secretion of chemokines in adipose tissue could stimulate immune cells to infiltrate into adipose tissue ,28. Thusrol mice ). For thol group ). SimilaH&E staining and immunohistology straining were then performed to check the CD14+ cells in the adipose tissue of lean mice. Results showed that the number of CD14+ monocytes was increased in the adipose tissues of UA treated mice as compared to that in the control mice -d).It was reported that chronic adipose tissue inflammation could impair insulin sensitivity and induce lipids metabolic disorder . Thus, wTlr4 was tested in both cultured adipocytes and adipose tissue. Results showed that UA could induce the expression of Tlr4 in a dose-dependent manner in 3T3-L1 adipocytes, as compared to the control group . Besideup (Tlr4 ). In vivrol mice ).Figure ERK/NFκB pathway was reported to be involved in the regulation of inflammation. Our data showed that the phosphorylation levels of both ERK and NFκB were upregulated by UA in 3T3-L1 adipocytes, as compared to the control group ,e). HoweAdipose tissue is the major organ for excess energy storage, as well as an endocrine organ which secretes inflammatory factors and chemokines. Here, we found that short-time treatment with UA induced the production of IL6, IL1β, MCP1, MCP3 and MIP2 in both adipocytes and adipose tissue, and subsequently stimulated the adipose tissue to recruit CD14+ monocytes. Besides, we also revealed that short-time UA treatment trended to impair glucose tolerance test in diet-induced obese mice. Our results are important for the functional study of UA.Tnfa, in both cultured adipocytes and mouse adipose tissue. Meanwhile, serum concentration of MCP1 was induced by UA treatment in both DIO mice and lean mice, while serum IL6 level was increased by UA treatment in lean mice. Previous studies have reported that UA inhibited inflammation in many tissues and cells, including liver, kidney, spinal cord, intestine and macrophages [Il6 and Il1b and chemokines Mcp1, Mcp3 and Mip2 in adipocytes and adipose tissue. It is interesting that only the expression of Il6, but not Tnfa, was elevated by UA in adipocytes. That might be because MCP1 could induce the expression of Il6, but not Tnfa [Adipose tissue secretes many cytokines, such as IL6, TNFα, MCP1 and MIP2, who play important roles in the regulation of metabolism and immunity ,29,30. Orophages ,23,26,31rophages ,15,20. Tnot Tnfa . It has not Tnfa ,34. ThusCd14 and the number of CD14 positive monocytes in adipose tissue, while the expression of macrophage marker genes F4/80 and Cd11 c were not changed. These data indicated that short-time UA administration stimulated the white adipose tissue to recruit CD14 positive monocytes, but not macrophages. CD14 has been reported to induced inflammation and insulin resistance in adipose tissue [Previous study indicated that adipocyte-secreted MCP1 could stimulate the infiltration of macrophages into adipose tissue via MCP1/CCR2 pathway . Besidese tissue ,38. Thuse tissue . The reaTNFα, IL6 and IL1β via the key inflammatory transcription factor NFκB in activated T cells, B cells and macrophages [Il6, Tnfa and Mcp1 expression through NFκB pathway in adipose tissue of ageing mice [Il6, Tnfa and Il1b expression by inhibiting TLR4/NFκB pathway in mouse macrophages [Tlr4 expression and phosphorylation level of NFκB in adipocytes. Therefore, UA might up-regulate the production of interleukins and chemokines through the TLR4/NFκB signalling pathway in adipocytes .et al. used BEAS-2B epithelial cells [et al., who found that UA stimulated ERK/NFκB pathway by activating Caspase 3, used human osteosarcoma cells [Il6 and Mcp1 in adipocytes [Foxo1 was increased by UA in our study. Thus, FOXO1 might also be involved in UA inducing the expression of interleukins and chemokines in adipocytes.Suppressed ERK/NFκB signalling was reported to mediate UA inhibiting inflammation in liver . Study oal cells . Wu et ama cells . Howeveripocytes . The expFat deposition is closely related to the expansion of adipose tissue mass. Overload of fat leads to adipose tissue hypertrophy, and even obesity . Thus, dMany studies have reported that UA decreased adipose accumulation in DIO mice and rats ,13,46–48SREBP1c [ACC1 and FASN expression [Foxo1 expression and decreased Srebp1c expression in adipocytes. Thus, UA might suppress lipid synthesis through FOXO1/SREBP1c pathway in adipocytes. Besides, IL6 was reported to inhibit Fasn expression in DIO mouse liver [Il6 expression was significantly upgraded by UA in adipocytes. Therefore, UA might also suppress lipids synthesis through IL6 pathway in adipocytes. However, the exact mechanisms need further studies to be elucidated.FOXO1 has been reported to suppress the expression of SREBP1c , a transpression ,50. Our se liver . Our resIn summary, our study indicated that short-term UA administration could stimulate adipose tissue to recruit CD14+ monocytes by inducing the adipocytes to produce interleukins and chemokines via the TLR4/NFκB pathway.Click here for additional data file.


The hedgehog (SHH) signaling pathway is primarily involved in embryonic gut development, smooth muscle differentiation, cell proliferation, adult tissue homeostasis, tissue repair following injury, and tissue polarity during the development of vertebrate and invertebrate organisms. GLIoma-associated oncogene homolog (GLI) family of zinc-finger transcription factors and smoothened (SMO) are the signal transducers of the SHH pathway. Both SHH ligand-dependent and independent mechanisms activate GLI proteins. Various transcriptional mechanisms, posttranslational modifications , and nuclear-cytoplasmic shuttling control the activity of SHH signaling pathway proteins. The dysregulated SHH pathway is associated with bone and soft tissue sarcomas, GLIomas, medulloblastomas, leukemias, and tumors of breast, lung, skin, prostate, brain, gastric, and pancreas. While extensively studied in development and sarcomas, GLI family proteins play an essential role in many host-pathogen interactions, including bacterial and viral infections and their associated cancers. Viruses hijack host GLI family transcription factors and their downstream signaling cascades to enhance the viral gene transcription required for replication and pathogenesis. In this review, we discuss a distinct role(s) of GLI proteins in the process of tumorigenesis and host-pathogen interactions in the context of viral infection-associated malignancies and cancers due to other causes. Here, we emphasize the potential of the Hedgehog (HH) pathway targeting as a potential anti-cancer therapeutic approach, which in the future could also be tested in infection-associated fatalities. Drosophila melanogaster, HH signaling is extensively used to explain the complex topic of cell segment number patterning. Advanced research identified its important morphogenic properties for cell proliferation, polarity, and differentiation . T. T128]. CDH1) expression during EMT in addition to enhancing the transcription of other EMT activators [Further evidence in human melanoma cells suggests that GLI2 is directly responsible for turning off the E-cadherin gene , chemokines CXCR4 and CXCR7 enhance the directional migration of breast cancer cells in lung metastasis by way of CXCL12 , which iGLI2 expression directly enhances tumorigenesis in a model of myofibroblastic cells representing reactive stromal prostate cancer cells . There iBesides controlling processes involved in embryogenesis, there are broader implications of HH signaling. Recent studies revealed that aberrant activation of HH signaling leads to pathological consequences . VirusesHere, we discuss influenza-A, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papillomavirus (HPV), Merkel cell polyomavirus (MCPyV), human T-cell leukemia virus, type 1 (HTLV-1), Epstein–Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) infection to highlight the plausible involvement of HH pathway players in the viral life cycle. We discuss the possible role of HH signaling in viral latency, virus reservoir maintenance, entry, replication, pathogenesis, infected cell proliferation, infection progression, pathogen recognition in the host, and the immune response of the host, exit of the pathogen and memory of the host immune mediators . HIV ,160, HCVHH signaling is a potential target for manipulating viruses, as this pathway plays a fundamental role in cell survival and proliferation ,189,190.Drosophila melanogaster has shown that the effect of the NS1 on the HH pathway is conserved between the species. A point mutation (A122V) was identified in Drosophila, which could reduce the HH-dependent activity of the NS1 in flies and transfected cells [Moreover, the HH receptor, PTCH1, is expressed in infiltrating and circulating lymphocytes (CD4 and CD8) at both protein and mRNA levels. It is evident that immune cells are equipped to respond to the HH ligand secreted from the inflamed area . HH signed cells . HoweverHIV , HCV 19, and HBVHH signaling also contributes to the epigenetic regulations. HH target genes are poised and are marked by an active (H3K4me3) and a repressive (H3K27me3) mark maintained by the H3K27 methyltransferase polycomb repressive complex 2 (PRC2) . SHH indHBV and HCV are associated with liver cirrhosis and hepatocellular carcinoma (HCC) worldwide . PereiraThe HH pathway maintains and controls stem cells and policies of hematopoiesis and lymphopoiesis to maintain immune cells ,225,226.HH regulates the expression of OPN in nonalcoholic steatohepatitis-related liver fibrosis as GLI directly interacts with the GLI-binding sites in the OPN promoter . A decreMerkel cell polyomavirus (MCPyV) is detected in approximately 80% of Merkel cell carcinoma (MCC), an aggressive neuroendocrine skin cancer mostly occurring in the elderly. Reactivation of HH signaling later in life can cause tumors. 29 MCPyV-positive and 21 MCPyV-negative MCCs were stained for SHH, IHH, PTCH1, SMO, GLI1, GLI2, and GLI3 and expression of the HH signal pathway players associated with MCPyV infection and prognosis of MCC . HPV oncogenes (E6/E7) and estradiol, major etiologic factors associated with cervical cancer, could induce GLI activity in the cervix and the skin in mice . TreatmeTumor cells of Hodgkin lymphoma (HL) are derived from mature B cells. The lineage infidelity of Hodgkin/Reed-Sternberg cells (HRSs) often causes diagnostic problems as HRS markers are also favorable for follicular dendritic cells (FDCs). Investigation of the expression of FDC markers in HL and anaplastic large cell lymphoma (ALCL) revealed GLI3, fascin (actin-bundling protein found in membrane ruffles), and TUBB3 (a member of the beta-tubulin protein family) as the most sensitive markers, which were diffusely positive in HL . A recenAnti-inflammatory lipoxin A4 treatment in PEL cells downregulated GLI1 expression . The decHTLV1 expression is activated by the interaction of a viral transactivator protein, TAX, and cellular transcription factor, CREB (cyclic AMP response element-binding protein), binds to the long terminal repeat (LTR). The human homolog of a member of the GLI oncogene family, GLI2 , helps HTLV1 infection progression as the simultaneous binding of hGLI2 and CREB seems critical for TAX protein to activate transcription . HIV 24, HCV 2442, HBV ,244, EBVHH signaling is also regulated by MAPK and NF-κB cascade and HH/GLI1, MAPK (KRAS-MEK-ERK) cascade and NF-κB cooperate to regulate growth and cell proliferation , and apoSalmonella enteritidis ST183 [Escherichia coli [Helicobacter pylori [Helicobacter pylori infection mediated inflammation and repair process involving macrophage recruitment activates transcription factor NF-κB and also upregulates HH proteins [Not only viruses, but bacterial infections such as is ST183  Escherichia coli , and Helr pylori  are alsor pylori ,287,288 proteins .Currently, small molecule modulators such as SMO and GLI1 inhibitors of HH signaling have been used in basic research to detect links between signaling and specific phenotypes of interest . Few inhAberrant HH signaling has been found responsible for chemo-resistance in aggressive cancers ,292. MucAmong SMO inhibitors, GDC-0449 and LDE-225 are in the clinic for the treatment of advanced BCC with aberrant HH activity due to loss of the functional allele of PTCH ,298 The IPI-926 (Saridegib), CUR61414 binds to SMO and prevents its activation, BMS-833923/XL139 (binds SMO), PF-04449913; Glasdegib (SMO inhibitor), PF-5274857 (SMO antagonist), TAK-441 (SMO inhibitor), LY2940680; Taladegib (SMO antagonist), MRT-92 , Jervine (binds to SMO and preventing its conversion to an active state), RU-SKI 43 (SHH Inhibitor), and SHH Monoclonal Antibody 5E1 (SHH Inhibitor) is SMO or SHH Inhibitor in clinical trials ,306.A well-known small molecule inhibitor, GANT61, reduces GLI1, GLI2, and PTCH1 mRNA expression in human colon carcinoma cell lines . GANT61 SHH pathway is highly activated in pancreatic CSCs and plays an essential role in cancer initiation, progression, and metastasis . SanguinGLI transcription factors interact with multiple signaling pathways. Therefore, many combination treatment modalities are under clinical trials, including PI3K inhibitor (BKM120 or BEZ235) and SMO inhibitor (LDE225), an mTOR inhibitor and GANT61, simultaneous targeting of GLI1 and HIF2α. LDE225/Sonidegib and GDC-0449/Vismodegib SMO inhibitors are FDA approved for treating basal cell carcinoma. Clinical trials are ongoing to evaluate the efficacy of this novel class of targeted therapy in various malignancies. The paracrine mechanism plays an essential role in upregulating the HH signaling in a tumor animal model, suggesting the need to consider the stroma-tumor interactions in HH/GLI signaling in the experimental models . HH sign


WNT-signaling controls important cellular processes throughout embryonic development and adult life, so any deregulation of this signaling can result in a wide range of pathologies, including cancer. WNT-signaling is classified into two categories: β-catenin-dependent signaling  and β-catenin-independent signaling , the latter can be further divided into WNT/planar cell polarity (PCP) and calcium pathways. WNT ligands are considered as unique directional growth factors that contribute to both cell proliferation and polarity. Origin of cancer can be diverse and therefore tissue-specific differences can be found in WNT-signaling between cancers, including specific mutations contributing to cancer development. This review focuses on the role of the WNT-signaling pathway in melanoma. The current view on the role of WNT-signaling in cancer immunity as well as a short summary of WNT pathway-related drugs under investigation are also provided. Wingless, a Drosophila segment polarity gene .,32.7,32]It should be noted that the transcriptional role of β-catenin extends beyond the TCF/LEF as β-catenin may be a partner of other transcription factors, e.g., sex-determining region Y (SRY) box-containing factors (SOX), mothers against decapentaplegic homolog (SMAD), octamer-binding transcription factor 4 (OCT4) and forkhead box class O family member proteins (FOXOs) .AXIN1, AXIN2, DKK1 and SFRP [WNT/β-catenin-signaling can be modulated by several antagonists at the ligand and receptor level, e.g., DKK proteins, secreted frizzled-related proteins (sFRPs), WNT inhibitory factor 1 (WIF1), WNT modulator in surface ectoderm (WISE), Kremen (KRM) and Cerberus protein (CER1) ,16,44. Tand SFRP ,46.CTNNB1 and comprises a flexible N-terminal domain , central armadillo (ARM) repeat domain  and a C-terminal transactivation domain  [The β-catenin-dependent WNT-signaling cascade  regulate~100 aa) . While N~100 aa)  such as n NTD; ~1 aa, cent~100 aa) , a conse2+-signaling pathway  A and WNT pathway B. The WN2+-signaling pathway is associated with the release of Ca2+ from intracellular stores. Interaction of WNT ligands with FZD activates phospholipase C (PLC) that hydrolyzes phosphatidylinositol -biphosphates (PIP2) to inositol -triphosphates (IP3) and diacylglycerol (DAG). DAG activates PKC kinase that in turn activates the small GTPase CDC42, while IP3 induces the release of Ca2+ ions from intracellular depots. Release of Ca2+ activates Ca2+/calmodulin dependent kinase II (CaMKII) and calcineurin (CaN). CaMKII phosphorylates TGFβ-activated kinase 1 (TAK1), which induces Nemo-like kinase (NLK) activation, which in turn inhibits transcriptional activity of canonical WNT-signaling. CaN via dephosphorylation activates nuclear factor of activated T-cells (NFAT) that translocates to the nucleus and regulates the expression of target genes. WNT/Ca2+ pathway activation plays an important role in the regulation of cell motility and cytoskeleton organization [The WNT/Canization ,54,55.APC in colorectal cancer and CTNNB1 in hepatocellular carcinoma, deregulations of several extracellular modulators of WNT-signaling e.g., DKKs, sFRPs and WIF1 also contribute to cancer development. These proteins antagonize canonical WNT-signaling by binding to LRP5/6 or inhibit the interaction between WNTs and their receptors [+-ATPase (v-ATPase), an electrogenic H+ transporter required for WNT-signaling activation may also trigger abnormal WNT/β-catenin-signaling and contribute to WNT-signaling-dependent tumorigenesis. High expression of v-ATPase subunits has been observed in colorectal, prostate, breast, ovarian and pancreatic cancer cells [As demonstrated in numerous studies, the aberrant activation of WNT-signaling contributes to malignant cell transformation and neoplastic proliferation with further metastatic dissemination and resistance to treatment ,56. Manyer cells . Furtherer cells . The preer cells ,59,60,61er cells . For thaer cells .APC and AXIN, but the gene encoding β-catenin, CTNNB1, is also frequently mutated in selected cancer types. High frequency of mutations causing hyperactivation of WNT-signaling can be detected for example in colorectal cancer [APC leading to APC loss of function have been found in about 70% of cases [Deregulations in the canonical WNT-signaling in cancer may result from diverse mutations and epigenetic mechanisms. Mutations in genes encoding distinct elements of the WNT pathway can cause (i) loss of function of the destruction complex, (ii) increase in nuclear localization of β-catenin resulting in β-catenin-mediated transactivation of several genes. Mutations are mostly detected in genes encoding components of the destruction complex such as l cancer ,64 and mof cases ,66,67.APC, AXIN1 and CTNNB1 are low, reaching according to cbioportal.org 10%, 2.9% and 5.9%, respectively  locus encodes two proteins: p16INK4a and p14(p19)ARF, regulating RB1 and p53 pathways, respectively. Inactivation of the genetic locus INK4α-ARF is important in overcoming the senescence barrier to oncogenesis [INK4ap16 expression can be silenced by activated β-catenin [INK4ap16 through a conserved TCF/LEF site in its promoter [high subpopulation of melanoma cells producing melanoma antigen recognized by T cells 1 (MART1) are less invasive, whereas when this differentiation antigen is lost melanoma cells acquire highly invasive properties [As melanoma cells utilize WNT/β-catenin-signaling for transformation and proliferation, whereas non-canonical signaling for metastasis , β-catenCTNNB1 . β-catenCTNNB1 ,103. TheCTNNB1 . SenesceCTNNB1 . The INKogenesis . In a vapromoter . While bpromoter ,94,107. promoter ,109,110.promoter ,112,113 promoter ,116,117.promoter . Recentlpromoter . In viewpromoter . In actipromoter ,118,121.promoter ,117 as ipromoter . It has promoter ,101. BRNpromoter . It has operties . Differeoperties . Therefooperties ,99. Arozoperties . When looperties . The rouWIF1 overexpression resulted in downregulation of WNT-signaling and suppression of melanoma cell proliferation [WIF1 expression have been observed between its level in the primary tumor and sentinel lymph node (SLN) metastases [Aberrant expression of WNT/β-catenin antagonists  is common in melanoma and is associated with elevated β-catenin level. Downregulation of DKKs has been detected in melanoma cell lines and tissue samples . Reducedferation . Differetastases . Similartastases . Reductitastases . Kaur ettastases . sFRP2 dtastases .2+-dependent release of exosomes by melanoma cells has been reported [high melanoma cells caused a reduction of the WNT5A level and induction of β-catenin [Non-canonical WNT-signaling, which is activated during melanoma progression, suppresses the WNT/β-catenin-signaling ,124. Thereported . Exosomereported . Using rreported . Further-catenin .2+-signaling [WNT5A can either inhibit or activate canonical WNT-signaling in diverse ways depending on the receptor context . Severalignaling . Calpainignaling . PKC actignaling . Furtherignaling ,133. WNTignaling ,134.WNT5A can act through SIAH2 via the GSK3-β-independent pathway to promote degradation of β-catenin ,136,137.FZD3 has been found to be associated with β-catenin-independent-signaling and upregulation of this receptor is associated with melanoma progression and a reduced patient survival, whereas down-regulation of FZD3 suppresses growth and metastasis of melanoma . OverexpThe complex interactions existing between diverse signaling pathways create the opportunity for a cancer cell to compensate for a blockade of a single pathway. The crosstalk consisting of multiple interactions and feedback loops is crucial for the cellular response to microenvironmental changes, including treatment. WNT-signaling is an important part of the crosstalk between key oncogenic pathways involved in melanoma development, progression and response to treatment. Elements of the WNT-signaling pathways both depend on and regulate activities of diverse pathways such as MAPK/ERK and phosphatidylinositol-3-kinase (PI3K)/AKT as well as transcriptional regulators including p53 and MITF. V600 enhanced the activity of β-catenin in the presence of WNT ligands [V600E and wild-type PTEN [WNT-signaling can interact with MAPK/ERK and PI3K/AKT-signaling in diverse ways. Moreover, growing evidence indicates that this crosstalk in melanoma depends on the genetic context and several mechanisms are considered as tumor stage-dependent or unique for melanoma. In an early study, it was shown that WNT/β-catenin and MAPK/ERK pathways synergized in order to induce melanoma without the need for p16INK4a mutations and β-catenin could cooperate with N-RAS . Next, i ligands . This co ligands . This ne ligands ,142. In  ligands . Full ac ligands . These r ligands . A most ype PTEN . Moreoveype PTEN ,143,144.ype PTEN . COX-2 iype PTEN .Transcription factors responding to diverse signal transduction pathways play a key role in integrating multiple signals to give rise to the optimal phenotype confronting the cancer developmental stage and microenvironmental insults . MITF, aThe WNT5A impact on p53 activity is another example of the influence of non-canonical WNT-signaling on the transcription factor-mediated phenotype of melanoma cells. As shown most recently, a high expression of WNT5A is observed in wild-type p53 melanomas representing 80% of this cancer, and more important, WNT5A can promote a slow-cycling phenotype of melanoma cells by increasing the half-life of wild-type p53 . WNT5A eCTNNB1 (8%) or loss of function mutations (11%) in genes encoding negative regulators of WNT/β-catenin-signaling have been detected in these metastases [Various studies indicate that WNT-signaling is associated with several aspects of immunity, and alterations in WNT-signaling can be connected with the deregulation of immune response against cancer, both innate and adaptive ,166,167.tastases . Genetictastases . This estastases . Interestastases .As activation of tumor-intrinsic WNT/β-catenin-signaling is enhanced in non-T cell-inflamed tumors, the WNT-signaling inhibitors may be useful for restoring immune cell infiltration to support immunotherapy.The important role of WNT-signaling in the development and adult life as well as its implication in a wide spectrum of diseases, including cancer has already attracted the attention of medical and biotech companies. Ongoing research is focused on the development of specific inhibitors of WNT/β-catenin-signaling for cancer therapies, and only very few modulators of non-canonical signaling are known. Based on the recent advances in cancer biology, targeting of WNT-signaling in cancer cells has to be put into perspective of its effectiveness either in reversing resistance to anticancer drugs or in inhibition of tumor evasion. It is important to underline that it is difficult to target only the WNT pathway without interfering with other signaling pathways. Diverse compounds of natural origin or synthetic, peptides and antibodies that are in preclinical studies are shown in Several agents influencing WNT-signaling have reached the clinical trials . Many ofCRD-BP), MITF and CCND1 [As shown in nd CCND1 . Pentoxind CCND1 .As demonstrated above, there is a large group of natural or synthetic compounds, peptides and antibodies that are capable of affecting, either inducing or inhibiting, the WNT-signaling pathways at various stages. Compounds that have been investigated in melanoma are shown in While inhibition of WNT-signaling represents an immensely appealing strategy for the development of anticancer therapeutics, none of drugs targeting WNT-signaling is yet available in clinical practice ,214.APC mutations in about 70% of colorectal cancer patients, mutations are not the major cause of these deregulations in melanoma. Moreover, transcriptionally active β-catenin is associated with less invasive disease and more favorable prognosis for melanoma patients, in contrast to other cancers, in which nuclear β-catenin is a driving force of both initiation and progression. It is thought that β-catenin-suppressed invasion occurs through a cell-type specific mechanism involving transcription factor MITF, one of the β-catenin target genes. On the other hand, low efficacy of immunotherapy is observed in melanomas with elevated level of β-catenin. The identification of therapeutic targets is further complicated by the crosstalk between WNT-signaling pathways and other signaling pathways crucial for melanoma development such as MAPK/ERK and PI3K/AKT, as well as the plasticity of melanoma cells in response to microenvironmental insults. Therefore, finding a therapeutic window for effective modulation of the WNT pathway in melanoma is a challenging task. A low number of clinical trials investigating WNT/β-catenin modulators in melanoma patients is the consequence of the controversial role of WNT-signaling in melanoma.WNT-signaling is extremely complex and context-dependent in cancer, including melanoma. Deregulation of WNT-signaling contributes to cancer initiation, progression, modulation of immune microenvironment and resistance to treatment. While WNT-signaling alterations start with