Ovarian cancer (OC) is the most lethal gynecological cancer, worldwide, largely due to its vague and nonspecific early stage symptoms, resulting in most tumors being found at advanced stages. Moreover, due to its relative rarity, there are currently no satisfactory methods for OC screening, which remains a controversial and cost-prohibitive issue. Here, we demonstrate that Papanicolaou test (Pap test) cervical scrapings, instead of blood, can reveal genetic/epigenetic information for OC detection, using specific and sensitive DNA methylation biomarkers.We analyzed the methylomes of tissues  and cervical scrapings , and integrated public methylomic datasets, including 79 OC tissues and 6 normal tubal epithelia. Differentially methylated genes were further classified by unsupervised hierarchical clustering, and each candidate biomarker gene was verified in both OC tissues and cervical scrapings by either quantitative methylation-specific polymerase chain reaction (qMSP) or bisulfite pyrosequencing. A risk-score by logistic regression was generated for clinical application.AMPD3, 0.79–0.85 of AOX1, 0.78–0.88 of NRN1, and 0.82–0.85 of TBX15. From this, we found OC-risk score, equation generated by logistic regression in training set and validated an OC-associated panel comprising AMPD3, NRN1, and TBX15, reaching a sensitivity of 81%, specificity of 84%, and OC detection accuracy of 0.91  in testing set.One hundred fifty-one genes were classified into four clusters, and nine candidate hypermethylated genes from these four clusters were selected. Among these, four genes fulfilled our selection criteria and were validated in training and testing set, respectively. The OC detection accuracy was demonstrated by area under the receiver operating characteristic curves (AUCs) in 0.80–0.83 of AMPD3/NRN1/TBX15. Further validation is warranted.Ovarian cancer detection from cervical scrapings is feasible, using particularly promising epigenetic biomarkers such as  Ovarian cancer (OC) is the fifth-leading cause of cancer death in the USA, and the most lethal female genital tract malignancy worldwide, with over 150,000 deaths in 2012 .With regard to therapies, while treatment advances have boosted survival outcomes for many types of cancer, over the past two decades, OC has seen slower progress. Thus, despite successful efforts in improving OC treatment, including surgery, cytotoxic chemotherapy, hyperthermic intraperitoneal chemotherapy, and targeted therapy, only marginal improvement has been seen , 5. TherOne much-studied, potential early detection approach, the use of the serum biomarker cancer antigen 125 (CA-125) and transvaginal ultrasound (TVU), was extensively examined in the Prostate, Lung, Colorectal, and Ovarian (PLCO) cancer screening trial, including 78,216 women, with a median follow-up up to 13 years. That study showed no mortality benefit across an OC screening and no screening arm. This diagnostic evaluation also yielded a high false-positive rate associated with surgical complications [To augment  traditional screening tools (TVU and CA-125), novel molecular biomarkers are now under intense study. To that end, the inclusion of additional blood-based protein biomarkers, such as HE4 or CA72-4, was found encouraging –10. EvenAPC, AKT1, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, PIK3CA, PPP2R1A, PTEN, and TP53, from Pap test specimens, was able to identify 41% of OCs (9 of 22), potentially opening OC detection to a new panel of molecular biomarkers found in cervical Pap smears [For the detection of cervical cancer, the Papanicolaou (Pap) test collects endocervical samples, although ovarian and endometrial cancers (ECs) are infrequently detected via abnormal cervical cytology. Recently, one study demonstrated that DNA mutational analysis of Pap samples was capable of detecting OCs and ECs. In that work, massive parallel sequencing of 12 exons of p smears .In addition to genetic events, epigenetic changes have been widely studied in cancer. For example, DNA hypermethylation-mediated silencing of tumor suppressor genes is common in overall carcinogenesis, such that research regarding epigenetic alterations in OC have also been associated with different histologies, grades, stages, response to chemotherapy or targeted therapy, relapse risk, and survival –21. Our http://www.kegg.jp/ or http://www.genome.jp/kegg/) and Reactome pathway databases showed enrichment in several signaling pathways, including maturity-onset diabetes of the young, peptide ligand-binding receptors, and the estrogen signaling pathway .We utilized these 151 HDM genes which were listed in detail  greater than 0.7, except MEGF11, were subject to further validation in the testing set. The distribution of the depicted plots represents the methylation levels, in terms of change in PCR threshold cycle  of each candidate gene, between normal and OC cervical scrapings in the training and testing sets, respectively. The results all reached statistically significant differences   × ΔCp of NRN1 + (− 0.57) × ΔCp of TBX15 + 6.38 resulted in a sensitivity of 80.7% and a specificity of 83.9%. Then, the cut-off value, 0.73, was applied to 42 cervical scrapings from testing set . Mucinous type has a lower OC-risk score  and listed in Additional file To translate the results of our findings for clinical application, we developed a mathematical equation for risk prediction of OC (OC-risk score), by integrating methylation levels of D3 + − 0.1 × ΔCp oOnly 25% of high-grade serous ovarian cancers are only diagnosed in early stages, underscoring an urgent need for practical means of screening. Prior large-scale efforts have assessed the efficacy of OC screening, using different modalities such as serum CA-125 levels and transvaginal ultrasound imaging, including the Prostate, Lung, Colorectal, and Ovarian Cancer (PULCO)  and UK CNRAS, CTNNB1, PIK3CA, FBXW7, APC, EGFR, BRAF, CDKN2A, PTEN, FGFR2, HRAS, AKT1, TP53), i.e., the “CancerSEEK” blood test, was reported to detect multiple cancers, including OC [Recently, the use of serum proteins  in combination with 13 cell-free (cf)-DNA amplicons . To simultaneously utilize such easily accessing approaches , for the discovery of OC detection biomarkers, is appealing. One study even illustrated that DNA mutational analyses of samples collected from cervical scrapings could detect ovarian and endometrial cancer . Due to AKT1, APC, BRAF, CDKN2A, CTNNB1, EGFR, FBXW7, FGFR2, KRAS, MAPK1, NRAS, PIK3CA, PIK3R1, POLE, PPP2R1A, PTEN, RNF43, and TP53, in addition to revealing chromosomally aneuploid OC cells, at a detection sensitivity of 33% [PTGDR, HS3ST2, POU4F3, and MAGI2 genes from cervical scrapings [AMPD3, NRN1, and TBX15 discovered increased sensitivity of 81%, and increased accuracy of 0.87–0.91.Recently, one study using a Pap brush, called PapSEEK detected 18 genetic mutations, including y of 33% . If, in y of 33% . Our precrapings . HoweverAMPD3 (adenosine monophosphate deaminase 3) encodes a member of the adenosine monophosphate (AMP) deaminase gene family, and its encoded protein belongs to a highly regulated enzyme that catalyzes the hydrolytic deamination of AMP to inosine monophosphate (IMP), in the adenylate cyclase catabolic pathway [AOX1  produces hydrogen peroxide, and can catalyze the formation of superoxide, under certain conditions. Much less is known about the physiological function of the enzymatic substrates/products of human AOX1, and other mammalian AOX isoenzymes [NRN1 (Neuritin 1), encodes a member of the neuritin family, which is expressed in postmitotic-differentiating neurons of the developmental nervous system. NRN1 participates in promoting migration of neuronal cells, and impacts microtubule stability [TBX15 (T-box-15), belongs to the T-box family of genes, which encode a phylogenetically conserved family of transcription factors that regulate a variety of developmental processes [The functional role of these genes in OC remains unexplored.  pathway . AOX1 . Third, epithelial OCs themselves are heterogeneous in histology types, with different etiologies. It raises challenges that epithelial OCs comprise of a large heterogeneity dividing into different subtypes according to their morphological, clinical, and molecular genetic characteristics. To solve these limitations before clinical application, further validation in population-based prospective clinical trial is warranted.Although promising, our study has several limitations. First, it is a discovery phase from a retrospective case-control study. The results here are not yet appropriate for dissemination to the general population. Second, confounding by other uterine or ovarian neoplasms, or disrupting anatomical location remains to be determined. According to our previous studies and literature , differeAMPD3, NRN1, and TBX15 as promising biomarkers for OC detection. Further, large-scale trials are needed to validate the potential of these procedures and the use of such promising biomarkers.The potential development of DNA methylation biomarkers, from cervical scrapings, expands the scope of the Pap test, a now-routinely used cytological exam especially prevalent in developed countries. The detection of female genital tract malignancies, including CC, EC, and OC, by combining cervical scrapings and molecular markers, is an attractive concept. Here, we revealed DNA methylation of the genes later® Stabilization Solution . We then liquated the cervical scrapings after vortexing for 1 min, followed by storage at − 80 °C, until DNA extraction. Age, histological type of tumor, International Federation of Gynecology and Obstetrics (FIGO) stage, and histological grade were tabulated in the hospital records for each anonymized participant. Ovarian tissues  and cervical scrapings  were utilized for methylomics analysis, respectively. We randomly selected cervical scrapings from 15 OCs and 15 normal controls for verification. Every 5 cervical scrapings from OCs or normal controls were put together as one DNA pool and depicted as one dot in Fig. We enrolled a total 205 participants, aged 20 to 90 years old, and collected 149 cervical scrapings and 50 malignant and 6 normal epithelial ovarian tissues. Participants signed informed consent for the study, between November 2014 and October 2017, at Shuang Ho Hospital and Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan. The study was conducted strictly according to a protocol approved by the Institutional Review Board of the Taipei Medical University, in accordance with the Declaration of Helsinki, 2000. Cervical scrapings were obtained in operation room or during an outpatient visit before initial surgery, using a cervical brush . Normal ovarian epithelial cells were obtained from participants diagnosed with uterine leiomyomas, after abdominal total hysterectomy combined with salpingo-oophorectomy. All specimens were collected and placed immediately in RNAFor validation, the samples size, estimated at AUC 0.75 for each candidate gene, compared with AUC 0.5 as the null hypothesis status, with 0.05 as the type I error (α), 0.2 as the type II error β, 1-power), and a 1:1 ratio of OC case numbers to normal groups. Accordingly, we assigned a ratio of the sample size of training set at 1.5-fold that of the testing set. Two samples were added to both the OC and normal groups to avoid a failed detection. The sample sizes of the training and testing sets were predicted to be 62 and 42, respectively. We enrolled participants between November 2014 and August 2016 for the training set, and from August 2016 to October 2017 for the testing set. Clinicopathological results and demographics are listed in Table -power, aU test with P < 0.01, HDM level > 0.2, and AUC > 0.85.For identifying highly differentially methylated (HDM) OC genes, we generated two methylomics profiles for tissues and cervical scrapings, respectively, and one public dataset. Taipei Medical University set A (TMU-A) ovarian tissues were analyzed for DNA methylomics profiles, using pull-down by the methyl-CpG-binding domain protein 2 (MBD2), followed by high-throughput, next-generation sequencing . We thenP value ≤ 0.01, the number of single nucleotide polymorphism (SNP) ≥ 2, genes annotated with NM-type RefSeq accessions, and excluded genes coded on sex chromosomes. We analyzed HDM probes by comparison with 79 primary serous-type OCs and 6 normal fallopian tubes from AOCS dataset. The fallopian tube epithelia rather than ovarian surface epithelia have been considered to be the origin of high-grade serous OC according to the previous epidemiologic studies , molecular genetic pathologic studies, and methylome analysis [U test with P < 0.05, AUC > 0.75, and the number of HDM probes at a promoter region of the closest gene ≥ 3.We also used another public methylome OC tissue dataset to assist discovery of potential OC-specific HDM biomarkers. The Australian Ovarian Cancer Study (AOCS)–tissue dataset was analyzed using the HumanMethylation450 BeadChip  and deposited in the NCBI’s Gene Expression Omnibus (GEO) with accession number GSE65820 . In the analysis , 37. SigTo identify OC-specific HDM genes by cervical scrapings, we assayed the Taipei Medical University set B (TMU-B) cervical scrapings dataset to construct methylomics profiles of 5 OC and 10 healthy control cervical scrapings, using the HumanMethylation450 BeadChip. Each pooled DNA contained equal amounts of DNA from 5 specimens. HDM genes were identified by including HDM level of each probe > 0.015, and the number of probes at a promoter region of the closest gene ≥3.https://sourceforge.net/projects/mev-tm4/) [For selecting candidate HDM genes, methylation profiles were grouped by unsupervised hierarchical clustering analysis, with complete-linkage and Euclidean distance methods performed using Multiple Experiment Viewer (MeV) version 4.9 (ev-tm4/) . One hunhttp://www.kegg.jp/ or http://www.genome.jp/kegg/) [, and Reactome [P ≤ 0.05 was used for enriched annotation   and KEGGp/kegg/) , and ReaReactome  pathway Genomic DNA was extracted from cervical scrapings and tissues using the QIAamp DNA Mini Kit , and its concentration detected using a Nanodrop 1000 . Pooled DNA contained DNA from five specimens. DNA was bisulfite-converted from 1-μg genomic DNA, using the EZ DNA Methylation Kit , according to the manufacturer’s recommendations of dissolution into 70-μl nuclease-free water. In the verification phase of methylation markers, we use DNA pools for reducing the expense of DNA’s amount, cost, and the time. It provides a rapid and cost-effective method. In the validation phase, we indeed analyzed these samples individually.For quantifying DNA methylation levels, we used bisulfite pyrosequencing and quantitative methylation-specific PCR (qMSP) assays. All primers are listed in Additional file COL2A1 as a reference. DNA methylation levels were estimated using the ΔCp-value and the following formula: (Cp of Gene) − (Cp of COL2A1). Test results of Cp of COL2A1 > 36 were defined as the absence of template DNA.qMSP assays were performed as described in our previous study . All bioU test and Kruskal–Wallis test were used to identify differences in methylation levels between ≥ 2 categories. All significant differences were assessed using a two-tailed t test with P < 0.05. For comparing the performance of each HDM gene, we calculated the sensitivity, specificity, and AUC by “closest.topleft” method and 200 bootstrapping iterations in the pROC package. For comparing the performance of combinations of HDM genes, we calculated the probability of logistic regression model for sensitivity, specificity, and AUC analysis. To translate the research results into clinical application and awareness, a logistic regression model, with ten-fold cross-validation and 200 replications, was utilized to generate a mathematical formula to predict the risk of having OC (OC-risk score). The unbiased optimism-adjusted estimates of the concordance statistic with similar absolute errors in the relatively smaller clinical dataset were generated by this method [i = ith gene, and ε is a variable with a value expected to be zero. For calculating ultimate estimator of the regression coefficients, ε, and βi, we repeated 200-times of 10-fold cross-validation, and analyzed the mean and median of all coefficients, sensitivity, specificity, and AUC. The aforementioned analyses and plots were performed using the statistical package in R (version 3.3.2) or MedCalc version 18 .The Mann–Whitney nonparametric s method . The forAdditional file 1. Figure S1. The differential methylation analysis on three datasets. Figure S2. The verification of HIST1H3E DNA methylation using bisulfite pyrosequencing in ovarian tissues Table S1. Clinicopatological features of clinical samplings for identification of DNA methylomics profiles Table S2. Summary of KEGG and Reactome pathways related to 151 differential methylation of candidate genes in ovarian cancer Table S3. The primers for quantitative methylation-specific PCR and bisulfite pyrosequencing Table S4. Summary methylation level of 151 DM genes in TMU-tissue set Table S5. Summary of differential methylation levels in eight genes from DNA pools of cervical scrapings Table S6. Comparisons of the methylation level between young and old cases using normal cervical scrapings.


CXC motif chemokine receptor type 4 (CXCR4) is associated with normal and abnormal development, including oncogenesis. The ligand of CXCR4 is stromal cell-derived factor (SDF), also known as CXC motif ligand (CXCL) 12. Through the SDF-1/CXCR4 axis, both homing and migration of hematopoietic (stem) cells are regulated through niches in the bone marrow. Outside of the bone marrow, however, SDF-1 can recruit CXCR4-positive cells from the bone marrow. SDF/CXCR4 has been implicated in the maintenance and/or differentiation of stemness, and tissue-derived stem cells can be associated with SDF-1 and CXCR4 activity. CXCR4 plays a role in multiple pathways involved in carcinogenesis and other pathologies. Here, we summarize reports detailing the functions of CXCR4. We address the molecular signature of CXCR4 and how this molecule and cells expressing it are involved in either normal (maintaining stemness or inducing differentiation) or abnormal (developing cancer and other pathologies) events. As a constituent of stem cells, the SDF-1/CXCR4 axis influences downstream signal transduction and the cell microenvironment. Chemokines are a large family of small, structurally related cytokines. There are two major subgroups of chemokines characterized by the arrangement of their first two cysteines, either adjacent to one another (CC subfamily) or separated by one amino acid (CXC subfamily). So far, 47 chemokines and 20 chemokine receptors have been identified in humans . CXC motOf note, Imitola et al. discovered that the migration of neural stem cells (NSCs) is mediated by the SDF-1/CXCR4 pathway during injury and inflammation. They noted that SDF-1/CXCR4 contributes to migration not only in bone marrow cells as previously described, but also in other cell types such as NSC . This idCXCR4 has been associated with several diseases, including human immunodeficiency virus (HIV) infection, cancer, warts, hypogammaglobulinemia, immunodeficiency, myelokathexis (WHIM) syndrome, pulmonary artery hypertension (PAH), and pulmonary injury. Several CXCR4 inhibitors have been developed to treat these diseases, especially cancer. Plerixafor is a CXCR4 antagonist that was approved by the Food and Drug Administration (FDA) in 2008. Many other CXCR4 antagonists that bind the CXCR4 receptor transmembrane domain have been developed . In thisCXCR4 was originally identified as the leucocyte-expressed seven-transmembrane domain receptor (LESTR)/fusin, a co-receptor for HIV. Recent reports concerning gene editing of CXCR4 demonstrated inhibition of HIV infection ,13,14, w−/− and SDF-1−/−, respectively, are similar, and the defect is observed in hematopoiesis. Notably, few mature B cells and T cells have been observed within the peripheral lymphoid organs [−/− mice display impaired vascularization in various organs, including the intestines, stomach, and heart. The latter is a ventricular septal defect that occurs during embryogenesis [SDF-1 can activate CXCR4-expressing cells and induce B cell proliferation and T cell recruitment. The phenotypes of the knock-out mice for the receptor and the ligand, CXCR4d organs ,20. In aogenesis . Additioogenesis . SDF-1 iogenesis . StudiesTBX-1 is one of the major genes responsible for forming the pharyngeal arch-derived structure [TBX-1 gene is not expressed in NC cells in pharyngeal arches. TBX-1 is expressed in pharyngeal ectoderm, endoderm, and mesoderm. The interactions of pharyngeal NC cells and the surrounding tissues such as pharyngeal ectoderm, endoderm, and mesoderm were studied in the context of TBX-1 signaling. Fibroblast growth factor 8, vascular endothelial growth factor, and more recently, SDF-1, were shown to be involved in this interaction [+ cells are present in the NC. Thus, CXCR4 may be involved in the migration of NC cells during normal development. Recently, however, SDF-1/CXCR4 was suggested to function in cardiovascular development in the context of second heart field to endothelial cells and not in TBX-1 haplosufficient arch artery phenotype [DiGeorge, or 22q11.2-deletion syndrome (DGS), is the most common human genetic deletion syndrome, and this disease results in mental disorders, craniofacial dismorphogenesis, thymus hypoplasia, and congenital heart defects caused by cardiac outflow anomalies. The responsible gene is located within 22q, which also includes T-box-containing transcription factor (TBX) 1. tructure ,24,25,26eraction ,28. Notaeraction . Intereshenotype . More st+ stem cell homing to collagen matrices [+ stem cell homing. Mesenchymal stem cells (MSCs) promote proliferation and differentiation of c-kit+ cardiac stem cells via SDF-1/CXCR4 signaling. If treated with the CXCR4 antagonist AMD3100, cardiac stem cells derived from a murine postnatal cardiac explant differentiate into cardiac myocytes [+ cardiac stem cells through SDF-1/CXCR4 signaling [+ cardiac stem cells are thought to rescue cardiac damage from injury such as ischemia [+ cells can act as cardiac stem cells [+ cells were utilized for cardiac repair, there were positive effects on cardiac differentiation and the enhancement of cardiac myocyte survival [+ cells and may contribute to the repair of tissues directly [SDF-1 is strongly expressed in bone marrow stromal cells. SDF-1 recruits stem cells and regulates their differentiation to repair injuries. Repair is mediated by growth factors and cytokines that include transforming growth factor-beta (TGF-β)  and vascmatrices . Additiomyocytes . Therefoignaling . The c-kischemia ,36,37. Cem cells ,39. Whensurvival ,41,42,43directly  or indirdirectly . Similarly, neural stem/progenitor cells are controlled by SDF-1/CXCR4 to maintain stemness ; however+/+ platelets could rescue lung regeneration, while SDF-1−/− platelets could not. Thus, pneumonectomized mice need SDF-1 from platelets for alveolar regeneration [It is established that after pneumonectomy, the left lung possesses greater capacity for respiratory function due to alveolar regeneration. Recently, platelets were reported to be involved in this regeneration through SDF-1. Without platelets, which are induced by thronbopoietin-deficient conditions (5% of wild type), regrowth of the lungs reduced markedly. SDF-1 could enhance this recovery significantly. Additionally, SDF-1neration . The recneration ,49. Furtneration . The nicneration . The resIn contrast to its normal role in stem cell function during development, CXCR4 is also expressed in various cancers . A recenVarious anticancer drugs that target the SDF-1/CXCR4 axis are under evaluation in clinical trials. Plexifor (AMD3100) induces mobilization of CD34+ cells in patients with multiple myeloma and non-Hodgkin’s lymphoma . Its useMSCs can contribute to cancer progression by becoming carcinoma-associated fibroblasts (CAFs) and are recruited to tumors through TGF-β and SDF-1 signaling ,63. CAFs+ and/or c-kit+ cells have recently been implicated in the development of PAH [+ cells were observed in a rat model of PAH and are thought to be expressed by the smooth muscle lineage [Pulmonary arterial hypertension (PAH) is a rare disease characterized by high blood pressure in the arteries of the lungs, caused by an endothelial cell disorder and the abnormal proliferation of smooth muscle cells. These associate with inflammation and finally lead to occlusion within the pulmonary artery, which in turn triggers right ventricle hypertrophy. CXCR4t of PAH ,70,71,72 lineage . CXCR4 i lineage . The conThe CXCR7 inhibitor CCX771 reportedly attenuates PAH . CXCR7 iA recent report revealed that hypoxia up-regulates interleukin (IL)-6 in the lung, and that IL-6 and IL-21 promote the differentiation of alveolar macrophages to M2 macrophages. Soluble factors such as CXCL12 can promote the proliferation of pulmonary artery smooth muscle cells, which can result in PAH . We haveSilybum marianum L. [We investigated the effect of the CXCR4 inhibitor, silibinin, in the treatment of PAH and obtained evidence of treatment benefit. Silibinin is derived from the seeds of the milk thistle plant ianum L. . Silibinianum L. . Anticanianum L. ,92,93,94ianum L. . Silibinianum L. ,85,86,95ianum L. . Silibinianum L. . SilibinSilibinin has been traditionally used in folk medicine and is considered relatively safe. The toxic effect of long-term (2 years) usage of silymarin was previously investigated in rats and mice. Silymarin is an extract from the milk thistle plant. The main component is silibinin . No signCXCR4 gene [CXCR4 mutations associated with this syndrome occur in the C-terminal region of the receptor. These mutations prevent CXCR4 internalization in response to SDF-1 binding, which impairs SDF-1-induced receptor down-regulation, and increases CXCR4 signaling [WHIM syndrome is a rare inherited primary immunodeficiency disorder caused by autosomal dominant gain-of-function mutations in the CR4 gene ,100,101.ignaling ,101. Sinignaling . BecauseCXCR4 is a seven-transmembrane G protein-coupled receptor. Its promoter activity is upregulated by c-Myc and downregulated by Yin-Yang . CXCR4 iRecently, a number of reports have revealed that heterodimers of CXCR4 and CXCR3 or CXCR7 possess numerous functions, including roles in cancer cell invasion. Jin et al. used colorectal cancer cell lines to investigate the association between metastatic behavior and CXCR3 and CXCR4heterodimerization. They found that CXCR3 could form heterodimers, and CXCR3 could cause CXCR4 to locate to the cell surface. Knock-down of CXCR3 reduced surface CXCR4 in vitro. They also confirmed in vivo that in SW620 cells, CXCR3 and CXCR4 are expressed highly, and knock-down of CXCR3-A and CXCR4 reduced cell metastasis . Song etThe role of the CXC chemokine family in lung cancer development has been previously investigated. A statistically significant decrease in the expression of CXCL4 and CXCL5 and a statistically significant increase in the expression of CXCL7 were observed; however, there was no significant difference in SDF-1 expression . ConversThe recent review by Messina et al. discussed the EMT/ mesenchymal-epithelial transition (MET) in terms of stemness, regeneration, and oncogenesis. The authors connected stemness and oncogenesis to the states of EMT and MET, and they also connected stem cells and CSCs . SDF-1/CTaken together, SDF-1/CXCR4 signaling can exert counteracting effects and may be active in the (micro)environment surrounding stem cells, such as niches, which are commonly used by both normal stem cells and CSCs. In the case of stem/progenitor cells, they may migrate from the niches (bone marrow) to the SDF-1-expressing damaged tissues. The cells may be involved in tissue repair or dysplasia, which may promote cancer, as summarized in + cells may be involved in inflammation/stem cell migration normally, and cancer/malignancy abnormally. Therefore, the amount and nature of binding of SDF-1 to CXCR4 may exert profound effects on downstream signal transduction, and these effects should be clarified in future studies.CXCR4 is an interesting molecule that is associated with HIV infection, cancer, stem cell proliferation, differentiation, and migration. In the early stages of development, SDF-1 and CXCR4 function in cellular migration. Throughout life, particularly in adulthood, SDF-1/CXCR4 signaling may influence stem cell migration from the bone marrow or niche to repair damaged tissues. Increases in the number of CXCR4


Rotator cuff disease is a widespread musculoskeletal pathology and a major cause of shoulder pain. Studies on familial predisposition suggest that genetic plays a role in the pathogenesis of rotator cuff disease. Several genes are responsible for rotator cuff disease. The aim of this study was to perform a systematic review on genetic association between rotator cuff disease and genes variations.A systematic review of the literature was performed, in accordance with the PRISMA guidelines. PubMed, Medline, CINAHL, Cochrane, Embase and Google Scholar databases were searched comprehensively using the keywords: “Rotator cuff”, “Gene”, “Genetic”, “Predisposition”, “Single-nucleotide polymorphism” and “Genome-wide association”.8 studies investigating genes variations associated with rotator cuff tears were included in this review. 6 studies were case-control studies on candidate genes and 2 studies were GWASs. A significant association between SNPs and rotator cuff disease was found for DEFB1, FGFR1, FGFR3, ESRRB, FGF10, MMP-1, TNC, FCRL3, SASH1, SAP30BP, rs71404070 located next to cadherin8. Contradictory results were reported for MMP-3.Further investigations are warranted to identify complete genetic profiles of rotator cuff disease and to clarify the complex interaction between genes, encoded proteins and environment. This may lead to individualized strategies for prevention and treatment of rotator cuff disease.Level IV, Systematic Review. Rotator cuff disease is a widespread musculoskeletal pathology and a major cause of shoulder pain . This diThe etiology of rotator cuff disease is multifactorial –7 and itSeveral genes are responsible for rotator cuff disease. Genetic susceptibility may affect the ultrastructure of the tendon. Achilles tendinopathy has been associated with polymorphisms of tenascin C and collagen type Va . SimilarThe aim of this study was to perform a systematic review on genetic association between rotator cuff disease and candidate genes.A systematic review of the literature was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines with a PRISMA checklist and algorithm.A comprehensive search of MEDLINE, PubMed, Cochrane, EMBASE, CINAHL, and Google Scholar databases using various combinations of the keywords: “Rotator cuff”, “Gene”, “Genetic”, “Predisposition”, “Genome-wide association”, “Single-nucleotide polymorphism” was performed. Three independent reviewers  conducted the search separately. All scientific journals were considered, and all relevant studies were analysed.In order to qualify, an article must have been published in a peer-reviewed journal All articles were initially screened for relevance. The three investigators separately reviewed each abstract and completed a close reading of all articles to minimize selection bias and error. According to the Oxford Centre of Evidence-Based Medicine, only Level I to Level IV articles in English were included in our study.We included articles that described genetic variations  associated with rotator cuff disease. Both case-control studies focused on candidate genes and Genome-wide association studies (GWASs) were included. Studies should clearly describe the criteria used for the diagnosis of rotator cuff disease, genes and SNPs investigated. Missing data relevant to these parameters warranted exclusion from this systematic review. We did not included studies about familial predisposition, genetic variants and gene expression patterns associated with rotator cuff disease and/or healing. Literature reviews, case reports, animal and cadaveric studies, technical notes, letters to the editor, and instructional courses were excluded.Finally, in order to avoid bias, the selected articles and their references, and the articles excluded from the study were reviewed, evaluated, and discussed by all the authors. All investigators independently extracted the type of study, number of cases and controls, diagnostic criteria of rotator cuff disease, investigated genes, mean age of cases and controls.We used the Coleman Methodology Score to assess the quality of the selected studies (CMS) , in whicThe subsections of the CMS are based on the subsections of the CONSORT statement outlined for randomized controlled trials, which have been modified to allow for other trial designs. Coleman criteria were subjected to modification in order to make them reproducible for this systematic review. Each study was scored by the three reviewers  independently and in triplicate.The literature search and the cross-referencing process resulted in 251 articles. 213 studies were assessed for relevance by title and abstract. 38 duplicates were removed and 200 were excluded because they did not meet inclusion criteria. After reading the remaining full-text articles, we rejected 3 studies about familial predisposition and 2 studies about genetic expression. Finally, 8 articles were included in the present review –26. The 8 studies investigating genes variations associated with rotator cuff tears were included in this review. 6 studies were focused on candidate genes –24 and 2DEFB1, DENND2C, ESRRB, FGF3, FGF10, FGFR1, MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, Col5A1, TNC, FOXP3, FCRL3. A significant association between SNPs and rotator cuff disease was found for DEFB1, FGFR1, FGFR3, ESRRB, FGF10, MMP-1, TNC, FCRL3. Contradictory results were reported for MMP-3 .DEFB1, DENND2C, ESRRB, FGF3, FGF10, and FGFR1) in 203 cases and 207 controls [DEFB1, FGFR1, FGFR3, and ESRRB. After adjustment by ethnic group and sex another association in FGF10 was revealed.Motta et al. assessed 23 SNPs in 6 candidate genes  transcription, and their interaction may be involved in tenocyte apoptosis [The association of variants in k et al. . They iding loss , stem ceing loss , and celing loss . ESRRB ipoptosis .TNC, Col5A1, and MMP-3) [Col5A1) [MMP-1) [MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, TIMP-1 that are specifically expressed in torn rotator cuff.Several studies selected candidate genes on the basis of pre-existing association analyses for Achilles tendon ruptures (d MMP-3) –37, tend[Col5A1) , rupture [MMP-1)  and matrMMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and Col5A1 genes while six SNPs in Tenascin-C (TNC) were associated with degenerative rotator cuff tears [MMP-3 and rotator cuff tearing that may be explained by the smaller number of individuals evaluated, nonpairing between cases and controls for age, and known risk factors such as high blood pressure, and racial and genetic characteristics of the population. Moreover, Assunção et al. studied a different polymorphism in MMP-1 (rs1799750) that was significantly associated with rotator cuff tearing.Kluger et al. found no differences in genotype and allele frequencies for SNPs in ff tears . Unlike ff tears  found a In accordance with to Kluger et al. , no signFCRL3 − 169 T > C) [FCRL3 is a glycoprotein of the immunoglobulin receptor superfamily, expressed in Treg cells that may play a role as a negative regulator of Treg function [As demonstrated in in-vivo studies, the immune cells play a key role in the pathophysiology of rotator cuff tears. Salles et al. found an increased risk of tendinopathy associated with Fc receptor-like 3 polymorphism (9 T > C) . FCRL3 ifunction –42.SASH1 (rs12527089) and SAP30BP (rs820218) [SASH1 is a tumor suppressor gene that is ubiquitous expressed and, therefore, may be a potential candidate for dysregulation in musculoskeletal tissue. SAP30BP is a ubiquitously present transcriptional regulator protein on chromosome 17. It may act as a transcriptional co-repressor of a gene related to cell survival [GWASs are a powerful tool to pinpoint genes that may contribute to the risk of developing rotator cuff disease. The GWAS by Tashjian et al. identified two significant SNPs associated with rotator cuff tears: SASH1 rs127089 andsurvival .In another GWAS, Ross et al. found a SNP located next to cadherin8 significantly associated with rotator cuff injury. It encodes a protein involved in cell adhesion .DEFB1, FGFR1, FGFR3, ESRRB, FGF10, MMP-1, TNC, FCRL3, SASH1, SAP30BP, rs71404070 located next to cadherin8.Studies on candidate genes and GWASs identified several genes variation associated with rotator cuff tears, such as Further investigations are warranted to identify complete genetic profile of rotator cuff disease and to clarify the complex interaction between genes, encoded proteins and environment. This may lead to individualized strategies for prevention and treatment of rotator cuff disease.


PP2C) gene family, known to participate in cellular processes, is one of the momentous and conserved plant-specific gene families that regulate signal transduction in eukaryotic organisms. Recently, PP2Cs were identified in Arabidopsis and various other crop species, but analysis of PP2C in cotton is yet to be reported. In the current research, we found 87 (Gossypiumarboreum), 147 (Gossypiumbarbadense), 181 (Gossypiumhirsutum), and 99 (Gossypiumraimondii) PP2C-encoding genes in total from the cotton genome. Herein, we provide a comprehensive analysis of the PP2C gene family in cotton, such as gene structure organization, gene duplications, expression profiling, chromosomal mapping, protein motif organization, and phylogenetic relationships of each species. Phylogenetic analysis further categorized PP2C genes into 12 subgroups based on conserved domain composition analysis. Moreover, we observed a strong signature of purifying selection among duplicated pairs  of Gossypiumhirsutum. We also observed the tissue-specific response of GhPP2C genes in organ and fiber development by comparing the RNA-sequence (RNA-seq) data reported on different organs. The qRT-PCR validation of 30 GhPP2C genes suggested their critical role in cotton by exposure to heat, cold, drought, and salt stress treatments. Hence, our findings provide an overview of the PP2C gene family in cotton based on various bioinformatic tools that demonstrated their critical role in organ and fiber development, and abiotic stress tolerance, thereby contributing to the genetic improvement of cotton for the resistant cultivar.The protein phosphatase ( Thus, it is involved in many biological processes, such as signal transduction, development, and environmental stimuli . The PKsArabidopsis, several members of PP2c such as PLL4 and PLL5 (POL-like gene) are known to adjust leaf development, though with no obvious functions within the meristem [PP2C are evolutionarily conserved from Archaea to higher plants that pointedly modulate stress signaling pathways and reverse the stress-induced PK cascades to complex environmental stimuli . From vameristem . Arabidopsis and rice, 112 and 132, respectively, candidate PP2C genes have been characterized into various groups [Arabidopsis PP2Cs , is accompanied by abscisic acid (ABA) signaling, group B is known to stimulate mitogen-activated protein kinase (MAPK) signaling, and group C is involved in the regulation of flower development [Arabidopsis [PP2C genes in plant development and environmental stresses. Hence, it is indispensable to probe into the identification and functional description of the PP2C gene family, which will cement the base for understanding its essential molecular mechanism in stress signaling.In s groups ,11. In pelopment ,15. In helopment . Proteinelopment ,18,19,20elopment . In reacelopment , such asbidopsis . Taken tGossypium whole-genome data in four different cotton species, such as Gossypium arboreum [Gossypium barbadense [Gossypium hirsutum [Gossypium raimondii [PP2C gene family based on bioinformatic tools. We further compared the PP2C genes between Gossypium and Arabidopsis, to explore and identify both shared and specific subgroups. Following the gene structure organization analysis, conserved protein motifs, and cis-elements, we traced the duplication gene pairs and their evolutionary divergence that likely resulted in the widespread extension of the PP2C gene family. In order to shed light on some critical PP2C genes, their associated indigenous functional roles were further exposed to heat, cold, drought, and salt stress conditions. Additionally, the transcriptional profiling of the PP2C genes for various tissues and fiber development and qRT-PCR analysis of 30 genes were analyzed and compared. Therefore, to our knowledge, this is the first systematic report of the genome-wide expression dynamics of the PP2C genes in cotton species, and it is necessary to carry out a comprehensive study to understand the regulation of phosphatases in cotton during stress and development.Cotton, as an oil crop and an important source of natural textile fiber, plays a crucial role in agriculture and industry all around the world. However, its production is mainly constrained due to various abiotic and biotic stress conditions. The release of arboreum , Gossypirbadense , Gossypihirsutum , and Gosaimondii  and theiArabidopsis 94 PP2C genes were obtained from TAIR (http://www.arabidopsis.org) with the help of Interpro scan domain (IPR001932) and then used as queries against G. arboreum, G. barbadense, G. hirsutum, and G. raimondii databases. This search resulted in 87, 147, 181, and 99 PP2C genes in Gossypium species i.e., G. arboreum, G. barbadense, G. hirsutum, and G. Raimondii, respectively. In the current study, we used the following nomenclature system for the PP2C genes to distinguish each PP2C from the homology of Arabidopsis and Gossypium species: GaPP2C (GaPP2C1–GaPP2C87) G. arboreum, GbPP2C (GbPP2C1–GbPP2C147) G. barbadense, GhPP2C (GhPP2C1–GhPP2C181) G. hirsutum, and GrPP2C (GrPP2C1–GrPP2C99) G. raimondii. We have studied different gene features of the PP2C genes including the chromosomal location, coding sequence length (CDS), protein length (aa), molecular weight (MW), isoelectric point (PI), grand average of hydropathicity (GRAVY), aliphatic index, and subcellular localization , a maximum likelihood (ML) tree was constructed with Arabidopsis using MEGA 7.0. The phylogenetic tree revealed that the PP2C genes can be further divided into 12 subgroups, as previously reported [GhPP2C genes were clustered into 12 subgroups (A–L) with Arabidopsis. In the phylogenetic tree, subgroup D had the most members of the PP2C genes (38), followed by subgroup E (27), and the least number of PP2C genes was observed in subgroup L, having 4 GhPP2C genes  ranging from 2–14 genes per chromosome and only 2 genes were found on the scaffold. Moreover, every chromosome showed variation in a number of genes, such as D04 exhibited the highest number of GhPP2C genes (14), followed by 12 genes in D02, and the least number of genes (2) were recorded for D07 (PP2C genes (2) were found on A13   a,b. To vhirsutum . The resPP2C genes, cis-acting elements play a critical role as stress-adaptive signaling in plants by interacting with their cognate transcription factor (TF). For instance, abscisic acid (ABA)-responsive elements (ABREs) are involved in salt, drought, and ABA signaling [G. hirsutum PP2C genes was executed by using the PlantCARE database and seven common cis-regulatory elements were briefly summarized (PP2C genes are recognized to show a major part in both biotic and abiotic stress phenomena. Most of the genes were highly (34.88%) responsive to light , followed by (24.32%) hormones , (18.61%) and other regulatory cis-elements , while the fewest genes (0.93% and 0.85%) were mainly counted in circadian and enhancer elements , respectively. In plants, the circadian cis-regulatory element is known to control the circadian rhythms. Moreover, these results indicate that PP2C genes are vital in various biotic-abiotic/hormone signaling which might be hypothesized by their diversity in nature.In the promoter region of ignaling . LTR is ignaling . Likewisignaling . The expmmarized . The resPP2C gene family. Moreover, the PP2C genes might have experienced functional discrepancy due to gene duplications, and few of them might have lost their unique functions, developed novel functions, or preserved partition of innovative functions [Ka/Ks ratios among selected genes from segmental and dispersed  and few of them were dispersed (12); thus, indicating that segmental duplication plays a major contribution to the expansion of the unctions ,32. Duriispersed . It was PP2C genes drastically varied in various tissues and most numbers of them were highly expressed. However, the expression of a few genes  did not show any striking expression in any tissues. Though, some of the PP2C genes were expressed in one or more tissues . Intriguingly, the tissue-specific clustering  (p = 0.05). Results showed a higher positive correlation among various specific tissues  exhibited a dynamic expression level. Majority of the xpressed a, but soxpressed . These fGhPP2C genes may participate in responses to heat, cold, drought, and NaCl stress tolerance. Moreover, the expression profiling of PP2C has been studied in various species after exposure to abiotic–biotic and hormones stresses [GhPP2C genes exhibited high transcript levels after exposure to multiple treatments, but a few of them were induced by one or more treatments. For instance, heat stress possesses the dominant portion of down-regulated genes (52%). However, cold stress showed 70% of the genes were up-regulated and 30% decreased in transcript level, followed by drought stress, which exhibited about 53% and 47% of up- and down-regulated GhPP2C genes, respectively. On the other hand, exposure to salt stress resulted in 56% up-regulation and 44% down-regulation in genes  events. For exploring new biological functions, evolutionary implications and its expansions are mainly based on gene duplications [Gossypium arboreum), 147 (Gossypium barbadense), 181 (Gossypium hirsutum), and 99 (Gossypium raimondii). Cotton species represent the high number of genes, specifying that the PP2C gene family have undergone extensive expansion during the evolution of cotton. In the current study, a comprehensive genome-wide analysis was executed, such as gene identification, gene structure organization, phylogenetic characterization, syntenic relationships, chromosomal localization, and gene duplications. Moreover, transcriptional profiling of 30 PP2C genes was also performed, after exposure to heat, cold, drought, and salt stress was also carried out [In this study, we systematically investigate the as maize , rice [1as maize , Arabidobidopsis , hot pepbidopsis , banana bidopsis , and Brastachyon . Thus, wications . Therefoications . The impications ,38,39. Hried out ,27. In this study, the subcellular predictions for most of the members of GaPP2C, GbPP2C, GhPP2C, and GrPP2C were mainly located in different organelles such as the chloroplast, nuclear region, mitochondria, cytoplasm, and others. In addition, the gene characteristics of GaPP2C, GbPP2C, GhPP2C, and GrPP2C drastically vary including the protein length (aa), molecular weight (MW), predicted isoelectric point (PI), and a grand average of hydropathicity (GRAVY), signifying that different PP2C proteins may play complex roles in variable microenvironments.Ka/Ks), provides useful information about purifying, positive, and neutral selections of gene pairs during the rate of divergence [Ka/Ks ratio is <1.00, this represents purifying selection, a Ka/Ks ratio of 1.00 indicates neutral selection, and/or Ka/Ks >1.00 depicts positive selection [PP2C genes with the help of the MEGA7.0 program. Thus, we estimated the selection pressure analysis of duplicated genes  pairs. The majority of the GhPP2C pairs showed Ka/Ks ratios of less than 1.00, indicating the purifying selection, and only two pairs showed values more than 1.00, speculating the positive selection [The estimate of evolutionary patterns, such as the rate of computing the selection pressure analysis  and fiber development . These results showed that most of the PP2C were highly varied, speculating the high diversity in their functions. However, few genes have shown tissue-specific expression, indicating their common importance to plant development. In GhPP2C genes, we also explored the promoter regions for identification of common conserved cis-regulatory elements. As a consequence, these results exhibit the participation of PP2C genes in various hormone signaling, including both biotic and abiotic stress factors. In the present study, to provide validation to our hypothesis, we also tested 30 candidate genes under various stresses using qRT-PCR. The majority of PP2C genes showed high striking transcriptional changes by exposure to heat, cold, drought, and salt stress, suggesting that PP2C genes might be crucial to stress tolerance in cotton. Noticeably, recent studies in Arabidopsis and rice also uncovered the pivotal role of PP2C candidate genes [Gossypium hirsutum. Plant productivity is always uncertain due to a range of climatic challenges such as heat, cold, drought, and salt stress being the major factors in limiting crop production. Previous studies reported that reatment . In Arabents too . These fte genes ,45, howeTherefore, taken together, the results of our study provided valuable insight, signifying that PP2C has provided a stepping stone to the molecular mechanism in cotton. Alongside, the differential expression profiling and gene duplications analysis might have experienced functional divergence, and their further study will help considerably in improving the crop yield and quality and cultivating new resistant varieties.PP2C genes in the Gossypium genome. Herein, we identified 87 (Gossypium arboreum), 147 (Gossypium barbadense), 181 (Gossypium hirsutum), and 99 (Gossypium raimondii) PP2C genes by bioinformatics analysis in cotton species. Gene synteny analysis showed that GhPP2C are highly conserved, while the gene duplications analysis reflected that only segmental duplication plays a starring role in the PP2C gene extension in cotton. Also, the results of the phylogenetic analysis categorized the PP2C genes into 12 subgroups. We further explored the previously published RNA-sequence (RNA-seq) data to compare the tissue-specific response of PP2C genes and their critical role in organ development and fiber. Various PP2C genes responded promptly to abiotic stresses, including heat, cold, salt, and drought, suggesting their crucial role in abiotic stress tolerance. As a consequence, our findings will facilitate advanced research on the functional analysis of PP2C genes regarding their critical role in tissues, fiber development, and abiotic stress tolerance.We analyzed the genome analysis, evolutionary rates, and molecular characterization of PP2C genes in Gossypium and other species, we utilized the Plaza 4.0 database (https://bioinformatics.psb.ugent.be/plaza/) with the help of InterPro PP2C domain “IPR001932”. The cotton genome sequences were downloaded from (https://www.cottongen.org/), and A. thaliana sequences were retrieved from TAIR (http://www.arabidopsis.org/). The domains of obtained GhPP2C proteins were further verified using the NCBI-Conserved Domain database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) search program and SMART databases (http://smart.embl-heidelberg.de/) [For identification of erg.de/) . Those pThe amino acid sequences of the GhPP2C proteins were used for further investigation, and multiple sequence alignment was performed by MUSCLE  using MEKa/Ks ratios were calculated for duplicated  gene pairs using MEGA 7.0 [The MEGA 7.0 . http://gsds.cbi.pku.edu.cn) [http://web.expasy.org/protparam/). The subcellular localization was predicted using the WOLF PSORT (https://wolfpsort.hgc.jp/) website.Conserved motif scanning of GhPP2C proteins was carried out through local MEME Suite Version 5.0.3. For this purpose, parameter settings were calibrated as follows: Maximum number of motifs 10, with a minimum width of 100 and a maximum of 150. The other parameters were set as default . For thehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [Every GhPP2C promoter sequence (selected as 2000 upstream bp) was imported in Generic File Format (GFF) file from the cotton genome. Then, the PlantCARE database (e/html/)  was utilPP2C genes was illustrated from top to bottom concerning their position in the genome annotation by using TBtools software [thaliana and Gossypium hirsutum. Circos (using TBtools software) program was applied to exhibit the syntenic relationships among the chromosomes of G. hirsutum and A. thaliana [The chromosomal location of software . For synthaliana . p-value) significance level. Pearson correlation analysis was performed with the help of Excel 2013 in order to evaluate the PCC values that were used for qRT-PCR according to a previously reported study . As wellG. hirsutum cv. Junmian 1 were grown in plastic pots containing a mixture of soil and vermiculite (3:1). The pots were then placed in an artificial growth chamber for five weeks. The growth conditions were as follows: The temperature was set to 24/16 ºC, the photoperiod was 16/8 h, and the relative humidity was 65%–70%. The two-week-old seedling was exposed to specific treatment, as follows: For heat and cold treatments, seedlings were exposed to 38 ºC and 4 ºC, respectively. For NaCl and drought stress treatments, the seedlings were cultured in a nutrient solution medium with 250 µM and 6000 PEG (w/v). All treatments were carried out in continuous time intervals of 1, 6, and 12 h, respectively. Additionally, for every specific treatment, five randomly chosen whole seedlings were pooled to form a biological replicate. After that leaf samples were quick-frozen in liquid nitrogen and stored at −80 °C for further use. In the present study, the germinated seeds of Gossypium hirsutum genome for confirmation. RT-PCR was performed according to the guidelines of previous studies [GhPP2C genes were analyzed based on a previous study [Total RNA was isolated from the treated frozen leaves with Trizol (Invitrogen) following the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using the Primer Script RT reagent kit  according to their instructions. Specific primers were designed using Becan Designer 7.9 and are presented in  studies . Relativus study ,56. The http://www.omicshare.com/) and TBtools software [High-throughput RNA-sequencing data  was utilsoftware .


The Wnt gene family members are known to participate regulating various normal and pathological processes including tumorigenesis. However, the association between Wnt ligands gene family and prognosis in hepatocellular carcinoma has not been systematically studied. Therefore, we evaluated the role of Wnt ligands gene family in hepatocellular carcinoma using publicly available data from The Cancer Genome Atlas (TCGA).Clinical information and RNA-Seq mRNA expression data were derived from TCGA hepatocellular carcinoma cohort. Differences in overall survival (OS) and disease-free survival (DFS) between increased and decreased expression groups  of Wnt ligands gene family were compared using Kaplan–Meier method and Cox regression model, with p-values calculated via log-rank test. Gene Set Enrichment Analysis (GSEA) was performed.Multivariate analysis adjusted for patient age, sex, BMI, tumor grade, and TMN stage revealed that Wnt1, Wnt3 and Wnt5B expressions were independent prognostic factors for OS and DFS . Furthermore, expression of Wnt1 and Wnt5B was significantly associated with TMN stage . GSEA showed that nucleotide excision repair was differentially enriched in Wnt1 low expression phenotype and aminoacyl trna biosynthesis and basal transcription factors were differentially enriched in Wnt5B low expression phenotype.Our results identified associations of several Wnt ligands with prognosis of HCC patients, indicating that these genes could serve as prognostic biomarkers of HCC.The online version of this article (10.1186/s12935-019-0743-z) contains supplementary material, which is available to authorized users. Liver cancer is one of the most commonly diagnosed cancer and the second most frequent cause of cancer-related deaths globally, with 854,000 incident cases and 810,000 deaths in 2015 . DetaileThe Wnt family of 19 secreted glycoproteins have crucial roles in the regulation of diverse processes, such as embryogenesis, differentiation, and tumorigenesis through canonical dependent and non-canonical pathways , 5. Numehttps://tcga-data.nci.nih.gov/tcga/) data portal. The clinicopathological characteristics of HCC patients, including age, sex, race, body mass index (BMI), tumor grade, tumor, node, metastasis (TNM) stage, overall survival time, overall survival status, disease free survival time and disease-free survival status, were collected. The methods of biospecimen collection, RNA isolation, and RNA sequencing were previously described by the Cancer Genome Atlas Research Network [The level-3 expression data (RNA-seqV2) and clinicopathological data of 360 HCC patients and 50adjacent normal liver samples were downloaded from The Cancer Genome Atlas  using the MSigDB C2 KEGG pathways gene sets, which contains 186 gene sets. Normalized enrichment score (NES), nominal p value and false discovery rate (FDR) were used to quantify enrichment magnitude and statistical significance, respectively [To determine whether a priori defined set of genes shows statistically significant, consistent differences between two biological states (Increased expression vs. Decreased expression), Gene Set Enrichment Analysis (GSEA) was performed by the JAVA program . Differences between groups were calculated by using Chi square test or Fisher exact test. Box-plots were used to visualize expression differences for Wnt ligands between tumor and adjacent non-tumor tissues. The optimal cut-off values for Wnt ligands expression were determined by X-tile software  . SubsequClinical information and Wnt ligands mRNA expression levels of 360 patients were obtained from TCGA. Among 360 patients, 310 patients were recorded with diseases free survival status. Detailed clinical characteristics of the 360 patients in the TCGA database are shown in Table All 19 members of the Wnt ligands family (Wnt1-Wnt16) had their expression analyzed in 360 HCC tumor tissues and 50 adjacent non-tumor tissues. The expression levels of Wnt2B, Wnt3A, Wnt6, Wnt8B and Wnt10B were significantly higher in primary liver tumor tissues than which in adjacent non-tumor tissues. However, the expression levels of Wnt2, Wnt5B, Wnt7A, Wnt7B, Wnt9A and Wnt11 were dramatically decreased in liver tumor tissues. Expression levels the remaining Wnt ligands, including Wnt1, Wnt3, Wnt4, Wnt5A, Wnt8A, Wnt9B, Wnt10A and Wnt16, remained insignificantly different between liver tumors tissues and adjacent non-tumor tissues. The box diagrams indicating the distribution of gene expression in HCC patients and adjacent normal for all Wnt ligands members are displayed in Additional file The expression levels of Wnt ligands were divided into increased expression group and decreased expression group according to the cut-off values determined by X-tile program. The univariate and multivariate analysis demonstrated that TNM stages , decreased Wnt1 expression , decreased Wnt3 expression , decreased Wnt5B expression , increased Wnt6 expression  and increased Wnt8A expression  were independent prognostic factors for overall survival  in enrichment of MSigDB Collection (c2.cp.kegg.v6.1.entrez). We selected the most significantly enriched signaling pathways based on their normalized enrichment score (NES) Fig. . The FigIn this study, we investigated the association between Wnt ligand family genes and HCC. We observed that the mRNA expression levels of several specific Wnt ligand family genes, such asWnt1, Wnt3 and Wnt5B, are associated with distinct OS and DFS. Moreover, we found that expression of Wnt1 and Wnt5B significantly correlated with TMN stage. Thus, Wnt ligand family genes-especially Wnt1, Wnt3 and Wnt5B-may serve as prognostic biomarkers of HCC and represent possible oncogenes that could serve as therapeutic targets of HCC.Wnt signals are known for regulation of diverse processes, including cell proliferation, survival, migration and polarization, embryonic development, specification of cell fate, and self-renewal in stem cells . It is nIn our study, it was found that increased expressions of Wnt 2B, Wnt3A, Wnt6, Wnt8B and Wnt10B in primary liver tumors and decreased expressions of Wnt2, Wnt5B, Wnt7A, Wnt7B, Wnt9A and Wnt11 in liver tumors tissues. These Wnts might have biomarker potential and could be utilized clinically in a diagnostic capacity. Interestingly, our study revealed that increased expressions of Wnt1, Wnt3, and Wnt5B and decreased expressions of Wnt6 and Wnt8A in HCC were associated with good OS probability. Increased expression of Wnt1, Wnt5A, Wnt5B and Wnt8B and decreased expression of Wnt3 were found to be associated with good DFS probability. The results showed that Wnt1, Wnt3 and Wnt5B were both independent prognostic factors affecting OS and DFS. Noteworthily, expression level of Wnt3 differed greatly for good OS and DFS probability. This reverse result could relate to the different cut-off values calculated by X-tile. As showed in Table Wnt1, one of the key ligands in β-catenin regulation, has been described for its prognostic role in several types of malignant tumors including non-small cell lung cancer , renal cAmong various Wnts associated with the canonical Wnt/-catenin signaling pathway, Wnt3 was previously reported being frequently up-regulated in human cancers , 32, 33.Wnt5B, an intermediately transforming or non-transforming Wnt family member, was reported to activate the noncanonical Wnt signals . SeveralIn recent years, a number of novel biomarkers, such as AFP , 42, glySince the prognostic value of Wnt3 expression for OS and DFS is discrepant, we merely performed GSEA analysis to further investigate the potential mechanism of Wnt1 and Wnt5B in HCC. GSEA analysis showed that nucleotide excision repair is differentially enriched in Wnt1 low expression phenotype and aminoacyl tRNA biosynthesis and basal transcription factors are differentially enriched in Wnt5B low expression phenotype. Nucleotide excision repair (NER) is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals, oxidative stress, etc. There have been a few reports demonstrating association of nucleotide excision repair with cancer , 53. IshNevertheless, the prediction of protein expression using mRNA was far from perfect. Because of limitations in our study design, the correlation between Wnts mRNA expression and Wnts protein expression could not be clearly assessed in this study. Further study in HCC is required.Our study identifies that several Wnt ligand family genes, such as Wnt1, Wnt3 and Wnt5B, may be potential prognostic biomarkers of HCC. Moreover, the nucleotide excision repair, aminoacyl tRNA biosynthesis and basal transcription factors may be the key pathway regulated by Wnts in HCC. Due to the small sample size and incomplete clinical information in this study, further experimental validation should be performed to prove the biologic impact of Wnts.Additional file 1: Figure S1. Comparison of gene expression levels of all 19 members of Wnt family genes in tumor tissue and adjacent non-tumor tissue.Additional file 2: Table S1. Univariate and multivariate analysis of disease-free survival using the Cox proportional hazard regression model.Additional file 3: Figure S2. TMN stage (A) and expression of Wnt1, Wnt3, Wnt5A, Wnt5B and Wnt8B (B–F) are associated with disease-free survival. Kaplan–Meier survival analysis and log-rank test were used to compare differences in overall survival between the groups classified using cut-off values determined by X-tile.Additional file 4: Table S2. Correlation of clinicopathologic variables and expression of several specific Wnts significantly associated with DFS.


Aurantiochytrium sp. (EEA) in the forced swimming test (FST)-induced depression in ICR mice. Imipramine, a commercially available tricyclic antidepressant drug, was used as positive control. Animals were administered EEA orally for 14 consecutive days and were subjected to the locomotor activity testing. Additionally, changes in gene expression in mice brain were assessed by real-time PCR and microarray assays to understand the molecular mechanisms underlying the effect of EEA. We found that the immobility time in FST was significantly reduced in the EEA-treated mice compared to that of in the control mice. Microarray and real-time PCR results revealed that EEA treatment induced changes in several genes in mice brain associated with pro-inflammation and dopaminergic, cholinergic, glutamatergic, and serotonergic synapses. It has previously been reported that several cytokines, such as IL-6 and TNF-α, which mediate neuroinflammation, are also responsible for indirectly altering brain neurotransmitter levels in neuropsychiatric disorders. Therefore, the regulation of the expression of pro-inflammatory genes in EEA-administered mice brain is considered to contribute to the enhancement of neurotransmitter systems-related gene expression in our study. Moreover, our in vitro study suggested that squalene, a component produced by Aurantiochytrium, was one of the active substances in EEA. In conclusion, our study provides the first evidence that Aurantiochytrium sp. can reduce neuroinflammation that may contribute to the modulation of the neurotransmitter systems, which could underlie its antistress and antidepressant effects.Although algae have been the focal point of biofuel research, studies on their biological activities have been limited. In recent years, however, the importance of algae as sources of functional ingredients has been recognized due to their health beneficial effects. In this study, we evaluated the antidepressant-like activities of ethanol extract of  Depression is a major cause of various psychiatric disorders worldwide. The World Health Organization predicts that depression will be one of the leading diseases by 2030 . DepressDifferent classes of antidepressant drugs are currently available on the market, such as tricyclic antidepressants, selective serotonin reuptake inhibitors, serotonin-noradrenaline reuptake inhibitors, monoamine oxidase inhibitors, and noradrenergic and specific serotonergic antidepressants . HoweverBotryococcus braunii by modulating neurogenesis and enhancing dopaminergic function [Falkowski and his colleagues have reported that microalgae were one of the earliest forms of life on the Earth that existed in Earth’s oceans more than 3 billion years ago, when the Earth’s environment was formed . The vasfunction .Aurantiochytrium is an oleaginous microorganism in the Thraustochytriaceae family that has attracted attention because of its ability to produce high levels of PUFAs and squalene. Recently, a research group of the University of Tsukuba isolated a novel strain of Aurantiochytrium sp. from the Okinawa prefecture in Japan, namely 18W-13a, which accumulates high amounts of squalene. The strain 18W-13a accumulated approximately 20% of squalene in glucose–peptone–yeast medium [Aurantiochytrium sp. [t medium . Squalent medium –15. Thusrium sp. .Aurantiochytrium sp. (EEA) using the forced swimming test (FST) in ICR mice and to further explore its possible molecular mechanism using DNA microarray analysis. We also focused our attention on changes in expression levels of genes associated with tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and brain-derived neurotrophic factor (BDNF) in mice brain. In addition, the neuroprotective effects of EEA and squalene were investigated using human neuroblastoma SH-SY5Y cells.The objectives of this study were to evaluate the antidepressant-like effects of the ethanol extract of Aurantiochytrium sp. was provided by Algae Biomass and Energy System (ABES) R&D Center, University of Tsukuba, Japan. The dry powder was extracted in the dark using 99.5% ethanol, at room temperature for two weeks. The mixture was shaken at least once a day. Finally, the liquid fraction (EEA) was collected and filtered through a 0.22 μm filter , and was used for in vitro assays. For the in vivo assay in ICR mice, EEA was concentrated using a SpeedVac  and the dried EEA was dissolved in milli-Q water.The dried algal powder was extracted following previous report . Dry powin vitro assays, Squalene was dissolved in the medium before further experiment.Squalene was purchased from Wako Co, Ltd. . For the in Vivo studies, male ICR mice (8 weeks old) with average body weight of 35–40 g were purchased from Charles River, Japan. All mice were housed individually. Animals were provided with free access to food and water, except when subjected to EEA administration or testing. The animal house was maintained at a 12-h light/dark period, and the temperature was kept at 22 ± 1°C throughout the study. This animal experiment was approved by the Ethics Animal Care and Use Committee of the University of Tsukuba (16–042).For the Botryococcus braunii to ICR mice to evaluate its antidepressant-like effects in the mouse FST [After one week of acclimatization to the laboratory conditions, animals were randomly assigned into three groups (8 mice per group): control group, imipramine-administered group , and EEA-administered group . In our previously reported study, we orally administered 100 mg/kg of In our study, imipramine  was used as positive control. It was dissolved in distilled water and was orally administered to mice at a volume of 20 mg/kg body weight for 14 days, as reported in our previous study .FST is a widely adopted behavioral animal model to investigate depression . The FSTFollowing the last FST trial on day 14, each mouse was sacrificed by cervical dislocation, and the whole brain was carefully isolated. The entire limbic area (100 mg) containing the cortex, hippocampus and amygdale was quickly dissected from mouse whole brain and washed with an ice-cold phosphate-buffered solution (PBS). The total RNA was purified using the ISOGEN kit  following the manufacturer’s instructions. The quantity and quality of total RNA was assessed with the NanoDrop 2000 spectrophotometer .in vitro transcription using the GeneChip 3´ IVT Express Kit . Briefly, purified aRNA was fragmented using the GeneAtlas 3´ IVT Express Kit and hybridized for 16 h at 45°C using the GeneChip MG-430 PM microarray . The chip was washed and stained in the Gene Atlas Fluidics Station 400 , and the resulting image was scanned using the GeneAtlas Imaging Station . Data analysis was performed using the Affymetrix Expression Console Software version and Visualization and Integrated Discovery (DAVID) software version 6.8 . Compared with the control (vehicle-treated group), fold-changes in the expression of genes in the imipramine- or EEA-treated groups were calculated and converted to linear data.DNA microarray analysis was conducted on isolated RNAs extracted from the limbic area of mice brains as reported previously . Double-12–18 primers were denatured at 65°C for 5 min and were subsequently chilled at 4°C. After addition of SuperScript III RT (200 U), the reaction mix was incubated at 42°C for 60 min, followed by another 10 min at 70°C. All primer sets and TaqMan probes for experimental genes were purchased from Applied Biosystems : mouse tumor necrosis factor-α (TNF-α) (Mm00447557_m1), mouse interleukin-6 (IL-6) (Mm00500992_m1), mouse brain-derived neurotrophic factor (BDNF) (Mm04230607_s1), and mouse GAPDH (Mm99999915_g1). For the mRNA quantification, TaqMan real-time PCR amplification reactions were carried out using an AB 7500 Fast Real-Time PCR system (Applied Biosystems). Amplifications were performed in 20 μL final volume, using 10 μL TaqMan Universal PCR Master Mix UNG (Applied Biosystems), 1 μL of the corresponding primer/probe mix, and 9 μL of template cDNA . Cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, and 40 cycles at 95°C for 15 s followed by 60°C for 1 min.On the basis of the results obtained from the microarray analysis, reverse transcription reactions were carried out with the SuperScript III Reverse Transcriptase (RT) kit . According to the manufacturer’s instructions, 1 μg of total RNA and 1 μL of oligo (dT) 2 incubator. A serum-free Eagle’s minimum essential medium  was used to culture the cells for the cell viability assay. The EEA used contained 20 mg/mL for in vitro assays.Human neuroblastoma SH-SY5Y cell line was obtained from American Type Culture Collection (ATCC). Cells were maintained in defined medium (DM) composed of Dulbecco´s modified Eagle´s medium/F12 medium (1:1 vol/vol)  supplemented with 15% heat-inactivated fetal bovine serum  and 1% penicillin (5000 μg/mL)-streptomycin (5000 IU/mL) (PS)  at 37°C in a 95% humidified air/5% CO5 cells·ml−1) cultured in 96-well plate (fibronectin-coated plate)  were treated with EEA  or squalene  and subsequently with 500 μM dexamethasone  for 48 h. After sample treatment, 100 μL of Opti-MEM and 10 μL of MTT (5 mg/mL) were added, and the cells were incubated further for 6 h. The MTT formazan formed was dissolved in 100 μL of 10% SDS (w/v), and the absorbance was measured using a micro titer plate reader .Cell viability was measured using the 3--2,5- diphenyltetrazolium bromide (MTT) method. SH-SY5Y cells . Statistical analysis of the results obtained in the FST was carried out using two-way ANOVA followed by Ryan-Einot-Gabriel-Welsch multiple range test. A one-way within subjects ANOVA (repeated measures) followed by the Ryan-Einot-Gabriel-Welsch multiple range test was also carried out. The statistical evaluation was performed using the Student’s t-test between control and corticosterone-treatment groups in the To determine whether EEA has antidepressant-like activity, its effect on FST-induced stress in mice was investigated. No death or sign of toxicity, such as significant loss of body weight, was observed in all the groups of mice (data not shown). As shown in On day 14, the average immobility time for the EEA-administered groups (30.2 ± 18.0 s) was similar to the average immobility time measured in the imipramine-administered group (26.3 ± 10.7 s), which represented our positive control. In mice, treatment with both imipramine and EEA induced a 3-fold reduction of the average immobility time compared to the vehicle-administered control mice (80.3 ± 6.4 s).Prkcd), adenylate cyclase 7 (Adcy7), phospholipase C, beta 4 (Plcb4), and son of sevenless homolog 1 (Sos1) genes associated with chemokine signaling pathway were downregulated in the EEA-administered groups. Moreover, the expressions of the following genes related to dopaminergic synapse were upregulated: protein kinase C, alpha (Prkca), adenylate cyclase 5 (Adcy5), inositol 1,4,5-trisphosphate receptor 1 (Itpr1), guanine nucleotide binding protein (G protein), gamma 7 (Gng7), adenylate cyclase 9 (Adcy9), glutamate receptor, ionotropic, AMPA2  (Gria2), protein phosphatase 3, catalytic subunit, alpha isoform (Ppp3ca), calcium/calmodulin-dependent protein kinase II alpha (Camk2a), transient receptor potential cation channel, subfamily C, member 1 (Trpc1), dopamine receptor D1 (Drd1), dopamine receptor D2 (Drd2), guanine nucleotide binding protein, alpha stimulating, olfactory type , and protein phosphatase 1, regulatory (inhibitor) subunit 1B (Ppp1r1b). In addition, the expressions of 10 genes associated with glutamatergic synapses were upregulated: Prkcd, Adcy5, Itpr1, Gng7, Adcy9, Gria2, Ppp3ca, glutamate receptor, metabotropic 3 (Grm3), glutamate receptor, ionotropic, kainate 3 (Grik3), and homer homolog 1 (Homer1). Further, we found the upregulation of 10 genes related to the cholinergic synapse: Prkcd, Adcy5, Itpr1, Gng7, Adcy9, Camk2a, Janus kinase 2 (Jak2), calcium/calmodulin-dependent protein kinase IV (Camk4), potassium voltage-gated channel, subfamily Q, member 5 (Kcnq5). Finally, the expressions of serotonergic synapse-related genes, e.g. Prkcd, Adcy5, Itpr1, Gng7, Kras, Trpc1, Rap guanine nucleotide exchange factor (GEF) 3 (Rapgef3), 5-hydroxytryptamine (serotonin) receptor 1B (Htr1b), and prostaglandin-endoperoxide synthase 2 (Ptgs2), were upregulated.To evaluate the molecular mechanism of the antidepressant-like effect of EEA, we investigated the changes in gene expression in the limbic area of the ICR mouse brain using microarray analysis. We found that the expressions of 28 genes were altered in the ICR mice administered with EEA compared to the control group . SpecifiOn the basis of the results obtained from the microarray analysis, we investigated the mRNA expression levels of major cytokines, TNF-α and IL-6, in the limbic area of ICR mouse of the four experimental groups. We also evaluated the mRNA expression levels of BDNF in ICR mouse brain as previous studies reported that cytokines interact with BDNF . Our resTo evaluate the cytotoxicity of EEA and squalene, SH-SY5Y cells were treated with EEA  and squalene  for 48 hours, and cell viability was measured by the MTT assay. EEA and squalene showed no toxicity at all on cell viability . InteresAurantiochytrium. In the present study, we evaluated the effect of Aurantiochytrium in animal models of FST-induced depression. The FST is widely used as a behavioral model to evaluate rodent depression in the screening of antidepressant drugs [Depression is a complex mood disorder and its heterogeneity suggests that various biological mechanisms underlie the clinical presentations of depression. As far as we know, there is no scientific report on the antidepressant effects of nt drugs . Specifint drugs , 18, we Aurantiochytrium as a sustainable source of PUFAs, such as docosahexaenoic acid [Aurantiochytrium sp. 18W-13a strain, which produces the highest level of useful hydrocarbon, squalene, compared with other algal strains [Recent researches have focused on oic acid –23. PUFAoic acid . However strains . We demoAdcy7, Plcb4, Prkcd, and Sos1, related to chemokine signaling pathways were downregulated in EEA-treated mice brain. It has been reported that Prkcd may regulate four inflammatory chemokines, including Ccl2, Mcp-1, Ccl7, Cxcl16, and Cx3cl1. It was also reported that Prkcd stimulates Ccl2 gene expression through the NF-B signaling [Sos1 may mediate Cxcl12-induced lymphocyte function-associated antigen 1 (LFA-1) activation, and LFA-1 affinity triggering to Cxcl12 is impaired by Sos1 downregulation [et al. reported in 1995 that depression is a disease caused by dysfunction of psychoneurotic immunity and activation of the inflammatory response system [Neuroinflammation is a major contributing factor to a broad range of neuropsychiatric and neurodegenerative disorders. Chemokines are known to be important modulators of the immune response and activators of neuroinflammation  associatignaling . Sos1 magulation . Therefogulation . Moreovee system . Becausee system , 34. TheAdcy5, Adcy9, Camk2a, Drd1, Drd2, Gria2, Gng7, Gnal, Itpr1, Prkca, Ppp1r1b, Ppp3ca, and Trpc1. Therefore, our study suggests that oral administration of EEA could enhance the dopamine pathway.The monoamine neurotransmission system, which includes dopamine, serotonin (5-HT), and norepinephrine systems, has long been recognized as critically involved in the pathogenesis of depression. Studies reported that depression can be attributed to the functional imbalance or deficiency of monoamine neurotransmitters . FurtherAdcy5, Adcy9, Gria2, Grik3, Grm3, Gng7, Homer1, Itpr1, Prkca, and Ppp3ca. Interestingly, the EEA-treated group showed a higher number of upregulated genes related to glutamatergic synapses compared to the imipramine-treated group. Therefore, EEA treatment is considered to activate glutamatergic synapses.Moreover, the involvement of glutamatergic synapses in mood disorders was proposed on the basis of preclinical studies of NMDA receptor antagonists . SeveralJak2, Kras, Adcy5, Adcy9, Camk2a, Camk4, Gng7, Itpr1, Kcnq5, and Prkca. Cholinergic neurons play a major role in the regulation of various CNS functions, such as excitation, attention, cognition, and memory. Impairment of cognitive function is often observed in major depressive disorders. Acetylcholine (Ach), restricted to cholinergic neurons, is detected at various sites outside of the central and peripheral nervous system, such as immune cells [Additionally, we confirmed the upregulation of several genes related to cholinergic synapse signaling in EEA-treated mouse brains, such as ne cells  and kerane cells , 43 and ne cells , 45. TheHtr1b, Kras, Rapgef3, Adcy5, Gng7, Itpr1, Ptgs2, Prkca, and Trpc1. Dysfunction of central serotonergic neurotransmission triggers the development of depressive symptoms. Serotonergic system is an important target of classical antidepressant drugs [In addition, we showed upregulation of several genes related to serotonergic synapse signaling in EEA-treated mice brains, such as nt drugs . Previount drugs . The resSeveral studies support the role of reduced BDNF activity in inflammatory cytokine-associated depression , 49. ThuAurantiochytrium may have therapeutic potential for the treatment of neuropsychiatric symptoms in neurodegenerative diseases.Development of new strategies for the prevention and treatment of psychological diseases is a major therapeutic challenge. Our results showed that the significant upregulation of several genes involved in the neurotransmitter systems, such as dopaminergic and serotoninergic synapses, through the significant reduction of proinflammatory-related genes contributes to the antidepressant-like effect of EEA. Altogether, our results suggest that


Indeed, these findings led to the clinical testing of globally acting CB1R blockers for obesity and various metabolic complications. However, their therapeutic potential was halted due to centrally mediated adverse effects. Recent observations that highlighted the key role of the peripheral eCB system in metabolic regulation led to the preclinical development of various novel compounds that block CB1R only in peripheral organs with very limited brain penetration and without causing behavioral side effects. These unique molecules, which effectively ameliorate obesity, type II diabetes, fatty liver, insulin resistance, and chronic kidney disease in several animal models, are likely to be further developed in the clinic and may revive the therapeutic potential of blocking CB1R once again.In this review, we discuss the role of the endocannabinoid (eCB) system in regulating energy and metabolic homeostasis. Endocannabinoids, via activating the cannabinoid type-1 receptor (CB Cannabis includes up to three strains, Cannabis sativa, Cannabis indica, and Cannabis ruderalis, each with a very long history of domestication [Cannabis sativa (marijuana) is considered one of the most resourceful plants, research investigations of which during the past half-century have led to the discovery of an important homeostatic system, the endocannabinoid (eCB) system, which plays a key role in human physiology  [8-tetrahydrocannabinol (Δ8-THC) [Throughout human history, plants have been used as a predominant source of medications. The genus tication . These stication . Regardiiewed in ). Curreniewed in . Of thesiewed in . The firiewed in , who isoiewed in ). Intereiewed in , Δ9-tetrrijuana) , Δ8-tetr(Δ8-THC) , cannabi(Δ8-THC) , cannabi(Δ8-THC) , and can(Δ8-THC) .9-THC in the brain and peripheral organs were identified, which were then termed as the cannabinoid-1 and -2 receptors  [i/Go proteins, despite the fact that they can also recruit Gs and Gq/11 proteins and facilitate G protein-independent molecular pathways [1R, primarily localized in the cell membrane, is the most widely expressed G-protein coupled receptor (GPCR) in the human brain [2R, on the other hand, is predominantly localized in immune cells and is moderately expressed in many peripheral tissues, with conflicting evidence regarding its expression in the central nervous system (CNS) [Cannabis, only Δ9-THC and its less abundant propyl analogue, Δ9-tetrahydrocannabivarin (THCV), have been shown to bind to CB1R and CB2R with high affinity . Regarding other cannabinoids, studies have shown their ability to bind to several different receptors, ranging from other GPCRs  to ion channel  channels) and nuclear receptors   ,16,17. Actively) ,19,20,21pathways . CB1R, pan brain , but it an brain . CB2R, oem (CNS) . Of the iewed in ); howeve1R and CB2R in mammalian cells prompted the discovery of their first endogenous ligand, arachidonoyl ethanolamide  [1R, and barely active at CB2R, 2-AG is known to activate both receptors with moderate-to-low affinity [N-acyl-phosphatidylethanolamine (NAPE) by NAPE-specific phospholipase D (NAPE-PLD) or via other means [The successful cloning and identification of CBndamide) , which wndamide) ,29. Wheraffinity ,31. Bother means . Their der means , whereaser means .The eCB system, acting both centrally and peripherally, is an important physiological system, comprising the cannabinoid receptors and their natural endogenous ligands as well as the enzymes/proteins involved in their biosynthesis, transport, and degradation. It is involved in many physiological and pathological conditions and functions as a regulatory homeostatic system in various tissues, such as the brain, skin, liver, cardiovascular system, bone, kidney, pancreas, adipose and muscle tissues, the digestive track, and many more (reviewed in ). Since Cultivated for millennia, marijuana still has a remarkable ability to alleviate different physical pathologies. To date, the U.S. Federal Food, Drug, and Cosmetic Act defines marijuana as a drug, taken by either smoking or consuming it orally for therapeutic purposes. As a drug, it may also cause harm and has toxic effects. Thus far, only limited reports related to the side effects of marijuana used for medical purposes have been reported. This contradicts the existing information regarding its recreational use, as well as in comparison to other drugs . Whereas the latter drugs may cause death when consumed inappropriately, mainly due to respiratory arrest ,40 and/o1R within appetite-related brain areas [th century firmly supported the ability of cannabis consumption to induce hyperphagia and snacking . Indeedrized in . Orally f hunger ,50,51, fd system . These cd system , individd system ,55,56,57d system  as well d system ,60.9-THC in various animal models [1R was identified in various brain regions, including the hypothalamus, which plays a key role in homeostatic regulation. Indeed, direct activation of CB1R by AEA has been shown to stimulate food intake [1R agonists were also reported to increase sucrose consumption [9-THC was reported in 1975 [1R [1R affects feeding behavior and subsequently induces weight loss in obese individuals.Accumulating basic evidence also supports the orexogenic effects of cannabinoids, demonstrating increased food intake by administering Δl models ,63,64,65d intake ,67,68,69sumption  and hypesumption . An inte in 1975 . In fact in 1975 . Similar in 1975 . Further in 1975 ). Intere1975 [1R ,78,79, w1975 [1R . In keep1R with the first-in-class synthetic CB1R inverse agonist rimonabant (SR141716A) does indeed reduce weight gain and food intake in a dose-dependent manner under both fasted and non-fasted conditions [1R are hypophagic and lean [1R blockade could be considered as a therapeutic tool against obesity and metabolic syndrome. Indeed, rimonabant was proven effective not only in decreasing food intake and body weight, but also in ameliorating obesity-induced insulin and leptin resistance, improving glucose homeostasis and dyslipidemias, as well as decreasing hepatic steatosis in obese/overweight individuals with metabolic syndrome [® (Sanofi-Aventis). However, growing evidence of anxiety, depression, and suicidal ideation, which was reported in a small but significant portion of individuals treated with rimonabant [1R blockers, and questions were raised regarding the therapeutic relevance of this class of molecules in modulating the eCB system for treatment of metabolic syndrome [Empirical studies in various animal models indicate that pharmacological blockade of CBnditions ,81,82,83nditions ,85. Thesand lean , led to syndrome ,91,92,93monabant , led to syndrome .1R blockade not only affects CNS-mediated energy homeostasis, but also regulates energy balance via peripheral mechanisms [1Rs are present not only in the CNS, but also in many peripheral organs. Their expression levels in adipose tissue, liver, skeletal muscle, kidney, and pancreas are elevated under obese/diabetic conditions [1R in liver, adipose tissue, kidney, pancreas, and skeletal muscle, studies have shown that CB1R modulates peripheral metabolic function. Interestingly, deletion of hepatic CB1R was sufficient to protect obese mice from hepatic steatosis and dyslipidemia, as well as insulin and leptin resistance [1R in adipocytes resulted in complete protection from diet-induced obesity in mice [1R-knockout mice are protected from high-fat/high-sugar diet-induced pancreatic dysfunction and inflammation [1R specifically located on the renal proximal tubule cells (RPTCs) [1R in the RPTCs gain weight and show metabolic impairment similar to their wild-type control animals, they remain completely protected from diabesity-induced renal dysfunction, inflammation, fibrosis, lipotoxicity, and mitochondrial function [1R plays in cellular/metabolic regulation in many peripheral organs suggest that targeting CB1R in peripheral organs by limiting brain access of CB1R blockers may improve their therapeutic efficacy via reducing their potential to cause CNS-mediated adverse effects. This idea was tested experimentally in numerous studies describing the contribution of the peripheral eCB/CB1R system to the development of obesity and its metabolic comorbidities, as well as the therapeutic potential of peripherally restricted CB1R antagonists to treat obesity and its sequelae.Despite having only a transient inhibitory effect on feeding, rimonabant was very efficacious in reducing body weight and adiposity, suggesting that CBchanisms . As mentnditions ,102,103.nditions ,110,111.sistance . A speci in mice . Beta ceammation , and itsammation . Recentl (RPTCs) ,116. Whefunction ,116. Tak1R antagonists devoid of brain penetration and CNS activity can be achieved by using two main paradigms: First, chemical modification of brain-penetrating CB1R blockers, such as rimonabant or other rimonabant-like compounds ; second, usage of computational or in vitro chemical tools to design and synthesize compounds that do not penetrate the blood–brain barrier (BBB), based on studies that characterize those properties responsible for brain penetration [1R blockers are well-described elsewhere [1R and limited BBB penetration have been designed and patented by different groups  [1Rs [Magel2 null mice, a well-established model of Prader–Willi syndrome [1R-, and type I diabetes-induced renal impairment [AM6545 was the first to undergo a detailed pharmacological, metabolic, and behavioral assessment in murine models of obesity. This molecule ameliorates hepatic steatosis, increases insulin sensitivity, and improves dyslipidemia in diet- and genetically induced obese mice . In addinabant®) . Its hypt®) [1Rs ,129. Morsyndrome , as wellpairment ,116.1R in the periphery have also been synthesized and characterized, although not to the same extent as AM6545 and JD5037. Among these, TM38837, also recently termed BPR0912, has a negligible impact on brain CB1R when tested in mice, primates, and healthy individuals [1R neutral antagonist whose structure is related to rimonabant, was found to be efficacious in ameliorating diet- and olanzapine-induced obesity and its metabolic abnormalities [1R blocker able to reduce food intake and body weight in rats [1R null mice [1R-dependent manner. With an IC50 value of 159 nM, Compound 1, described by Son and colleagues in 2010, was found to be less brain-penetrating and efficacious than rimonabant in ameliorating food intake and obesity in mice [1Rs [1R inverse agonist program of the same group, reduced glucose levels without centrally mediated behavioral effects and a reduction in food intake or body weight [1R, improved dyslipidemia, glucose homeostasis, and fat mass in obese mice without affecting their food intake [1Rs located in the periphery can be potentially considered as clinically relevant targets for therapeutics against obesity and its comorbidities, thus warranting further preclinical development and clinical testing of the peripherally restricted CB1R blockers. Of note, in December 2017, the U.S. Food and Drug Administration (FDA) cleared the Investigational New Drug (IND) Application for JD5037 to begin Phase 1 clinical trials. However, it remains to be seen if this novel compound will clear the way for other molecules that target peripheral CB1Rs to be fully translated into use for humans and rekindle the spark for discovering new blockbuster therapies against metabolic syndrome.A few other novel molecules that mostly target CBividuals ,131,132,ividuals ,134 and ividuals . NESS06Smalities ,137. LH- in rats ,139,140,ull mice . URB447 ull mice , probablull mice ,144 in a in mice . With a  in mice . Althougice [1Rs . Compouny weight . Lastly,d intake . Overall1Rs play in orexigenic signaling via central modulation of energy balance and feeding behavior. However, the influence of the eCB/CB1R system on energy utilization and homeostasis cannot be solely explained by central mechanisms. Indeed, data show that this system also acts peripherally to modulate adipose tissue metabolism, kidney function, hepatic lipogenesis, muscle activity, and pancreatic homeostasis. Being tonically overactivated during obesity, the eCB/CB1R system contributes to impairment in hormonal/metabolic function, propelling CB1R forward as a potential therapeutic target for obesity. Whereas the globally acting CB1R blocker rimonabant once held tremendous promise in ameliorating the metabolic abnormalities of obesity, its CNS-mediated adverse effects limited its clinical use. Targeting the eCB system using novel compounds that block CB1Rs in periphery with negligible brain penetration still holds promise for future therapy for obesity and its sequelae.The appetite-stimulating ‘side effect’ of marijuana has been recognized for centuries. Mounting evidence supports the key role that CB