RANK Signaling Research Articles

Comparison of miRNA Cargo within Wharton's Jelly mesenchymal stem cell-derived exosomes manufactured in FBS and hPLT expansion media

Background & Aim Wharton's Jelly Mesenchymal Stem Cell (WJMSCs)-derived exosomes have been studied extensively in animal models of degenerative and chronic diseases. Thus, the development of a large-scale manufacturing procedure to harvest clinical-grade exosomes is required for the translation of exosome to the clinic. We have developed an exosome production technique to manufacture large quantities of exosomes using the quantum bioreactor (Terumo BCT) with both fetal bovine serum (FBS) and human platelet lysate (hPLT) supplemented expansion media. AWe aimed to compare the miRNA cargo of exosomes isolated from FBS-expanded and PLT-expanded WJMSC to determine if growth media is a significant contributor to exosome product preparation. Methods, Results & Conclusion WJMSCs were isolated from Wharton's Jelly and expanded to passage 1. After harvest, 30 × 106 cells were loaded into the quantum bioreactor and expanded for 1 week to achieve maximum confluence. Expansion media was washed and replaced with serum-free aMEM media. Conditioned media was collected every 24 hours for 120 hours and exosomes were precipitated by sequential centrifugation and ultracentrifugation. NanoSight analysis of the final products revealed a production of 15.0 × 1012± 0.32 × 1012 total particles with an average mode size of 111±4.78 nm. Surface protein analysis revealed positive expression of exosome markers CD9, CD63, and CD81. Exosome products were subjected to miRNA isolation, sequencing, and miRNA identification for bioinformatics analysis with the Qiagen IPA software. miRNA analysis was completed using 3 WJMSC donors grown in 20% FBS or 5%PLT supplemented media. Sequencing results identified 157 miRNAs in the PLT group and 154 miRNAs in the FBS group (with a 100-copy minimum expression cut off). FBS and PLT comparison revealed 142 common miRNAs with 15 unique to PLT and 12 unique to FBS. Further RNA target analysis revealed 68 miRNAs that target 1024 mRNA in the PLT group and 64 miRNA that target 1048 mRNA in the FBS group. The two groups share 998 common mRNA targets with 27 unique to PLT and 51 unique to FBS. Canonical pathway analysis of the unique targets reveals the top pathway hits, serotonin receptor and JAK/Stat signaling for PLT and IL-7 and RANK signaling for FBS. We found both expansion medias to be effective for large scale expansion and downstream exosome production. miRNA sequencing revealed small variations in the total number of predicted mRNA targets, suggesting a conserved therapeutic effect between the two groups. Wharton's Jelly Mesenchymal Stem Cell (WJMSCs)-derived exosomes have been studied extensively in animal models of degenerative and chronic diseases. Thus, the development of a large-scale manufacturing procedure to harvest clinical-grade exosomes is required for the translation of exosome to the clinic. We have developed an exosome production technique to manufacture large quantities of exosomes using the quantum bioreactor (Terumo BCT) with both fetal bovine serum (FBS) and human platelet lysate (hPLT) supplemented expansion media. AWe aimed to compare the miRNA cargo of exosomes isolated from FBS-expanded and PLT-expanded WJMSC to determine if growth media is a significant contributor to exosome product preparation. WJMSCs were isolated from Wharton's Jelly and expanded to passage 1. After harvest, 30 × 106 cells were loaded into the quantum bioreactor and expanded for 1 week to achieve maximum confluence. Expansion media was washed and replaced with serum-free aMEM media. Conditioned media was collected every 24 hours for 120 hours and exosomes were precipitated by sequential centrifugation and ultracentrifugation. NanoSight analysis of the final products revealed a production of 15.0 × 1012± 0.32 × 1012 total particles with an average mode size of 111±4.78 nm. Surface protein analysis revealed positive expression of exosome markers CD9, CD63, and CD81. Exosome products were subjected to miRNA isolation, sequencing, and miRNA identification for bioinformatics analysis with the Qiagen IPA software. miRNA analysis was completed using 3 WJMSC donors grown in 20% FBS or 5%PLT supplemented media.

Drivers in AF colocate to sites of electrogram organization and rapidity: Potential synergy between spectral analysis and STAR mapping approaches in prioritizing drivers for ablation.

Stochastic trajectory analysis of ranked signals (STAR) mapping has recently been used to ablate persistent atrial fibrillation (AF) with high rates of AF termination and long-term freedom from AF in small, single-arm studies. We hypothesized that rapidity and organization markers would correlate with early sites of activation (ESA). Patients undergoing persistent AF ablation as part of the STAR mapping study were included. Five-minute unipolar basket recordings used to create STAR maps were used to determine the minimum-cycle length (Min-CL) and CL variability (CLV) at each electrode to identify the site of the fastest Min-CL and lowest CLV across the left atrium (LA). The location of ESA targeted with ablation was compared with these sites. Dominant frequency was assessed at ESA and compared with that of neighboring electrodes to assess for regional gradients. Thirty-two patients were included with 83 ESA ablated, with an ablation response at 73 sites (24 AF termination and 49 CL slowing ≥30 ms). Out of these, 54 (74.0%) and 56 (76.7%) colocated to sites of fastest Min-CL and lowest CLV, respectively. Regional CL and frequency gradients were demonstrable at majority of ESA. ESA colocating to sites of fastest Min-CL and lowest CLV were more likely to terminate AF with ablation (odds ratio, 34 and 29, respectively, P = .02). These showed a moderate sensitivity (74.0% Min-CL and 75.3% CLV) and specificity (66.7% Min-CL and 76.9% CLV) in predicting ESA with an ablation response. ESA correlate with rapidity and organization markers. Further work is needed to clarify any role for spectral analysis in prioritizing driver ablation.

Two signals of social rank: Prestige and dominance are associated with distinct nonverbal displays.

Converging evidence suggests that high rank is communicated through various nonverbal behaviors (e.g., expansiveness), but prior studies have not examined whether 2 distinct forms of high rank-known as prestige and dominance-are communicated through distinct nonverbal displays. Given the divergent messages that prestigious and dominant leaders need to send in order to attain and retain their place in the social hierarchy, theoretical accounts would suggest that individuals use distinct sets of nonverbal behaviors to communicate these 2 forms of high rank. In the present research, we tested this hypothesis in 7 studies, using carefully controlled experimental designs (Studies 1, 2, 3, 4a, and 4b) and the assessment of spontaneously displayed nonverbal behaviors that occurred during a lab-based group interaction (Study 5) and a real-world political contest (Study 6). Results converged across studies to show that prestige and dominance strategies are associated with distinct sets of nonverbal behaviors, which are largely consistent with theoretical predictions. Specifically, prestige, or the attainment of rank through earned respect, and dominance, or the use of intimidation and force to obtain power, are communicated from different head positions (i.e., tilted upward vs. downward), smiling behaviors (i.e., presence vs. absence of a symmetrical smile), and different forms of bodily expansion (i.e., subtle chest expansion vs. more grandiose space-taking). These findings provide the first evidence for 2 distinct signals of high rank, which spontaneously emerge in social interactions and guide social perceptions and the conferral of power. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

The Non-Bone-Related Role of RANK/RANKL Signaling in Cancer.

RANK ligand (RANKL) is a member of the tumor necrosis factor alpha superfamily of cytokines. It is the only known ligand binding to a membrane receptor named receptor activator of nuclear factor-kappa B (RANK), thereby triggering recruitment of TNF receptor-associated factor (TRAF) adaptor proteins and activation of downstream pathways. RANK/RANKL signaling is controlled by a decoy receptor, osteoprotegerin (OPG), but also has additional more complex levels of regulation. Itis crucial for the differentiation of bone-resorbing osteoclasts and is deregulated in disease processes such as osteoporosis and cancer bone metastasis. Cells expressing RANK and RANKL are commonly found in the tumor environment. In many tumor types, the RANK/RANKL pathway is overexpressed, and this is in most cases correlated with poor prognosis. RANK signaling plays an important role in the innate and adaptive immune response, generates regulatory T (Treg) cells, and increases the production of cytokines. It is also involved in chemo resistance in vitro. Recent evidence suggests that RANKL blockade improves the efficacy of anti-CTLA-4 antibodies against solid tumors and experimental metastasis. Therefore, there is increasing interest to use RANKL inhibition as an immunomodulatory strategy in an attempt to make immune-resistant tumor responsive to immune therapy.

Acanthopanax senticosus aqueous extract ameliorates ovariectomy-induced bone loss in middle-aged mice by inhibiting the receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis.

Acanthopanax senticosus (Ciwujia) has broad-spectrum pharmacological activities, including osteoprotective effects. However, the mechanisms underlying these effects remain unclear. We investigated whether Acanthopanax senticosus aqueous extract (ASAE) ameliorates ovariectomy-induced bone loss in middle-aged mice through inhibition of osteoclastogenesis. In vitro, ASAE significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclast differentiation and formation of F-actin rings by downregulating the expression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), c-Fos, and osteoclastogenesis-related marker genes and proteins, including c-Src, tartrate-resistant acid phosphatase (TRAP), cathepsin K, β3-integrin, and matrix metallopeptidase-9 (MMP-9). This was achieved by inhibiting RANK signaling pathways, including p65, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and p38 in osteoclast precursors. In vivo, ASAE markedly ameliorated bone loss in ovariectomized (OVX) middle-aged mice. ASAE significantly inhibited the serum levels of tartrate-resistant acid phosphatase 5b (TRACP-5b) and RANKL, whereas it increased those of osteocalcin, procollagen 1 N-terminal peptide (P1NP), and osteoprotegerin in OVX mice. ASAE significantly inhibited the OVX-induced expression of osteoclast-specific proteins and genes in the femur. In conclusion, ASAE prevents ovariectomy-induced bone loss in middle-aged mice by inhibiting RANKL-induced osteoclastogenesis through suppression of RANK signaling pathways and could be potentially used in mediated treatment of osteoclast-related diseases (e.g., osteoporosis).

Hybrid Sparse Array Beamforming Design for General Rank Signal Models

The paper considers sparse array design for receive beamforming achieving maximum signal-to-interference plus noise ratio (MaxSINR) for both single point source and multiple point sources, operating in an interference active environment. Unlike existing sparse design methods which either deal with structured environment-independent or non-structured environment-dependent arrays, our method is a hybrid approach and seeks a full augumentable array that optimizes beamformer performance. This approach proves important for limited aperture that constrains the number of possible uniform grid points for sensor placements. The problem is formulated as quadratically constraint quadratic program (QCQP), with the cost function penalized with weighted l_1-norm squared of the beamformer weight vector. Simulation results are presented to show the effectiveness of the proposed algorithms for array configurability in the case of both single and general rank signal correlation matrices. Performance comparisons among the proposed sparse array, the commonly used uniform arrays, arrays obtained by other design methods, and arrays designed without the augmentability constraint are provided.

STAR mapping method to identify driving sites in persistent atrial fibrillation: Application through sequential mapping.

IntroductionThe optimal way to map localized drivers in persistent atrial fibrillation (AF) remains unclear. The objective of the study was to apply a novel vector mapping approach called Stochastic Trajectory Analysis of Ranked signals (STAR) in AF.Methods and ResultsPatients having persistent AF ablation were included. Early sites of activation (ESA) identified on global STAR maps created with basket catheters were used to guide AF ablation post‐pulmonary vein isolation (PVI). All patients also had sequential STAR maps created with ≥10 PentaRay recordings of 30 seconds. These were validated offline in their ability to identify the ESA targeted with a study‐defined ablation response (AF termination or cycle length [CL] slowing of ≥30 ms).Thirty‐two patients were included in whom 92 ESA were identified on the global STAR maps, with 73 of 83 targeted sites demonstrating an ablation response (24 AF termination and 49 CL slowing). Sixty‐one out of 73 (83.6%) ESA were also identified on the sequential STAR maps. These showed greater consistency (P < .001), were seen pre‐ and post‐PVI (P < .001) and were more likely to be associated with AF termination on ablation (P = .007). The sensitivity and specificity of sequential mapping for the detection of ESA with an ablation response was 84.9% (95% confidence interval [CI] = 74.6‐92.2) and 90.0% (95% CI = 55.5‐99.8), respectively. During a follow‐up of 19.4 ± 3.7 months, 28 (80%) patients were free from AF/atrial tachycardia.ConclusionsSTAR mapping consistently identified ESA in all patients and the ablation response was compatible with ESA being driver sites. Mechanistically important ESA were successfully identified using sequential recordings.

Weak target echo detection based on low rank signal in multi-static passive radar

This paper considers weak target echo detection in multi-static passive radar system. A new detector adopting a test statistic based on the cross correlation of the principal left singular vectors of the reference and surveillance signal-plus-noise matrices is devised. Due to the exploitation of the low-rank structure of transmitted signal, it offers better performance than traditional detector. Simulation results verify the effectiveness of the proposed detection method.

Proteasome inhibition suppress microgravity elevated RANK signaling during osteoclast differentiation

Microgravity (µXg) induces bone loss in astronauts during space missions. Therefore, it is necessary to delineate the underlying mechanisms which leads to bone loss for developing countermeasures. Osteoclasts (OCLs) are multinucleated cells, which resorb bone. Previously, we have demonstrated that simulated µXg enhances OCL formation. However, control of osteoclast bone resorption activity under μXg remains unclear. The OCL formation has been shown to be regulated by ubiquitin–proteasome pathway. Hence, we hypothesized that proteasome inhibition could regulate osteoclast differentiation under μXg. In this study, we identified that RAW264.7 preosteoclast cells treated with proteasome inhibitor (MG-132) suppress RANK receptor expression essential for OCL differentiation, but no effect on TRAF-6. We identified that MG-132 treatment abolished K48-linked poly-ubiquitination under μXg. Immunostaining confirms inhibition of protein ubiquitination and RANK expression in preosteoclast cells. Furthermore, proteasome inhibition suppresses the expression of SQSTM1/p62 under both the ground based Xg and μXg conditions. Also, confocal microscopy using Lyso-Tracker demonstrated that proteasomal inhibition suppress the co-localization of p62 and lysosomes. MG-132 inhibited RANKL induced proteasome activity. RAW264.7 cells treated with the proteasome inhibitor showed an increased level of p-c-Jun activity in control cultures, however decreased under µXg. In contrast, c-Fos and NFATc1 expression was decreased. In-addition, mouse bone marrow cultures treated with MG-132 suppress OCL formation and bone resorption activity. Thus, our findings suggest that proteasome inhibition represents a novel therapeutic approach for bone loss under µXg in space environment.

Water extract of Cnidii Rhizoma suppresses RANKL-induced osteoclastogenesis in RAW 264.7 cell by inhibiting NFATc1/c-Fos signaling and prevents ovariectomized bone loss in SD-rat

BackgroundCnidii Rhizoma is the dried root stem of Cnidium officinale Makino. Cnidii Rhizoma (CR) has been used to treat menstrual irregularity, menstrual pain, and menopause in Korea. However, the effects and mechanisms of CR on RANKL-induced osteoclastogenesis pathway remain to be elucidated. In this study, we investigated the effects of CR on the inhibition of bone resorption of osteoclast and its mechanism RANK signaling pathway.MethodsThe anti-osteoclastogenesis of water extract of CR was measured using RAW 264.7 cell. Tartrate-resistant acid phosphatase (TRAP) assay, pit assay, reverse transcription polymerase chain reaction (RT-PCR) and western blot were performed. Moreover, the effects of CR were determined with an in vivo model using ovariectomized (OVX) rats.ResultsCR extract suppressed osteoclastogenesis, its activity and bone resorption activity through decreasing gene of osteoclast-related such as nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-Fos, etc. Moreover, CR extract prevented the bone loss in OVX rats.ConclusionThese results show that CR has a positive effect on menopausal osteoporosis by suppressing osteoclastogenesis.

TRAF6 maintains mammary stem cells and promotes pregnancy-induced mammary epithelial cell expansion

Receptor activator of nuclear factor (NF)-κB (RANK) signaling promotes pregnancy-dependent epithelial cell differentiation and expansion for mammary gland development, which requires NF-κB pathway-dependent Cyclin D1 induction and inhibitor of DNA binding 2 (Id2) pathway-dependent anti-apoptotic gene induction. However, the roles of tumor necrosis factor receptor-associated factor 6 (TRAF6) remain unclear despite its requirement in RANK signaling. Here we show that TRAF6 is crucial for both mammary stem cell maintenance and pregnancy-induced epithelial cell expansion. TRAF6 deficiency impairs phosphoinositide 3-kinase (PI3K)/AKT and canonical NF-κB pathways, whereas noncanonical NF-κB signaling remains functional. Therefore, we propose that TRAF6 promotes cell proliferation by activating PI3K/AKT signaling to induce retinoblastoma phosphorylation in concert with noncanonical NF-κB pathway-dependent Cyclin D1 induction. Furthermore, TRAF6 inhibits apoptosis by activating canonical NF-κB signaling to induce anti-apoptotic genes with the Id2 pathway. Therefore, proper orchestration of TRAF6-dependent and -independent RANK signals likely establishes mammary gland formation.

Oxidation derivative of (-)-epigallocatechin-3-gallate (EGCG) inhibits RANKL-induced osteoclastogenesis by suppressing RANK signaling pathways in RAW 264.7 cells

Tea consumption has positive effects on the skeletal system and prevents postmenopausal osteoporosis, mainly by inhibiting osteoclastogenesis. In green tea, (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and active compound and has been shown to inhibit RANKL-induced osteoclast formation. Taking into account the highly oxidizable and unstable nature of EGCG, we hypothesized that EGCG oxidation product exhibits greater anti-osteoclastogenesis potential than EGCG. In this study, we successfully isolated and identified an EGCG oxidation derivative, (-)-gallocatechin gallate (compound 2), using a chemical oxidation strategy. We then compared the ability of compound 2 and EGCG to inhibit RANKL-induced osteoclastogenesis in RAW 264.7 cells. The results of TRAP staining and F-actin ring immunofluorescent staining showed that compound 2 exhibits stronger inhibition of RANKL-induced osteoclast differentiation and F-actin ring formation, respectively, than EGCG. Additionally, quantitative real-time PCR (qRT-PCR) and western blotting analyses showed that compound 2 significantly and more strongly inhibited the expression of osteoclastogenesis-related marker genes and proteins, including c-Src, TRAP, cathepsin K, β3-Integrin, and MMP-9, compared with EGCG. Furthermore, compound 2 significantly suppressed RANKL-induced expression of NFATc1 and c-Fos, the master transcriptional regulators of osteoclastogenesis, more strongly than EGCG. Mechanistically, molecular interaction assays showed that compound 2 binds to RANK with high affinity (KD = 189 nM) and blocks RANKL–RANK interactions, thereby suppressing RANKL-induced early RANK signaling pathways including p65, JNK, ERK, and p38 in osteoclast precursors. Taken together, this study demonstrates for the first time that an oxidation derivative of EGCG (compound 2) inhibits RANKL-induced osteoclastogenesis by suppressing RANK signaling pathways in RAW 264.7 cells.

Airway M Cells Arise in the Lower Airway Due to RANKL Signaling and Reside in the Bronchiolar Epithelium Associated With iBALT in Murine Models of Respiratory Disease.

Microfold (M) cells residing in the follicle-associated epithelium of mucosa-associated lymphoid tissues are specialized for sampling luminal antigens to initiate mucosal immune responses. In the past decade, glycoprotein 2 (GP2) and Tnfaip2 were identified as reliable markers for M cells in the Peyer's patches of the intestine. Furthermore, RANKL–RANK signaling, as well as the canonical and non-canonical NFκB pathways downstream, is essential for M-cell differentiation from the intestinal stem cells. However, the molecular characterization and differentiation mechanisms of M cells in the lower respiratory tract, where organized lymphoid tissues exist rarely, remain to be fully elucidated. Therefore, this study aimed to explore M cells in the lower respiratory tract in terms of their specific molecular markers, differentiation mechanism, and functions. Immunofluorescence analysis revealed a small number of M cells expressing GP2, Tnfaip2, and RANK is present in the lower respiratory tract of healthy mice. The intraperitoneal administration of RANKL in mice effectively induced M cells, which have a high capacity to take up luminal substrates, in the lower respiratory epithelium. The airway M cells associated with lymphoid follicles were frequently detected in the pathologically induced bronchus-associated lymphoid tissue (iBALT) in the murine models of autoimmune disease as well as pulmonary emphysema. These findings demonstrate that RANKL is a common inducer of M cells in the airway and digestive tracts and that M cells are associated with the respiratory disease. We also established a two-dimensional culture method for airway M cells from the tracheal epithelium in the presence of RANKL successfully. This model may be useful for functional studies of M cells in the sampling of antigens at airway mucosal surfaces.

Clinical Impact of RANK Signalling in Ovarian Cancer.

Ovarian cancer (OC) is a gynaecological malignancy with poor clinical outcome and limited treatment options. The receptor activator of nuclear factor-κB (RANK) pathway, activated by RANK ligand (RANKL), critically controls bone metabolism, tumourigenesis and tumour immune responses. Denosumab, a monocloncal RANKL antibody, exerts tumour-suppressive effects in mice and humans. Here, we investigated the relevance of RANK signalling in OC. RANK, RANKL and OPG expression in 192 epithelial OC tissues was compared to expression in 35 non-malignant control tissues and related to clinico-pathological characteristics. Findings were validated in a cohort of 563 OC patients from The Cancer Genome Atlas (TCGA). The expression of RANK, RANKL and OPG was studied in four OC cell lines and the impact of RANK ligation or blockade on OC cell proliferation was determined. RANK, RANKL and OPG were expressed in epithelial and stromal cells in OC. RANKL expression was elevated in OC tissue, particularly in BRCA1/2 mutated tumours. High RANKL expression independently predicted reduced progression-free (PFS, p = 0.017) and overall survival (OS, p = 0.007), which could be validated in the TCGA cohort (PFS, p = 0.022; OS, p = 0.046, respectively). Expression of RANK and OPG in OC cells was induced by inflammatory cytokines IL-1β and TNFα. Neither recombinant RANK ligation nor denosumab treatment affected OC cell proliferation. Our study independently links RANKL expression with poor clinical outcome in two unrelated OC cohorts. These findings implicate RANK signalling in the immunopathogenesis of OC and warrant clinical trials with denosumab in OC.

Specific RANK Cytoplasmic Motifs Drive Osteoclastogenesis.

Upon receptor activator of NF-κB ligand (RANKL) binding, RANK promotes osteoclast formation through the recruitment of tumor necrosis factor (TNF) receptor-associated factors (TRAFs). In vitro assays identified two RANK intracellular motifs that bind TRAFs: PVQEET560-565 (Motif 2) and PVQEQG604-609 (Motif 3), which potently mediate osteoclast formation in vitro. To validate the in vitro findings, we have generated knock-in (KI) mice harboring inactivating mutations in RANK Motifs 2 and 3. Homozygous KI (RANKKI/KI ) mice are born at the predicted Mendelian frequency and normal in tooth eruption. However, RANKKI/KI mice exhibit significantly more trabecular bone mass than age- and sex-matched heterozygous KI (RANK+/KI ) and wild-type (RANK+/+ ) counterparts. Bone marrow macrophages (BMMs) from RANKKI/KI mice do not form osteoclasts when they are stimulated with macrophage colony-stimulating factor (M-CSF) and RANKL in vitro. RANKL is able to activate the NF-κB, ERK, p38, and JNK pathways in RANKKI/KI BMMs, but it cannot stimulate c-Fos or NFATc1 in the RANKKI/KI cells. Previously, we showed that RANK signaling plays an important role in Porphyromonas gingivalis (Pg)-mediated osteoclast formation by committing BMMs into the osteoclast lineage. Here, we show that RANKL-primed RANKKI/KI BMMs are unable to differentiate into osteoclasts in response to Pg stimulation, indicating that the two RANK motifs are required for Pg-induced osteoclastogenesis. Mechanistically, RANK Motifs 2 and 3 facilitate Pg-induced osteoclastogenesis by stimulating c-Fos and NFATc1 expression during the RANKL pretreatment phase as well as rendering c-Fos and NFATc1 genes responsive to subsequent Pg stimulation. Cell-penetrating peptides (CPPs) conjugated with RANK segments containing Motif 2 or 3 block RANKL- and Pg-mediated osteoclastogenesis. The CPP conjugates abrogate RANKL-stimulated c-Fos and NFATc1 expression but do not affect RANKL-induced activation of NF-κB, ERK, p38, JNK, or Akt signaling pathway. Taken together, our current findings demonstrate that RANK Motifs 2 and 3 play pivotal roles in osteoclast formation in vivo and mediate Pg-induced osteoclastogenesis in vitro.

Oleanolic acid inhibits RANKL-induced osteoclastogenesis via ER alpha/miR-503/RANK signaling pathway in RAW264.7 cells

Oleanolic acid (OA) has recently become a research hotspot in the treatment of many human diseases, especially osteoporosis and arthritis. However, the mechanisms are not elucidated completely. We aimed to elucidate the target and the mechanism via which OA inhibited osteoclast differentiation. We used TRAP staining and toluidine blue dye to test OA effect on osteoclastogenesis and bone resorption respectively. We detected the expression level of osteoclast differentiation related genes, estrogen receptor alpha (ERα) and miR-503. We blocked ERα with its specific blocker, methylpiperidino pyrazole (MPP). We antagonized the function of miR-503 with antagomir-503-5p. RT-PCR and ELISA kits were used to investigate the effects of OA on miR-503 formation and maturation-relevant enzymes Dicer and Drosha at gene and protein levels. The data suggested that OA inhibited osteoclastogenesis and bone resorption. OA upregulated ERα and miR-503 expression levels, inhibited RANK expression. MPP significantly attenuated the OA effect including inhibiting osteoclastogenesis, inhibiting bone resorption and up-regulating miR-503 expression. It showed that ERα was the target of OA and OA up-regulated miR-503 expression through ERα. Antagomir-503-5p inhibited the function of miR-503 and attenuated the inhibition of OA on osteoclastogenesis, suggesting that OA inhibited osteoclast by up-regulating miR-503 expression. In addition, OA up-regulated miR-503 by up-regulating Dicer expression. In conclusion, OA inhibits RANKL-induced osteoclastogenesis via ERα/miR-503/RANK signaling pathway in RAW264.7 cells.

Deciphering the pharmacological mechanism of Guan-Jie-Kang in treating rat adjuvant-induced arthritis using omics analysis.

Traditional Chinese medicine (TCM) formulas have attracted increasing attention worldwide in the past few years for treating complex disease including rheumatoid arthritis. However, their mechanisms are complex and remain unclear. Guan-Jie-Kang (GJK), a prescription modified from "Wu Tou Decoction," was found to significantly relieve arthritis symptoms in rats with adjuvant-induced arthritis after 30-day treatment, especially in the 24 g/kg/day group. By analyzing 1749 targets related to 358 compounds in the five herbs of GJK, we identified the possible anti-arthritis pathways of GJK, including the calcium signaling and metabolic pathways. Bone damage levels were assessed by micro-computed tomography, and greater bone protective effect was observed with GJK treatment than with methotrexate. Receptor activator of nuclear factor κB ligand (RANKL)-RANK signaling, which is related to calcium signaling, was significantly regulated by GJK. Moreover, a target metabolomics assay of serum was conducted; 17 metabolic biomarkers showed significant correlations with treatment. An integrated pathway analysis revealed that pyruvate metabolism, purine metabolism, and glycolysis metabolism were significantly associated with the effects of GJK in arthritis treatment. Thus, this study establishes a new omics analytical method integrated with bioinformatics analysis for elucidating the multi-pathway mechanisms of TCM.

Ablation in Persistent Atrial Fibrillation Using Stochastic Trajectory Analysis of Ranked Signals (STAR) Mapping Method.

The aim of this study was to demonstrate that a stochastic vector-based mapping approach could guide ablation of atrial fibrillation (AF) drivers as evidenced by ablation response and long-term follow-up outcomes. The optimal method for mapping and ablation of AF drivers is yet to be defined. Patients undergoing persistent AF ablation were recruited. Patients underwent pulmonary vein isolation (PVI) with further ablation guided by the stochastic trajectory analysis of ranked signals (STAR) mapping method. The proportion of the time an electrode's activation was seen to precede its neighboring electrodes activation was used to determine early sites of activation (ESA). A positive ablation response at ESA was defined as AF termination or cycle length slowing of≥30ms. Clinical outcome was defined as recurrence of AF/atrial tachycardia (AT) during a follow-up of 12months. Thirty-five patients were included (AF duration of 14.4 ± 5.3months). After PVI, an average of 2.6 ± 0.8 ESA were ablated per patient with study-defined ablation response achieved in all patients. Of the 86 STAR maps created post-PVI, the same ESA was identified on 73.8 ± 26.1% of maps. ESA that resulted in AF termination were more likely to be identified on both pre- and post-PVI maps than those associated with cycle length slowing (23 of 24 vs. 16 of 49; p<0.001). During a follow-up of 18.5 ± 3.7months, 28 (80%) patients were free from AF/AT. The ablation response at ESA suggests they may be drivers of AF. Ablation guided by STAR mapping produced a favorable clinical outcome and warrants testing through a randomized controlled trial. (Identification, Electro-mechanical Characterisation and Ablation of Driver Regions in Persistent Atrial Fibrillation [STAR MAPPING]; NCT02950844).

Supplemental Material for Two Signals of Social Rank: Prestige and Dominance Are Associated With Distinct Nonverbal Displays

Supplemental Material for Two Signals of Social Rank: Prestige and Dominance Are Associated With Distinct Nonverbal Displays

Development, in vitro validation and human application of a novel method to identify arrhythmia mechanisms: The stochastic trajectory analysis of ranked signals mapping method.

IntroductionStochastic trajectory analysis of ranked signals (STAR) is a novel method for mapping arrhythmia. The aim was to describe its development and validation as a mapping tool.Methods and ResultsThe method ranks electrodes in terms of the proportion of the time they lead relative to neighboring electrodes and ascribes a predominant direction of activation between electrodes. This was conceived with the aim of mapping atrial fibrillation (AF) drivers. Validation of this approach was performed in stages. First, in vitro simultaneous multi‐electrode array and optical mapping were performed on spontaneously fibrillating HL1 cell cultures, to determine if such a method would be able to determine early sites of activation (ESA). A clinical study acquiring unipolar electrograms using a 64‐pole basket for the purposes of STAR mapping in patients undergoing atrial tachycardia (AT) ablation. STAR maps were analyzed by physicians to see if arrhythmia mechanisms could be correctly determined. Mapping was then repeated during atrial pacing. STAR mapping of in vitro activation sequences accurately correlated to the optical maps of planar and rotational activation. Thirty‐two ATs were mapped in 25 patients. The ESA accurately identified focal/micro‐reentrant ATs and the mechanism of macro‐reentrant ATs was effectively demonstrated. STAR method accurately identified four pacing sites in all patients.ConclusionsThis novel STAR method correlated well with the gold standard of optical mapping in vitro and was able to accurately identify AT mechanisms. Further analysis is needed to determine whether the method might be of use mapping AF.