Airway remodeling (AR) is a progressive pathological feature of the obstructive lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). The pathology manifests itself in the form of significant, progressive, and (to date) seemingly irreversible changes to distinct respiratory structural compartments. Consequently, AR correlates with disease severity and the gradual decline in pulmonary function associated with asthma and COPD. Although current asthma/COPD drugs manage airway contraction and inflammation, none of these effectively prevent or reverse features of AR. In this review, we provide a brief overview of the features and putative mechanisms affecting AR. We further discuss recently proposed strategies with promise for deterring or treating AR. Furthermore, the gradual deleterious transformation in lungs can affect airways of all sizes along the bronchial tree. Although the strategies for reversing airway contraction and mitigating airway inflammation have been mainstays of asthma therapy, AR has been clinically intractable. Consequently, a pressing need exists for defining the fundamental pathways contributing to AR pathology and for empowering both basic and clinical research to address this problem. For a comprehensive understanding of the conceptual and practical challenges in AR research, readers are encouraged to review the official research statement of the American Thoracic Society2. In this current report, we provide an overview of the limitations of currently approved anti-asthma/chronic obstructive pulmonary disease (COPD) drugs in addressing AR and further describe the therapeutic potential of recently proposed approaches for targeting AR.Airway remodeling (AR) can be defined as a progressive pathological reorganization of the cellular and molecular constitution of the airway wall. While the onset and rate of progression of structural changes in the airways have been subjects of immense debate, AR has been associated with each of the asthmatic phenotypes3. Numerous subsequent clinical investigations have revealed that AR encompasses broad structural changes in the airway that includes thickening of the airway wall, airway smooth muscle (ASM) hyperplasia and hypertrophy, edema, subepithelial fibrosis, increased extracellular matrix (ECM) deposition, immune cell and fibroblast accumulation, angiogenesis, altered matrix composition, goblet cell metaplasia, and mucus hypersecretion2. A consensus has emerged that multiple cell types  contribute to the development of AR in asthma and COPD5  suggest that reducing ASM area is sufficient to improve outcomes in asthmatics27.Physiological ASM function is crucial for maintaining adequate airflow. Changes in both ASM responsiveness and morphology occur with asthma, which affects airway resistance and airflow. A critical feature of AR is an increase in ASM mass that contributes significantly to asthma pathology29. Beyond contributing to increased airway wall thickness, ECM components can modulate cellular proliferation and migration. However, the role of fibroblast and ECM components in AR in the context of obstructive lung diseases is not fully understood.Fibroblasts can contribute to AR through increased secretion of ECM32. A correlation between AR and disease severity has been established, but the clinical consequences of AR are yet to be fully understood38. This lack of knowledge also impacts drug discovery efforts. In the subsequent sections, we review the efficacy of current therapeutics in blunting or reversing AR and discuss novel therapeutic approaches to regulate progression of AR.The structural changes may contribute toward a gradual decline in lung function and potentially in loss of pulmonary elasticity, leading to hyperinflation and air trapping in lungs. Moreover, remodeling reduces effectiveness of bronchodilatory treatmentsCurrent management of asthma focuses on reversing ASM contraction and mitigating airway inflammation. None of these approaches directly addresses the progressive pathology that causes remodeling in the lung .40. This procedure has been shown to significantly reduce collagen deposition in the basement membrane. Although bronchial thermoplasty has been shown to improve quality of life for severe asthmatics in the short term, the cost of the procedure, post-procedure exacerbations, and questions regarding long-term efficacy have limited its application41.As noted earlier, bronchial thermoplasty has been shown to reduce ASM mass in conducting airways of some, but not all, severe asthmatics undergoing the procedure5. Signaling through cysteinyl leukotriene receptors (CysLTRs) and muscarinic acetylcholine receptors (mAChRs) has been established to promote outcomes that contribute to AR44. Antagonists of both receptors have shown some utility in preventing AR. Treatment with the CysLTR antagonist montelukast reversed ovalbumin-induced AR by decreasing goblet cell metaplasia, ASM mass, and subepithelial collagen deposition46. In a cohort of mild asthmatics, montelukast treatment showed reduced accumulation of myofibroblasts in the airway wall, suggesting some potential to mitigate AR47. Similarly, the long-acting mAChR antagonist, tiotropium, has demonstrated a robust ability in preventing AR in rodent (guinea pig and mouse) models of ovalbumin-induced asthma and lipopolysaccharide-induced COPD52. Overall, although some evidence suggests that mAChR and CysLTR antagonists may have utility in deterring AR, additional studies in humans are necessary to establish the true effectiveness of these drugs in preventing or reversing AR5.Among pharmacological options, β-agonists are the drug of choice for evoking bronchorelaxation in attempting to reverse an acute asthma attack or for providing bronchoprotection when used in combination with an inhaled corticosteroid as a maintenance therapy. However, there is no compelling evidence that β-agonists deter or reverse AR54.In vitro, multiple corticosteroids have been shown to significantly inhibit fibroblast proliferation either alone or in combination with β-agonists56. Similar anti-proliferative effects have also been reported with corticosteroids in ASM cells stimulated with distinct mitogenic agents58. However, others have shown that corticosteroids have no effect on ASM proliferation60. While corticosteroids inhibit growth factor–stimulated proliferation of ASM cells sourced from healthy controls, this effect was lacking on ASM cells from asthmatics59. In animal models, dexamethasone has been shown to reduce goblet cell metaplasia; however, this treatment showed no effect on ASM mass and subepithelial fibrosis45. In humans, in conjunction with limiting inflammation and airway hyperresponsiveness, corticosteroid treatment can also reduce mucin secretion and limit ECM deposition and AR64. However, others have shown that corticosteroids have a mixed effect on the resolution of subepithelial fibrosis70. Collectively, studies to date indicate a need for developing better therapeutic drugs for targeting AR pathology in obstructive lung diseases.Persistent asthma is commonly treated with inhaled corticosteroids either as a monotherapy or in combination with a β-agonist or mAChR antagonist. In epithelial cells, corticosteroids limit the inflammatory response and induce apoptosisIn recent years, basic science research has begun to provide insight into the mechanisms, mediated by multiple cell types, that promote AR and these studies help to inform potential strategies for managing AR. Certain approaches that show promise in mitigating features of AR have recently been proposed .G protein–coupled receptors (GPCRs) play a substantial role in numerous normal physiological functions. Unsurprisingly, they can contribute towards the pathophysiology of various diseases. As noted earlier, GPCR agonists (of the β2-adrenergic receptor) and antagonists (of the mAChRs and CysLTR) are principal drugs in the management of asthma and COPD. In this section, we provide a brief overview of novel targets, the drugs that modulate them, and the potential of such drugs to address AR pathology.E-prostanoid receptor agonists. The role of prostaglandin E2 (PGE2) and E-prostanoid (EP) receptor subtypes in mitigating AR has been a subject of recent research. Early studies demonstrated that autocrine PGE2, generated as a consequence of cytokine-induced cyclooxygenase-2 induction, significantly suppresses mitogen-induced ASM proliferationin vitro72. Furthermore, studies of cultured human ASM in our laboratory have demonstrated that exogenous PGE2 shows relatively superior anti-mitogenic activity in comparison to multiple β-agonists with anti-mitogenic effects corresponding to drug efficacy in activating the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) axis74. Application of PGE2 in humans has been hindered by the ability of PGE2 to signal through multiple receptor subtypes (EP1–4)79, which contributes to undesirable side effects. It is now known that the cough response is mediated by PGE2 activation of the EP3 receptor on vagal sensory nerves80. Additionally, studies in our lab have shown that EP3 receptor signaling has a pro-mitogenic role in ASM74. To overcome the heterogeneity of PGE2 signaling, the development of EP receptor subtype–specific modulators that specifically promote Gs-cAMP-PKA axis activity (via EP2 and EP4 subtypes) has been purposed114. Currently, our lab is evaluating various strategies of targeting specific EP receptor subtypes in pre-clinical models of allergen-induced asthma84.Bitter taste receptors. Recently, our laboratory showed that bitter taste receptor (TAS2R) agonists can limit proliferation of ASM cellsin vitro82. Mechanistically, TAS2R agonists restrict ASM proliferation by inhibiting (1) the growth factor–activated protein kinase B (Akt) phosphorylation; (2) transcription factors AP-1, STAT3, E2 factor, and NFAT; and (3) genes associated with cell cycle progression.83. Specifically, treatment with bitter compounds inhibited the expression of calponin, smooth muscle alpha-actin, and smooth muscle myosin heavy chain in lungs. Furthermore, levels of matrix metallopeptidase-8 (MMP-8) (neutrophil collagenase), pro-MMP-9 (gelatinase), and MMP-12  were significantly reduced in lungs following treatment with TAS2R agonists. Finally, allergen-induced expression of pro-fibrotic cytokine transforming growth factor-beta (TGF-β) as well as phospho-mothers against decapentaplegic homolog 2 (pSmad2) and fibronectin in the lung tissue was also curtailed by TAS2R agonists. Collectively, these studies indicate that TAS2R agonists, unlike current GPCR ligands used to treat asthma, address multiple features of asthma pathology, including AR. Advancements in the development of selective ligands for TAS2R subtypes will allow for a refined therapeutic approach in the near future.The anti-mitogenic effects of TAS2R agonists further translate to pre-clinical asthma models as well. In a chronic allergen  challenge model, treatment with bitter taste compounds (chloroquine and quinine) significantly reversed remodeling features115. Specifically, the motile cilia on human airway epithelia express TAS2Rs . The organization of TAS2Rs on cilia with the distribution of the signaling machinery along the ciliary shaft and within the attached epithelial cell presents an interesting mechanical apparatus for signal transduction. Stimulation of TAS2Rs with bitter tastants induces transient Ca2+ flux within the epithelial cells and increases ciliary beat frequency. Functionally, promoting increased ciliary movement could be beneficial in the removal of excess mucus from the airways.Bitter tastants have also been shown to modulate function of ciliated epithelial cells117. Specifically, a role for autophagy/mitophagy in regulating mitochondrial function in pathophysiology of obstructive lung diseases is emerging. As noted earlier, our studies show that activation of TAS2Rs can promote anti-mitogenic activities. Further explorations into the mechanisms that underlie the anti-mitogenic effects of TAS2R agonists have uncovered an interesting role for bitter tastants in altering mitochondrial function and inducing autophagy82. TAS2R agonists can induce changes in mitochondrial membrane potential, increase reactive oxygen species generation, and promote mitochondrial fragmentation. These observations provide insight into the broader therapeutic potential of targeting mitochondrial function and promoting autophagy to restrict cellular proliferation.In recent years, the role of mitochondrial dysfunction in disease states, including obstructive lung diseases, has become increasingly clear118. Consequently, in recent years, therapies that target specific cytokines or immune cells to disrupt immune networks responsible for asthma pathology have gained significant interest. Targeting key cytokines with specific antibodies—biologics, including antibodies targeting interleukin-4 (IL-4), IL-5, and IL-13—can significantly limit recruitment of inflammatory cells to the lungs or blunt their pleiotropic effects. For instance, anti–IL-5 treatment can significantly reduce the number of circulating eosinophils in asthmatics and improve lung function121. Anti–IL-5 treatment has been shown to prevent the development of subepithelial fibrosis in a murine model of asthma and reduce incorporation of proteoglycans in the human airway wall86. However, antibodies targeting other cytokines or their receptors have not been studied in the context of AR122. Collectively, the data on the effects of biologics on AR are lacking and this is possibly due to the relatively recent development of these drugs. Future longitudinal studies that evaluate biologics in the context of remediation of AR features are needed to address the utility of these drugs as anti-AR agents.Asthma pathology is orchestrated by multiple immunologic mediators (cellular and secreted)123. Biologics that block the interactions of IgE antibodies to the high-affinity FcεRI receptors on mast cells and antigen presenting cells can curtail the sensitization profile in asthmatics and reduce exacerbations. In severe asthmatics evaluated for 36 months, blocking IgE activity was sufficient to reduce the thickening of the reticular lamina, thereby having an impact on AR87.Allergen-specific immunoglobulin E (IgE) isotype antibodies can cross-link on mast cells and basophils, causing degranulation and release of histamine, cytokines, and growth factors126. Application of cytokine-specific antibody therapy is limited by the heterogeneity of asthma phenotypes127. Non-atopic asthmatics are also not suitable for certain therapy . Biologics can also cause side effects such as hypersensitivity reactions, although the underlying mechanisms are unclear. Finally, there is also significant cost associated with the use of biologics.Although biologics have become an increasingly important tool in the management of severe asthma, their application in the clinic has some drawbacks128. In this section, we provide a brief overview of drugs that target distinct kinases. For a more comprehensive discussion of targeting kinases in the context of obstructive lung diseases, the reader is referred to129.Kinase enzymes modulate multiple cellular functions by regulating various signaling networks, including those regulating cellular proliferation and growth. Consequently, inhibitors that target various kinases have received increasing attention. Modulators of kinase functions account for one third of drugs in the development pipeline, and the majority of these represent cancer therapeuticsMitogen-activated protein kinase inhibitors. Mitogen-activated protein kinases (MAPKs) have been studied extensively for their contribution to inflammatory gene expression and activation of multiple networks that contribute to the pathophysiology of obstructive lung diseases130. Extracellular signal-regulated kinases (ERK1/2) are particularly interesting given that they are activated in multiple cell types that contribute to asthma and COPD pathology132. Inhibition of ERK kinase  which is upstream of ERK1/2 can significantly reduce mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) expression in cultured human bronchial epithelial cells subjected to chronic mechanical stress at the air-liquid interface89. Other MAPKs, such as p38, c-Jun N-terminal kinase (JNK), and transforming growth factor beta-activated kinase 1 (TAK1), are activated in asthma and COPD129, and inhibitors of these targets can mitigate various features of AR in both cell and animal asthma models95. However, to date, no studies in humans have evaluated these inhibitors. Another limitation of kinase inhibitors is that these compounds are predominantly inhibitors of ATP-competitive and catalytic sites and block all enzymatic activity, including MAPK functions important for normal physiological activity in cells. Given the ubiquitous functions of the MAPK signaling pathway, development of substrate-selective MAPK inhibitors with the goal of targeting specific kinase functions associated with disease, while preserving kinase functions in normal cells, appears necessary to overcome limitations of off-target effects.Receptor tyrosine kinase inhibitors. Receptor tyrosine kinases (RTKs) occupy a central role in critical signaling networks that promote asthma pathology, including remodeling133. With inflammation, distinct RTKs and their ligands  are upregulated in human asthmatic airways and show a strong correlation with disease severity143. RTKs can stimulate pathophysiological functions in ASM and epithelial cells. Thus, significant interest in advancing tyrosine kinase inhibitors for targeting RTKs has developed.144. It is also responsible for sustaining oxidative damage in the epithelial compartment through recruitment of neutrophils in a TGF-β–dependent manner148. EGFR inhibitors tyrphostin AG1478 and BIBX1522 have been evaluatedin vitro and in animal models of lung inflammation101. Collectively, these studies report significant reductions in expression of mucus-associatedMUC5AC gene and mucin secretion. More importantly, there is a concomitant reduction in collagen deposition and ASM proliferation98. Although these observations are encouraging, some inhibitors of EGFR have failed to produce similar outcomes in clinical studies149. Activation of platelet-derived growth factor receptor (PDGFR) has been shown to stimulate ASM proliferationin vitro andin vivo151. Multiple drugs targeting PDGFR can mitigate ASM proliferationin vitro, although animal and human studies that address AR are lacking104. In severe corticosteroid-dependent asthmatics, treatment with the tyrosine kinase inhibitor mastinib has shown improved outcomes; however, some adverse effects, including skin rash and edema, have also been reported152. During airway inflammation, multiple cell types (immune and resident) can be stimulated to secrete angiogenic factors, including vascular endothelial growth factor (VEGF)159. These angiogenic factors can further stimulate increase in formation of new blood vessels from endothelial cells in the subepithelial mucosa162. Although the contribution of neovascularization in AR is unclear, it has been suggested that newly formed vasculature is permeable, thus contributing to edema and limiting airflow164. An antagonist of VEGFR-1 and VEGFR-2 (SU5416) has been shown to limit inflammatory responses in animals165, although its impact on AR is unknown and studies in humans are lacking.Activation of epidermal growth factor receptor (EGFR) is essential for mucus secretion and goblet cell metaplasia166. This approach could be useful in targeting multiple redundant RTK networks that contribute to AR. Finally, inhibitors of non-RTKs, such as stem cell growth factor receptor (c-kit), spleen tyrosine kinase (SYK), the proto-oncogene tyrosine-protein kinase Src, and Janus kinase (JAK), have also been investigated in rodent models of asthma but have not yet progressed to studies in humans171.Because multiple RTKs contribute to pathology of AR, a novel strategy that targets multiple RTKs has gained momentum. In a pre-clinical murine model of ovalbumin-induced asthma, treatment with nintedanib—a small-molecule inhibitor that targets multiple RTKs —significantly improved indices of remodeling and airway inflammationOther kinase inhibitors. Diverse stimuli  can activate the transcription factor nuclear factor-kappa B (NF-κB) in multiple airway cell types. This transcription factor plays a key role in orchestrating immune responses and thus multiple intra- and inter-cell inflammatory signals129. Although inhibitors that target activation of NF-κB have been shown to suppress certain synthetic functions of ASM172 and modulate pro-inflammatory outcomes in epithelial cells173, specific NF-κB inhibitors have not translated into clinical trials for asthma and this is due to their multiple side effects129. Inhibitors of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI-3K) that regulate cellular lipids and coordinate inflammatory pathways have undergone extensive investigation in asthma and COPD175. However, data assessing AR indices are lacking. TGF-β plays an important role in cellular proliferation and differentiation and its expression increases in asthmatic airways, especially in the submucosal compartment178. TGF-β has also been implicated in AR and can promote proliferation in ASM180. TGF-β activates TGF-β receptor type I (T-βRI) kinase, which in turn activates Smad-dependent signaling that regulates expression of various genes. Small-molecule inhibitors of (T-βRI) kinase have yielded mixed results in studies assessing their effects on mechanisms mediating AR. T-βRI kinase inhibitors have been shown to diminish collagen deposition in lungs of rats challenged repeatedly with an allergen108.In vitro, T-βRI inhibitors have demonstrated the ability to limit ASM proliferation, although their use in animal studies failed to inhibit TGF-β–induced increases in ASM mass181. Clinical application of these inhibitors has been limited due to adverse effects, including cardiotoxicity in clinical trials for cancer therapy182. Protein kinase C (PKC) is another target of interest given its ability to promote contractile signaling in ASM following activation of Gq-coupled GPCRs. PKC is also relevant to AR because of its role in ASM proliferation183 and mucus secretion in epithelium148. Owing to severe toxicity, non-selective inhibitors of PKC have not progressed to clinical trials184. Selective inhibitors of PKC isoforms have been developed for clinical studies for treatment of cancer, metabolic diseases, and psychiatric disorders, although adverse effects have been problematic and no clinical trials have been conducted for treatment of obstructive lung diseases in humans184. Finally, Rho-associated protein kinase (ROCK) inhibitors have been shown to mitigate multiple features of asthma, including AR in guinea pig and murine models185. Although ROCK inhibitors have been approved for certain indications, clinical trials in asthma or COPD are lacking129 as issues regarding selectivity and toxicity have limited progression of these inhibitors to clinical application in airway diseases129.in vitro studies of pharmacological inhibition of multiple kinases that contribute to dysfunction in ASM and epithelium have yielded promising results186. However, certain limitations have stalled progression of many drugs for clinical use. Specificity, efficacy, solubility issues, and poor pharmacokinetic profiles plague drug development187. With chronic inhibitor treatment, compensatory signaling by other kinases may limit drug efficacy; this appears to be the case with p38 isoform inhibitors188. Inhibition of any widely expressed kinase runs the risk of adverse effects. For example, given that NF-κB is crucial for mounting an immune response to microbial pathogens, blocking its activation could render patients susceptible to life-threatening infections129. Current challenges in developing effective and safe kinase inhibitors hinge on improving the poor solubility, selectivity, and targeting of the current versions of these drugs129.In summary,110. Inhibition of PDE3 (but not PDE4) has anti-proliferative action on mitogen-activated human ASM cellsin vitro, but as with most potential anti-AR drugs, usefulin vivo data are lacking111. More recently, an inhibitor of PDE8 (PF-04957325) has been shown to regulate proliferation of ASM cells by enhancing cAMP accumulation generated specifically from the β2-AR/AC6 pathway189.Phosphodiesterase (PDE) inhibitors have a beneficial effect of promoting ASM relaxation by increasing intracellular cAMP resulting in PKA-mediated ASM relaxation. In murine models of asthma, PDE inhibitors have also been shown to curtail inflammation and reduce ARin vitro and mitigate inflammation and ARin vivo in ovalbumin-challenged mice190, yet no clinical studies for asthma or COPD have been reported.The rapamycin derivative SAR-943 has been shown to limit the mitogen-induced proliferation of human ASM cells The correlation between AR and obstructive lung disease severity suggests a strong pathogenic role of AR in these diseases. Thus, remediation of AR appears critical for improving the severity and progression of these diseases. A growing arsenal of small-molecule inhibitors and biologics in conjunction with non-pharmacological interventions such as bronchial thermoplasty has shown promise in addressing this unmet clinical need. As our understanding of mechanisms underlying AR improves, so will the drug development approaches as well as the phenotyping capabilities that accurately assess AR in humans. These advances will undoubtedly fulfill our need for more refined, efficacious, and safer drugs that enable us to finally control the entire spectrum of asthma pathology.2-AR, beta 2 adrenergic receptor; ASM, airway smooth muscle; cAMP, cyclic adenosine monophosphate; c-kit, Stem cell growth factor receptor; COPD, chronic obstructive pulmonary disease; CysLTR, cysteinyl leukotriene receptor; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; EP, E prostanoid; ERK1/2, extracellular signal-regulated kinases 1/2; GPCR, G protein–coupled receptor; FGFR, fibroblast growth factor receptor; ICS, inhaled corticosteroids; IgE, immunoglobulin E; IL, interleukin; JAK, Janus kinase; JNK, c-Jun N-terminal kinase; mAChR, muscarinic acetylcholine receptor; MAPK, mitogen-activated protein kinase; MEK1, mitogen-activated protein kinase kinase 1; MMP, matrix metalloproteinase; MUC5AC: mucin 5AC, oligomeric mucus/gel-forming; NF-κB, nuclear factor-kappa B; PDE, phosphodiesterase; PDGFR, platelet-derived growth factor receptor; PGE2, prostaglandin E2; PI-3K, phosphatidylinositol-4,5 bisphosphate 3-kinase; PKA, protein kinase A; PKC, protein kinase C; ROCK, Rho-associated proteinase kinase; RTK, receptor tyrosine kinase; Smad2: Mothers against decapentaplegic homolog 2; Syk, spleen tyrosine kinase; TAK1, Transforming growth factor-β-activated kinase 1; TAS2R, bitter taste receptor; TGF-β, transforming growth factor-beta; T-βRI, transforming growth factor-beta receptor type I kinase; VEGFR; vascular endothelial growth factor receptorAR, airway remodeling; β


Androgen receptor splice variant V7 (AR-V7) was recently identified as a valuable predictive biomarker in metastatic castrate-resistant prostate cancer. Here, we report a new, sensitive and accurate screen for AR-V7 mRNA expression directly from circulating tumor cells (CTCs): We combined EpCAM-based immunomagnetic CTC isolation using the IsoFlux microfluidic platform with droplet digital polymerase chain reaction (ddPCR) to analyze total AR and AR-V7 expression from prostate cancer patients CTCs. We demonstrate that AR-V7 is reliably detectable in enriched CTC samples with as little as five CTCs, even considering tumor heterogeneity, and confirm detection of AR-V7 in CTC samples from advanced prostate cancer (PCa) patients with AR-V7 detection limited to castrate resistant disease status in our sample set. Sensitive molecular analyses of circulating tumor cells (CTCs) or circulating tumor nucleic acids present exciting strategies to detect biomarkers, such as AR-V7 from non-invasive blood samples, so-called blood biopsies. The insight that cancers, even of the same type, show strong inter- and intra-patient heterogeneity has emerged in recent years. Tumor biomarkers are most commonly conceptualized as specific cellular, biochemical or molecular alterations that characterize heterogeneous subcategories of cancers. Consequently, patient management increasingly relies on the detection of such tumor biomarkers to predict prognosis, guide therapies and monitor treatment response as well as the development of specific resistance mechanisms . The numSome serum proteins, such as the prostate specific antigen (PSA) in prostate cancer (PCa) and carcinoembryonic antigen (CEA) in colorectal cancer, are well established biomarkers and clinically widely used, although their correlation with other disease progression parameters is not always ideal ,3. More Traditionally, such molecular biomarkers are examined in excised tumor tissue or fine needle biopsies at a single timepoint in the disease course. However, such biopsies are not always available or informative . To progIn PCa, genetic changes in the androgen receptor (AR), such as point mutation and gene amplification, render the receptor independent of upstream testosterone levels and cause resistance against androgen deprivation therapy (ADT), which is the predominant first line therapy for advanced disease . Thus faHere, we present development of a reliable droplet digital PCR based method to detect AR-V7 and total AR expression in PCa patient CTCs enriched by the IsoFlux system. Our assay has high specificity and sensitivity and detects AR-V7 expression in as little as five AR-V7 PCa cells spiked to produce a modeled CTC sample, and was confirmed in PCa patient CTCs.Droplet digital PCR (ddPCR), utilizing Taqman PCR principles, is a novel and sensitive method to detect rare mutations, gene expression and copy number variations in samples with limited amounts of nucleic acid templates . To optiTo test assay specificity we determined total AR and AR-V7 expression in a cohort of PCa cell lines: 22Rv1, VCaP, C4-2, LNCaP, C4-2B, LAPC4, and PC3 and the b-lymphocyte line WME-099 . AmongstOne mammalian cell contains approximately 10–30 pg of total RNA dependent on cell type and physiological state . To testp = 0.008).The ddPCR assay was validated by testing twenty six CTC samples from twenty-four PCa patients after IsoFlux CTC isolation for expression of AR-V7. AR-V7 was detected in 30.8% of CTC samples (8/26): no AR-V7 was detected in any (0/10) of the HSPC (hormone sensitive prostate cancer) samples  whereas AR-V7 was detected in 50% 8/16) of CRPC samples. Detection of AR-V7 in CTC positive patient samples is within the range reported by others for qPCR AR-V7 assays (29%–55%) and above that found by immunocytostaining 18%)  of CRPC ,18,27. A%  of CRP2 at 37 °C.The human PCa cell lines LNCaP, VCaP, C4-2, C4-2B, PC3, LAPC, 22Rv1, as well as the human b-lymphocyte cell line WME-099, were either recently purchased  or authenticated . Cells were maintained in RPMI media  supplemented with 10% FBS  in a humidified incubator with 5% COTwenty-four patients diagnosed with high risk PCa and positive for CTCs were recruited at Liverpool Hospital. Of the twenty-four patients, twenty-two had metastatic disease, with the remaining two having biochemical recurrent disease (Gleason grade 7 or greater). All patients and healthy blood donors gave informed consent to participate in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the South Western Sydney Local Health District Ethics Committee . Whole blood from patients or healthy donors was drawn in 9 mL K3E K3EDTA-tubes  after discarding the first 2 mL blood to avoid contamination with epithelial skin cells. Patients were considered to have CRPC if they had experienced new metastases, progression of metastases or a rise in PSA despite adequate castration serum testosterone levels <1.7 nmol/L .CTC enrichment was performed with the IsoFlux CTC enrichment kit  according to manufacturer’s instructions. Briefly, PBMCs were separated from 8 mL of blood using 50 mL SepMate tubes and Lymphoprep  according to the manufacturer’s instructions. PBMCs were once washed with PBS and transferred into a 1.5 mL Low-Protein-Bind tubes  with 40 µL anti-human EpCAM conjugated immunomagnetic beads and 40 µL Fc blocker and incubated at 4 °C with slow agitation for 1.5 h. Cells were then loaded into isolation cartridges and CTCs were isolated using the IsoFlux CTC enrichment system and run using the standard separation protocol (Fluxion). Isolated CTCs were either enumerated or frozen at −80 °C for later down-stream analysis.CTCs were immunocytostained for the presence of cytokeratin, CD45 and nuclei by Hoechst dye using the IsoFlux CTC enumeration kit (Fluxion) according to the manufacturer’s instructions. CTCs mounted in 24-well glass bottom plates were visualized and scanned with Olympus Ix71  mounted with an automated stage ProScan III (PRIOR) (10× objective). The exposure time for Hoechst, CD45 and cytokeratin are 2, 200 and 400 ms, respectively. CD45 negative cells with positive Hoechst and cytokeratin staining were considered to be CTCs and counted manually from scanned images. Total cell numbers  were also determined in the Hoechst channel using the Olympus CellSens Software “count and measure” plugin .2O. RNA quality and quantity were measured using the NanoDrop 2000 . cDNA synthesis was performed from 1 µg total RNA with the SensiFAST cDNA Synthesis kit . Total RNA from IsoFlux CTC samples or healthy control PBMCs was extracted with the RNA purification Micro kit  and double-eluted in a total volume of 30 µL RNase-free H2O. 15 µL of this RNA was converted into cDNA with the SensiFAST cDNA Synthesis kit (Bioline). Healthy donor PBMCs consisted of 4000 cells to mimic IsoFlux CTC samples.Total RNA from cell lines was extracted with the ISOLATE II RNA Mini Kit  and any residual genomic DNA contamination was removed by on-column DNAse I treatment for 15 min. RNA was eluted in 50 µL RNase-free HPrimers and Taqman probes were designed using NCI primer software . ddPCR sSingle cells were isolated using the CellCelector  as described before . In brieTo establish the relationship between hormone sensitivity and AR-V7 detection, we performed a Fisher’s Exact Test using SPSS Statistics (IBM) software package version 23 .We developed a specific and sensitive ddPCR-based assay to determine AR-V7 expression in PCa CTC samples that can reliably detect AR-V7 expression from CTC positive patient samples. The advantage of the ddPCR based assay is that it determines not only AR-V7 positivity but actual transcript copy numbers and that allows in some instances to detect heterogeneity of AR-V7 gene expression without further separation of individual CTCs from residual lymphocytes and other CTCs after immunomagnetic CTC isolation using the IsoFlux platform. Although we were able to detect AR-V7 from a single cell, we conservatively estimate that AR-V7 can be reliably determined in samples with at least five CTCs, as this takes into account heterogeneity of AR-V7 expression as evident in two of our patient samples (patient 18 and 19). In our small proof of concept patient set, association of AR-V7 detection was significantly associated with development of CRPC and we are currently screening larger patient cohorts to confirm the robustness of our approach and association with clinical parameters. Our assay is likely adaptable for CTCs isolated by other methods or for circulating tumor RNA, which can be isolated from processed plasma samples.


P < 0.01). The multivariate analysis indicated that the posterior AR jet was independently associated with the AML/PML area (P < 0.01). 3D TEE depicted geometric differences in the MV apparatus between the different types of AR jet directions. These results may be helpful in understanding the mechanism of MV leaflet remodeling in chronic AR.Chronic aortic regurgitation (AR) induces mitral valve (MV) leaflet enlargement, although, its mechanism still remains unclear. This study aimed to clarify the influence of AR jet directions on the MV apparatus in patients with chronic AR. This study included 69 consecutive patients with severe chronic AR and 17 controls who underwent three-dimensional (3D) transesophageal echocardiography (TEE). The anterior mitral leaflet (AML), posterior mitral leaflet (PML) and MV annulus areas were measured at mid-diastole. All AR patients were classified into the posterior  or non-posterior  group based on the AR jet directions. Both two groups revealed the increased total leaflet areas compared with the controls. No significant differences in the left ventricular volumes, PML or MV annulus area were observed between Group A and B; however, Group A had the larger AML area and greater AML/PML area ratio than Group B (both  AR is characterized by the reflux of blood from the aorta into the left ventricle (LV). The overall prevalence of AR detected by color Doppler echocardiography in adults has been reported 4.9% in the Framingham Heart Study1 and 10% in the Strong Heart Study2. These studies have showed that age is an independent predictor of AR; hence, the increased prevalence of AR is presumed in developed countries2. Mitral valve (MV) enlargement in patients with chronic AR has been previously reported3. Beaudoin et al.4 demonstrated that MV leaflet enlargement in patients with chronic AR decreased functional mitral regurgitation (MR) and speculated that LV chamber dilatation was the main cause of MV leaflet growth as an adaptation for MV annulus enlargement7. In fact, patients with AR are often observed less MR considering their large LV chamber sizes. However, the detailed mechanisms of MV apparatus morphological changes in patients with chronic AR remain unclear. We often observe the asymmetrical growth of the MV in patients with AR, particularly in patients with severe eccentric posterior jets. According to these experiences, we assumed that the MV remodeling in patients with AR should be varied by the directions of AR jets. Today, three-dimensional (3D) transesophageal echocardiography (TEE) can provide a detailed quantification of the MV apparatus, including MV annulus area, anterior mitral leaflet (AML) and posterior mitral leaflet (PML) areas, and MV opening area13. We hypothesized that the directions of AR jets affect the MV apparatus and trigger AML enlargement in patients with chronic AR. Here, this study aimed to investigate the MV apparatus by using 3D TEE to understand the mechanism of MV morphological changes in patients with chronic AR.Aortic valve diseases, such as aortic regurgitation (AR) and aortic stenosis, have become relatively common in aging developing countriesThis study consisted of 103 consecutive patients with severe AR and 17 normal controls who underwent two-dimensional (2D) and 3D TEE between February 2012 and November 2016. The exclusion criteria included insufficient TEE image qualities (n = 9), MV organic pathology , acute AR caused by infective endocarditis and aortic root dissection (n = 8), post-balloon aortic valvuloplasty (n = 4), and prosthetic mitral and aortic valves (n = 1). Ultimately, 69 patients and 17 controls were registered  for quantifying MV geometry. The MV leaflets and annulus were traced at mid-diastole and the area of each leaflet and the MV annulus were quantified in all patients  displaying both 2D and real-time 3D images. Under topical anesthesia in the pharynx and intravenous sedation with propofol, the transducer was advanced into the esophagus. The directions of AR jets from the mid-esophageal position with a 2D long-axis view (approximately 135°) of the aortic valve and MV were evaluated. The directions of AR jets were visualized using color Doppler 2D TEE and classified into the following two groups, the posterior jet (Group A) or non-posterior jet (Group B) group  was calculated by the following formula: [(end-diastolic volume – end-systolic volume)/end-diastolic volume] ×10016. The peak early and late diastolic velocities of LV inflow (E and A velocity), deceleration time of early diastolic velocity, and peak early diastolic velocity on the septal corner of the mitral annulus (E’) were measured in the apical four-chamber view. The severity of AR was assessed by an integrate approach using TTE according to the ASE guidelines18: effective regurgitant orifice area (EROA) > 0.30 cm2 by the proximal isovelocity surface area (PISA) method, vena contracta (VC) width > 6 mm, and and/or LV end-diastolic diameter (LVDd) > 55 mm were defined as severe AR. The PISA method successfully measured EROA in 38 patients (55%) and VC width in 58 patients (84%). When these quantifications were not feasible by some reasons, the patients who had visually significant AR with LV dilatation (LVDd > 55 mm) and holo-diastolic reverse flow of the descending aorta were identified as having severe AR.Comprehensive transthoracic echocardiography was performed in all patients using a commercially available ultrasound system within 3 months before or after TEE. The 2D and Doppler images were recorded according to the guidelines described by American Society of Echocardiography (ASE)t-test, chi-squared test, and Fisher exact test as appropriate. The categorical data were compared with the χ2 test and Fisher exact test. P-values < 0.05 were considered to indicate significant differences. The univariable and multivariable analyses using single and multiple regression models were performed to assess the influence on the AML and PML areas and the MV opening area/MV annulus area ratio. In the multivariable analysis, the factors possibly affecting MV remodeling were selected . The intraobserver and interobserver variabilities of the MV annulus area, AML and PML areas, and MV opening area were assessed using an intra-class correlation coefficient for absolute agreement (ICCa) and Bland-Altman methods. Data analyses were performed using JMP® 10 .All continuous variables were presented as mean ± standard deviation; the categorical data were presented as number and percentage. The data for the posterior and non-posterior groups were compared using the Student’s Baseline characteristics are shown in Table P = 0.06 and P = 0.13). Both posterior and non-posterior groups revealed the increased total leaflet area compared with the controls (10.6 ± 2.1 vs 9.9 ± 2.8 vs 8.8 ± 1.9 cm2). The AML area was significantly larger in Group A than Group B  was 66 ± 13 bpm. The results of MV analysis using 3D TEE are shown in Table P < 0.001). In the multiple regression analysis, age and posterior AR jet were independently associated with the AML/PML area ratio (P < 0.001). Meanwhile, the posterior AR jet was also associated with the MV opening area/MV annulus area ratio , 0.95 for AML area , 0.92 for PML area , and 0.93 for MV opening area . The interobserver variabilities on these areas were 0.96 , 0.97 , 0.95 , and 0.93 . The Bland-Altman method showed that interobserver and intraobserver variabilities were −19.9 ± 56.5 and −36.0 ± 54.5 mm19. Some studies7 have already demonstrated that patients with chronic AR, particularly those with lower EF, have a larger total mitral leaflet area and this phenomenon probably reduces MR4. Our study population also had the significant correlations between significant MR and MV total leaflet area and total leaflet area/MV annulus area ratio. Although the detailed mechanism of leaflet enlargement in AR patients remains unknown, an adaptation to increased LV chamber and distorted MV annulus shape are considered as possible factors. Some of the earlier conducted studies using 3D transthoracic echocardiography failed to quantify each leaflet area. Nowadays, the MV apparatus depicted by 3D TEE allows software quantifying the MV annulus area, total leaflet area, and AML and PML areas, individually10. The present study using 3D TEE demonstrated the geometric changes of the MV apparatus in patients with chronic AR according to the directions of AR jets. In our study, the MV total leaflet area was larger in both posterior and non-posterior groups than the controls reported in the earlier studies7. Furthermore, we found that the posterior AR jets induced AML growth, while PML remained unchanged. These results support the theory that MV remodeling could prevent functional MR in both posterior and non-posterior groups. Although relatively symmetrical remodeling was observed in the patients with non-posterior AR jets, MV remodeling in those with posterior AR jets was asymmetrical. Accordingly, we presumed that the mechanisms of MV remodeling might be different based on the directions of AR jets. AML growth in the patients with posterior AR jets might also be affected by the multiple factors. These speculations were due to mechanical stretch induced by MV annulus dilatation and chronic tethering caused by LV remodeling7. In fact, the patients with posterior AR jets in our study tended to have greater LV volume and larger MV annulus area. One study has described that LV dilatation did not affect AML length20, although, it still remains controversial. In the present study, the patients with posterior AR jets had the larger AML area than those with non-posterior AR jets, whereas the PML areas did not significantly differ among these groups. These results support the prevalence of asymmetrical remodeling of the MV leaflet in chronic AR patients with posterior jets and this phenomenon cannot be solely explained by mechanical stretch of annulus dilatation caused by LV remodeling. Meanwhile, AML enlargement is triggered by mechanical stretch caused by AR jets. Shear stress and mechanical pressure on the heart valves induce the biosynthesis of extracellular matrix materials, such as collagen and proteoglycan, and valvular remodeling25. The collagen synthesis of the heart valves is also found to be dependent on the degree and duration of mechanical stretch26. The posterior jets in patients with chronic AR cause cyclic stress towards the AML over the long period of time. The results of our study thus suggested that the mechanisms of MV remodeling might differ based on the directions of AR jets. Recently, leaflet remodeling has become a focus of attention as one of the important factor to prevent the occurrence of MR27; however, the mechanism of leaflet remodeling has not been fully investigated. The earlier studies speculated mechanical stretch induced by MV annulus dilatation and chronic tethering caused by LV remodeling. Our study results additionally demonstrated the other mechanism, AML remodeling triggered by mechanical stretch from the AR jets. The present study also demonstrated the restricted MV opening at mid-diastole in patients with posterior AR jets. Although this finding was suggested for a long time28, the quantitative assessment using 3D has not been performed. The influence of MV opening restriction at mid-diastole on the hemodynamics has not been fully elucidated; however, all the trans-mitral flow data, such as peak A velocity, E’, E/E’, and deceleration time, were not significantly different between the AR-jet groups in our study. These findings suggested that MV closing induced by a AR jet during mid-diastole might not affect functional mitral stenosis. These transformations of mitral leaflet area in patients with chronic AR also provide important information to determine treatment policy. Firstly, the changes of the MV apparatus should be considered when assessing AR patients for a MV repair. One study20 suggested that AML length should be used as a reference of annular dilatation because AML enlargement in patients with posterior AR jets requires an adequate MV ring size. The other study reported that AML length was a predictor of MV reparability29. Thus, the careful assessment should be provided to these patients while discussing MV repair. Secondly, AML enlargement may occasionally cause systolic anterior motion (SAM) of the MV. Maron and his colleagues30 demonstrated that an AML length/LV outflow tract (LVOT) diameter was associated with subaortic obstruction in patients with hypertrophic cardiomyopathy. They speculated that the elongated AML leaflet might have the potential for mitral-septal contact and create LV outflow obstruction. Several studies also reported that long AML and a mismatch of AML/PML length were the predictors of SAM and LVOT obstruction in patients with LV hypertrophy32. Some reports34 have described the possibility of LVOT obstruction in patients with large AML without LV hypertrophy in some situations, such as dehydration, during exercise, and inotropic treatment. Although most of patients with chronic AR have no concentric LV hypertrophy, patients with AML enlargement may suffered from LVOT obstruction in these situations.LV chamber remodeling induces mitral leaflet growthThe present study has several limitations. This study was a retrospective single center study; the numbers of subgroups were relatively small and varied. A large populated and multicenter study is thus warranted. The present study did not assess the histological appearance. Since the surgeons were not able to approach the LV and MV while performing isolated aortic valvuloplasty and replacement, the MV tissues were not obtained. The severity of AR was not equally evaluated by the quantitative methods, such as the PISA method and VC width. The selections of mid-diastolic frame in the MV images were not strictly the same in all patients, although, the frame rate was enough to decrease the measurement errors in HR. In the present study, the influence of MV annulus shape due to aortic root dilatation on the severity of MR was not completely excluded; however, its influence was not significant in the analyses. We only excluded the patients with severe calcification of the MV annulus in the present study; the influence of mild MV annulus calcification on MR degree and MV Doppler parameters was not completely ruled out. Although this study included the patients with normal TEE findings as controls, they might not be truly normal because they had indication of TEE for suspected intracardiac thrombus and infective endocarditis. The selection bias possibly remained in this study. Meanwhile, the controls in the earlier studies conducted on MV geometry also had the similar prevalence of these comorbidities. Finally, the duration of AR and morphological changes were not assessed in this study. Further studies are thus required to investigate more detailed associations between the disease duration of chronic AR and MV morphological changes.3D TEE identified geometric differences in the MV apparatus between the two different AR jet directions. The present study demonstrated that an eccentric posterior AR jet enlarged the AML and restricted MV opening to a greater degree than a non-posterior jet. These results would be helpful in understanding the mechanisms of MV morphological changes in chronic AR.


The principal source of this Fe is thought to be dust transported from southern mid-latitude deserts. However, uncertainty exists over contributions to CO2 sequestration from complementary Fe sources, such as the Antarctic ice sheet, due to the difficulty of locating and interrogating suitable archives that have the potential to preserve such information. Here we present petrographic, geochemical and microbial DNA evidence preserved in precisely dated subglacial calcites from close to the East Antarctic Ice-Sheet margin, which together suggest that volcanically-induced drainage of Fe-rich waters during the Last Glacial Maximum could have reached the Southern Ocean. Our results support a significant contribution of Antarctic volcanism to subglacial transport and delivery of nutrients with implications on ocean productivity at peak glacial conditions.Marine sediment records suggest that episodes of major atmospheric CO 2 sequestration during the last glacial period remain uncertain. Here, based on the biogeochemical analysis of subglacial calcites, the authors propose Antarctic volcanism, via subglacial drainage of Fe-rich waters, as a key contributor.Contributions of iron sources to Southern Ocean CO Datable material capable of recording the chemical, microbial and hydrological signatures of subglacial environments is required, and previous approaches that involved drilling subglacial lakes and sediments suffered from a lack of precise age controlThe presence of subglacial carbonates in an area confirms the existence of steady liquid water production at the ice-bedrock interface17202 of ice-free metamorphic, non-carbonate bedrock  before the year 2000 (b2k). Boggs Valley translucent columnar calcite sparite (Cs) crusts yielded ages from 26.951±0.824 to 17.054±0.332 ka, and their high U concentration  necessary to sustain basal temperature (Tb) conducive to wet-based conditions during the LGM was modelled by using a thermal conductivity of ice (Kice) of 2.4 W m−1 K−1, a mean annual air temperature (Ts) of −29.4 °C, 9.5 °C (ref. G) of 120.9 mW m−2 inferred from magnetic data−2 hypothesized from modellingFor basal meltwater production to be sustained in a polar glacier during glacial maxima, the ice must have been thick enough to limit dispersion of geothermal heat flux to the atmosphere. The glacier thickness  than in the Lake Vostok region950Transantarctic Mountain (TAM) volcanic activity, from Mount Melbourne to Mount Erebus, to the Pleiades , is char46We used Icelandic examples9ca. 17.1±0.3 ka (ca. 18 ka) documented in areas of strong ice streaming in East Antarctica53At 1±0.3 ka  calcite 2+) from the dissolution of silicates and pyrite oxidation56572+ may have formed complexes with the natural organic matter in the basal meltwater or remained as dissolved species (<<1 nm), both of which could have been adsorbed onto reactive calcite surfaces. Thus, the amount of Fe2+ released can be estimated from the concentration of Fe incorporated in the clean sparite crystals. Using a conservative concentration of 100 p.p.m., the Fe2+ dissolved in the LGM basal waters was calculated by extrapolating the experimental distribution coefficient between solid calcite and Fe in solution (DFe2+)Fe2+ is 3.6 for precipitation rates of 1.8–6.6 μmol m−2 h−1 estimated for Cs . Assuming that the LGM subglacial waters had a Ca2+ concentration of 0.5 mmol l−1 (ref. 60), the divalent Fe concentration would have been ∼0.32 μmol l−1, a value similar to that reported for Antarctic basal iceUnder today's global warming scenario, meltwaters discharged from supraglacial environments interacting with subglacial sediments and bedrock, deliver dissolved Fe to the ocean7ca. 23 ka that are not paralleled by an increase in dust flux recorded by ice cores . Epifluorescence was excited by light at 250–350 nm wavelength and the most common emission band was in the 450–550 nm.Thin sections of subglacial calcites were observed by optical and epifluorescence microscopy. One millimetre thick wafers of age-equivalent subglacial crusts were polished and ultrasonically cleaned. Imaging at nm-scale resolution on uncoated surfaces was carried out by field emission scanning electron microscopy with a ZEISS Sigma Variable Pressure field emission scanning electron microscopy equipped with Bruker EDS system in backscattered electron mode at the Electron Microscope and X-ray unit of the University of Newcastle, Australia. Semi-quantitative chemical microanalyses were carried out Powder X-ray diffraction on clean calcite and Dm was carried out on a PANalytical h-h diffractometer equipped with a Cu X-ray tube operating at 40 kV and 40 mA. Scans were performed over the range 3–80° with an integrated step size of 0.017° and a counting time of 100 s per step. Identification of minerals, and cell parameter determination were performed using High Score Plus and the ICSD database .pre-quem age of 26.307±0.204 ka, and a date for the top Dm precipitated on partially dissolved Cs of 23.524±0.446 ka. It is reasonable to reconstruct, on the basis of correlation with dated Cs+Dm in BV9a2a, an age of ca. 25.5 ka (25.135±0.537 ka) for the commencement of Cs+Dm precipitation in BV9b  or correlative conformities (continuously growing columnar Cs). This implies that the stratigraphy of Boggs Valley subglacial calcites reflects accommodation space available on the substrate (or between substrate and ice) and Cc (+ Dm in BV9a2a and BV9b) formed on topographic highs relative to the lows were sedimentation of clast occurred.−5 mbar. Maps were acquired by raster scanning with a step of 2 μm in both directions. XRF was stimulated with a monochromatic beam and was collected in the horizontal plane, using a large solid state detector. This geometry, together with the linear polarization of the incident X-ray beam and the vacuum conditions, minimize background signals from elastic and inelastic scattering. The micro-XRF mapping was carried out in an area of 20 × 5 mm to include all microstratigraphic components identified by microscopy. Excitation energy was set at 7.5 keV and 2.6 keV to maximize the XRF yield of the elements of interest and avoid the excitation of Ca, which would easily saturate the XRF detector. XRF spectra were batch-fitted using the PyMca software package.Micro X-ray fluorescence (XRF) mapping was carried out at beamline ID21 at the European Synchrotron Radiation Facility . Double-polished 300 μm thick wafers were ultrasonically cleaned and loaded in the sample chamber operating at a pressure of 10Micro X-ray Absorption Near Edge Structure (μXANES) investigation was carried out by tuning the exciting energy around the S absorption K-edge (from 2.45 to 2.55 keV) and the Fe K-edge (from 7.1 to 7.2 keV) utilizing a Si(220) double-crystal monochromator and an energy resolution of 0.25 eV ref. . Calibra−1. Background measurement and analyses of the NIST612 standard were carried out periodically for drift corrections. Data were calibrated to the NIST612 standard and converted to absolute concentrations using 43Ca as internal standard. Background was subtracted and baseline drift corrected by linear interpolation of the standard counts. Because of mass interferences, S and P were quantified by EDS using an internal standard for the regions where these elements were detected by synchrotron-radiation-based micro-XRF.For quantitative geochemical analysis we used a Varian 810 quadrupole ICP-MS equipped with helium excimer laser system . The helium excimer produces an ultraviolet light beam (24 mm × 8 mm) with wavelength of 193 nm and pulse length of 24 ns. Typical scan speed is 20 μm s2 evolved from the reaction was admitted into the ion-source chamber under vacuum in an ultra-high-purity helium gas stream. Sample isotopic ratios were standardized to the VPDB scale using an in-house standard of Carrara Marble (NEW1) and two international reference materials, NBS19 and NBS18. Analytical reproducibility for C and O was better than 0.05 and 0.10‰, respectively.Stable-isotope analyses were conducted using a GV Instruments GV2003 continuous-flow isotope ratio mass spectrometer at The University of Newcastle, Australia. Samples of ∼0.75 mg were acidified in evacuated septum-capped vials with 0.05 ml of 105% phosphoric acid. The COBacterial amplicon sequencing was undertaken on BV8a, BV9b and BV11a, representative of clear sparite, a sequence of sparite and Dm, and Dm, respectively . Contami6867Our DNA data are publically available on the QIITA database: study ID 10766: ‘The Influence of Antarctic Subglacial volcanism on the global Fe cycle during the Last Glacial Maximum'. DNA sample list is supplied in How to cite this article: Frisia, S. et al. The influence of Antarctic subglacial volcanism on the global iron cycle during the Last Glacial Maximum. Nat. Commun.8, 15425 doi: 10.1038/ncomms15425 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Table and Supplementary Referenceslist of Boggs Valley subglacial calcite samples analysed for DNA. Sample BV11, consisting of angular clasts cemented by microsparite, is sterile (see text for details.)OTU .txt file relative to the DNA data for Boggs Valley calcite samples BV8a and BV9b. BV 11 is sterile.OTU data in .Biom format relative to samples BV8a and BV9b.


We demonstrated that knockdown of GGA1 and GGA2 by shRNA and siRNA significantly reduced the cell surface expression of inducibly expressed α2B-AR and arrested the receptor in the perinuclear region. Knockdown of each GGA markedly inhibited the dendritic expression of α2B-AR in primary cortical neurons. Consistently, depleting GGA1 and GGA2 attenuated receptor-mediated signal transduction measured as ERK1/2 activation and cAMP inhibition. Although full length α2B-AR associated with GGA2 but not GGA1, its third intracellular loop was found to directly interact with both GGA1 and GGA2. More interestingly, further mapping of interaction domains showed that the GGA1 hinge region and the GGA2 GAE domain bound to multiple subdomains of the loop. These studies have identified an important function and revealed novel mechanisms of the GGA family proteins in the forward trafficking of a cell surface GPCR.The molecular mechanisms that control the targeting of newly synthesized G protein-coupled receptors (GPCRs) to the functional destinations remain poorly elucidated. Here, we have determined the role of Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins 1 and 2 (GGA1 and GGA2) in the cell surface transport of α G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface receptors and their functions are highly regulated by intracellular trafficking processes. As compared with well-characterized internalization, recycling and degradation pathways125679101112131415171819202122Golgi-associated, γ-adaptin homologous, ARF-interacting proteins (GGAs) are well known adaptor proteins for clathrin-coated vesicles. There are three GGA isoforms, namely GGA1, GGA2 and GGA3, in humans which have been well characterized to have similar trafficking function that is to facilitate the transport of cargo proteins from the TGN to the endosomal compartment. All three GGAs have identical domain organizations, containing the N-terminal VHS  domain followed by the GAT (GGAs and TOM1) domain, the hinge region and the C-terminal GAE (γ-adaptin ear) domain. Each domain of GGAs has been shown to interact with specific proteins to coordinate their trafficking functions. Specifically, the N-terminal VHS domain interacts with the DxxLL-type sorting motifs of cargo proteins which cycle between the TGN and the endosomal compartment2425262728293031323334353738394041424344452B-adrenergic receptor (α2B-AR), a prototypic member of the GPCR superfamily, and that the function of GGA3 in modulating α2B-AR export is mediated through its VHS domain interaction with the receptor, providing the first evidence implicating a role of the GGA family proteins in GPCR trafficking2B-AR cell surface export and elucidate the underlying mechanisms. We have found that all three GGAs are equally important in regulating the cell surface export of α2B-AR and more interestingly, three GGAs physically associate with the receptor via distinct domains. These studies have revealed novel mechanisms of the GGA-mediated cell surface GPCR trafficking.Our laboratory is interested in dissecting the mechanisms of anterograde trafficking of GPCRs. We have recently demonstrated that GGA3 is required for the TGN-to-cell surface transport of α2B-AR in HEK293 cells and utilized these inducible cells to define the function of GGA3 in the cell surface transport of newly synthesized α2B-AR2B-AR at the cell surface was quantified by intact cell ligand binding assays using the cell nonpermeable radioligand [3H]-RX821002 after doxycycline induction for different time periods. shRNA-mediated knockdown of GGA1 and GGA2 similarly inhibited the cell surface expression of α2B-AR at each time point and the maximal inhibition was observed by about 30% after doxycycline induction for 20 h (We have generated stable cell lines by using the Tet-On 3G inducible expression system to drive the expression of HA-αfor 20 h .2B-AR. α2B-AR was tagged with green fluorescent protein (GFP) and its subcellular distribution was visualized by confocal microscopy in cells transiently transfected with shRNA targeting GGA1 and GGA2. α2B-AR was clearly arrested in the perinuclear region, unable to transport to the cell surface, by GGA1 and GGA2 shRNA as compared to control cells in which α2B-AR was robustly expressed at the cell surface  ERK1/2 and the reduction of cAMP production in HEK293 cells. Consistent with the reduction of the cell surface expression of α2B-AR, ERK1/2 activation in response to UK14304 stimulation was significantly inhibited by approximately 50% by shRNA targeting GGA1 and GGA2 as compared to cells transfected with control shRNA (2B-AR-mediated inhibition of cAMP production in response to forskolin stimulation (To determine if GGA-mediated αol shRNA . Consistmulation .2B-AR and the interaction is mediated through the VHS domain of GGA3 and the third intracellular loop (ICL3) of the receptor2B-AR export, we determined if GGA1 and GGA2 could interact with the receptor in co-immunoprecipitation and GST fusion protein pulldown assays. HEK293 cells stably expressing α2B-AR were transiently transfected with myc-GGA1 or myc-GGA2 followed by immunoprecipitation using α2B-AR antibodies. GGA2 was clearly detected in the immunoprecipitates of α2B-AR antibodies, whereas GGA1 was undetectable in the immunoprecipitates . One possible explanation for this phenomenon is that there are multiple pathways to direct α2B-AR export trafficking and GGAs mediate only one of these pathways. It should be pointed out that the data described here do not provide sufficient evidence indicating that α2B-AR transport from the Golgi/TGN to the cell surface is direct. As proteins destined for the plasma membrane can be transported either directly from the Golgi to the plasma membrane or from the Golgi through the recycling endosomal compartment to the plasma membrane482B-AR targeting to the cell surface passes through the recycling endosomal compartment. In support of this possibility, GGA3 was shown to modulate the transport of internalized Met receptor tyrosine kinase from the recycling endosomes2B-AR in the dendrites of primary cortical neurons, implicating that GGAs may play a more important role in the dendritic transport of α2B-AR in the native neurons. Fourth, consistent with the reduction of cell surface receptor expression, knockdown of individual GGAs suppressed α2B-AR-mediated signaling measured as ERK1/2 activation and cAMP reduction, suggesting that GGAs modulate not only the cell surface trafficking but also the function of the receptor.There are several interesting points regarding the regulation of cell surface transport of α2B-AR. It has been well described that the function of GGAs in sorting proteins into the TGN-to-endosome pathway is tightly controlled by their VHS domain interaction with the DxxLL-type motifs of cargo proteins. These proteins include cation-dependent and cation-independent mannose 6-phosphate receptors, sortilin, sorting-protein-related receptor, low-density lipoprotein receptor-related proteins and β-secretase242526272829303132332B-AR. Here we found that GGA1 and GGA2 also interacted with the ICL3 of α2B-AR in GST fusion protein pulldown assays. Although the VHS domains are highly conserved amongst three GGAs, the VHS domains of GGA1 and GGA2 did not interact with α2B-AR. Interestingly, the α2B-AR-binding domains were identified as the hinge domain of GGA1 and the GAE domain of GGA2 domain. Although our data have clearly shown that GGA2 and GGA3 were able to form complexes with α2B-AR in co-immunoprecipitation assays, GGA1 did not under the same experimental condition, suggesting that interactions of three GGAs with α2B-AR have different regulatory properties. Nevertheless, our previous and current studies indicate that the GGA1 hinge domain, the GGA2 GAE domain and the GGA3 VHS domain are responsible for the interaction with α2B-AR . We have used the progressive deletion strategy to successfully map the binding sites of both GGA1 and GGA2 to the regions R285-C326 and L339-Q358. As the GGA-binding subdomains R285-C326 and L339-Q358 do not possess the D/EFGXØ and DxxLL-type motifs and thus, they may contain novel, yet unidentified, GGA-binding signals. Our unpublished data showed that deletion of the fragment R285-Q358 significantly reduced the interaction of α2B-AR with GGA2 in coimmunoprecipitation assays. However, the truncated receptor was unable to export to the cell surface. These data suggest that, in addition to binding to GGAs, the ICL3 fragment R285-Q358 may contain other signals important for receptor transport to the cell surface. Nevertheless, these data strongly demonstrate differential interactions of three GGAs with α2B-AR which are mediated through different domains/motifs in individual GGAs and the receptor  was purchased from Perkin Elmer Life Sciences . All other materials were obtained as described elsewhere4765Full length GGA1 and GGA2 tagged with myc at their N-termini were generously provided by Dr. Juan S. Bonifacino . Arrestin-3 was obtained from Dr. Jeffrey L. Benovic (Thomas Jefferson University). Antibodies against GGA1 were purchased from Abcam Inc. . Antibodies against GGA2, GM130 and p230 were from BD Transduction Laboratories . Antibodies against GFP, myc and phospho-ERK1/2 were obtained from Santa Cruz Biotechnology, Inc. . Anti-ERK antibodies detecting total ERK1/2 expression and GM130 were from Cell Signaling Technology, Inc. . Alexa Fluor 594-labeled antibodies were from Molecular Probes, Inc. . Anti-His antibodies and UK14304 were obtained from Sigma-Aldrich . -RX821002 as described652B-AR were cultured on 6-well dishes and transiently transfected as described above for 12 h. The cells were split into 12-well plates and cultured for an additional 24 h. After induction with doxycycline at a concentration of 40 ng/ml for different time periods, the cells were incubated with DMEM plus [3H]-RX821002 at a concentration of 20 nM in a total volume of 400 μl for 90 min at room temperature. The non-specific binding of α2B-AR was determined in the presence of rauwolscine (10 μM). The binding was terminated and excess radioligand eliminated by washing the cells with ice-cold DMEM. The retained radioligands were then extracted by digesting the cells in 1 M NaOH for 2 h. The radioactivity was counted by liquid scintillation spectrometry. All radioligand binding assays were performed in triplicate. For measurement of α2B-AR internalization, HEK293 cells stably expressing α2B-AR were cultured on 6-well dishes and transfected with control or GGA shRNA together with 1 μg of arrestin-3 for 24 h. After starvation for 3 h, the cells were stimulated with epinephrine at a concentration of 100 μM for different time periods. The cells were washed three times with cold phosphate buffered saline (PBS) and α2B-AR expression at the cell surface was measured by intact cell ligand binding at 4 °C as described above.The cell surface expression of α2-AR expression was measured by flow cytometry as described previously2B-AR were suspended in PBS containing 1% fetal calf serum at a density of 4 × 106 cells/ml and permeabilized with 0.2% Triton X-100 in PBS for 5 min on ice. The cells were then incubated with high affinity anti-HA-fluorescein (3F10) at a final concentration of 2 μg/ml at 4 °C for 30 min. After washing with 0.5 ml of PBS twice, the cells were resuspended and the fluorescence was analyzed on a flow cytometer .Total α2B-AR was carried out as described previously2B-AR-GFP together with 400 ng of GGA shRNA. The coverslips were mounted with prolong antifade reagent (Invitrogen) and images were captured using a LSM510 Zessis confocal microscope equipped with a 63× objective. To visualize the localization of α2B-AR in primary neurons, the neuronal cultures were transfected with α2B-AR-GFP with or without co-transfection with GGA siRNA for 2 days. The neurons were fixed, permeabilized and stained with GGA-isoform specific antibodies at a dilution of 1:500. The amounts of α2B-AR-GFP signal pixels in the dendrites were determined by using NIH Image J software as described previouslyFluorescence microscopic analysis of the subcellular localization of αHEK293 cells were cultured on 6-well dishes and transfected with 2 μg of GGA shRNA as described above. The cells were starved for at least 3 h before stimulation with 1 μM UK14304 for 5 min. Stimulation was terminated by addition of 1 × SDS-loading buffer. After solubilizing the cells, 20 μl of total cell lysates were separated by 12% SDS-PAGE. ERK1/2 activation was determined by measuring the levels of phosphorylation of ERK1/2 with phospho-specific ERK1/2 antibodies by immunoblotting2B-AR with or without GGA shRNA. The cells were then split into 96-well plates at a density of 1 × 104 cells/well. After starvation for 1 h, the cells were stimulated with forskolin (1 μM) plus or minus different concentrations of UK14304 (1 to 1000 nM) in the presence of 0.1 mM isobutylmethylxanthine for 5 min at 37 °C. The reactions were stopped by aspirating the medium and the cells were lysed with 100 μl of lysis buffer. 50 μl of cell lysate was transferred into microtitre plates and cAMP concentrations were measured according to the protocol provided by the Kit.cAMP concentrations were measured by using the cAMP Direct Biotrak Enzyme Immunoassay System  as described previously2B-AR were cultured on 100-mm dishes and transfected with 10 μg of myc-GGA1 or myc-GGA2 for 24 h. After incubation with doxycycline (40 ng/ml) for 24 h, the cells were washed twice with PBS, harvested, and lysed with 1 ml of lysis buffer . After gentle rotation for 1 h, samples were centrifuged for 15 min at 14,000 × g and the supernatant was incubated with 50 μl of Dynabeads Protein G for 1 h at 4 °C to remove nonspecific bound proteins. Samples were then incubated with 2 μg of anti-α2B-AR antibodies overnight at 4 °C with gentle rotation followed by incubation with 50 μl of Dynabeads Protein G for 4 h. The beads were washed 3 times with lysis buffer without SDS. Immunoprecipitated proteins were eluted with 30 μl of SDS-gel loading buffer and separated by SDS-PAGE. Myc-GGA and HA-α2B-AR in the immunoprecipitates were detected by immunoblotting using myc and α2B-AR antibodies, respectively.HEK293 cells inducibly expressing HA-α2 and 0.5% bovine serum albumin at 4 °C for 4–6 h. The resin was washed four times with 0.5 ml of binding buffer and the retained proteins were solubilized in SDS-gel loading buffer and separated by SDS-PAGE. Proteins bound to GST fusion proteins were detected by immunoblotting.The GST fusion proteins were expressed in bacteria and purified using a glutathione affinity matrix as described previously652B-AR, GGA1 and GGA2 were tagged with the epitope His at their N-termini in the pET-28 vector and purified by using His SpinTrap kit  as described previouslyTo determine if GGAs could directly interact with αt test, and p < 0.05 was considered as statistically significant. Data are expressed as the mean ± S.E.Differences were evaluated using Student’s How to cite this article: Zhang, M. et al. Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2. Sci. Rep.6, 37921; doi: 10.1038/srep37921 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The SDs of MA and FFA were prepared using microwaves irradiation (MW) technique. Different carriers such as Pluronic F127® (PL), Eudragit EPO® (EPO), polyethylene glycol 4000 (PEG 4000) and Gelucire 50/13 (GLU) were used for the preparation of SDs. Prepared MW irradiated SDs were characterized physicochemically using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform infra-red (FT-IR) spectroscopy, powder X-ray diffraction (PXRD) and scanning electron microscopy (SEM). The physicochemical characteristics and drug release profile of SDs were compared with pure drugs. The results of DSC, TGA, FT-IR, PXRD and SEM showed that SDs were successfully prepared. In vitro dissolution rate of MA and FFA was remarkably enhanced by SDs in comparison with pure MA and FFA. The SDs of MA and FFA prepared using PEG 400 showed higher drug release profile in comparison with those prepared using PL, EPO or GLU. The dissolution efficiency for MA-PEG SD and FFA-PEG SD was obtained as 61.40 and 59.18%, respectively. Optimized SDs were also evaluated for in vivo anti-inflammatory effects in male Wistar rats. The results showed significant % inhibition by MA-PEG (87.74% after 4 h) and FFA-PEG SDs (81.76% after 4 h) in comparison with pure MA (68.09% after 4 h) and pure FFA (55.27% after 4 h) (P<0.05). These results suggested that MW irradiated SDs of MA and FFA could be successfully used for the enhancement of in vitro dissolution rate and in vivo therapeutic efficacy of both drugs.The present studies were undertaken to develop solvent-free solid dispersions (SDs) for poorly soluble anti-inflammatory drugs mefenamic acid (MA) and flufenamic acid (FFA) in order to enhance their  Thus, the drug solubility plays an important role in the therapeutic achievement of a drug. Moreover, the preferred drug concentration in systemic circulation and the pharmacological response depend on the drug solubility [Solubility of drug molecules affects the lubility , 2. The lubility .The majority of the recently formulated drugs exhibit low bioavailability due to their poor aqueous solubility and dissolution rate . The disMicrowave (MW) irradiation is relatively a novel method used for solubility and bioavailability enhancement of thermo-stable and drying materials , 17. MW N-phenylanthranilic acid derivatives [® (PL), Eudragit EPO® (EPO), polyethylene glycol 4000 (PEG 4000) and Gelucire 50/13 (GLU).Mefenamic acid (MA) and flufenamic acid (FFA) are non-steroidal anti-inflammatory drugs (NSAIDs) that are ivatives . The molivatives –23. Theyivatives . They beivatives . Thus thivatives –31. HoweMA (purity 99.3%), FFA (purity 99.5%) and PL (purity 99.6%) were obtained from “Sigma Aldrich ”. GLU (purity 99.2%) was obtained as a kind gift sample from “Gattefosse ”. EPO (purity 99.1%) was obtained as a kind gift sample from “Evonik ”. PEG 4000 (purity 99.0%) was obtained from “E-Merck ”. All other chemicals used were of high purity and analytical grade.In this particular study, different SDs of MA and FFA were prepared using domestic MW irradiation (Samsung Model ME0113M1) , 17. EacTGA analysis was performed using “Pyris 1 TGA ” apparatus outfitted with auto sampler to analyze the thermal stabilities of pure chemicals . The experiments were carried out in the heating range of 25–250°C with accelerated rate of 10 C/min. Each sample weighed approximately 3.0 mg and analyzed under a nitrogen purge of 20 ml/min. The TGA results were evaluated according to the weight loss of the treated samples subtracted from the original one.DSC analysis of pure MA, FFA and eight different formulations were determined using “DSC apparatus ” over the temperature range of 25.00°C to 250.00°C at 10.00°C/min and recorded under a nitrogen purge of 20 ml/min. This instrument was equipped with auto sampler and chiller. The samples of interest were accurately weighed (around 3.0 mg) and placed in an aluminium pan and hermetically sealed. This instrument was equipped with Pyris manager software “” to evaluate the solid state characterization and thermal stability of each sample.-1 wavenumber using 3 scans and 2 cm-1 resolution [The FTIR spectra of pure MA, FFA, carriers and their MW SDs were obtained using a “Perkin Elmer FTIR spectrum BX ”. The materials were prepared as KBr pellets and spectra were collected in the range of 4400 to 350 cmsolution , 33. TheThe changes in the shape and surface morphology of the pure drugs and SD samples were evaluated using SEM. Photomicrographs of the samples were taken using SEM microscope . The samples were fixed on stubs using both side adhesive carbon tape  and coated under vacuum with gold in a Q150R sputter coater unit from Quorum Technologies Ltd.  in an argon atmosphere at 20 mA for 60 sec.PXRD of pure drugs and SD samples were evaluated by Ultima IV diffractometer  over the 3−60° 2θ range at a scan speed of 1°/min. The tube anode was Cu with Ka = 0.1540562 nm mono chromatized with a graphite crystal . The pattern was collected at 40 kV of tube voltage and 40 mA of tube current in step scan mode . This study was set to determine the crystallinity of the pure drugs in the SD system.® UPLC, Waters Inc., Bedford, MA) with UV detection at 278 nm (for MA) and 286 nm (for FFA). Briefly, separation employed reverse-phase isocratic elution using a mobile phase consisting of a mixture of 0.05 M ammonium acetate buffer (adjusted to pH = 2 with phosphoric acid) and acetonitrile (65:35% v/v) run at flow rate of 0.3 ml/min and injection volume was 1 μl. The PDA detector was set to acquire 3D data from 210 to 400 nm while the 2D channel was recorded at 336 nm. The column  was used and temperature was set at 40°C. The linearity was excellent with R2 = 0.999 and the peak was resolved at a retention time of 1.02 min (for FFA) and 1.3 min (for MA). This method was employed in determination of the drug content in dissolution samples.The analysis of MA and FFA was performed using a validated ultra performance liquid chromatography-ultra-violet (UPLC-UV) method. The amount of MA and FFA was determined by injecting samples into a UPLC  using an automated dissolution tester . The SDs were filled in hard gelatin capsules which were placed in a dissolution medium of 900 ml 0.05 M phosphate buffer (pH = 7.4) for 2 h maintained at 37°C. The samples of 5 ml each were withdrawn at predetermined time intervals and replaced with freshly prepared dissolution medium. The samples were filtered and analyzed by injection into UPLC as mentioned above in the previous section. The mean value of the percentage of drug release was plotted against the time interval.in vivo anti-inflammatory studies. Therefore, in vivo anti-inflammatory studies were performed on SDs MA/PEG and FFA/PEG in order to compare their anti-inflammatory effects with pure MA and pure FFA. Thirty male Wistar rats (weighing from 200–250 g) were obtained from “Animal Care and Use Center of College of Pharmacy at King Saud University, Riyadh, Saudi Arabia”. Approval to carry out in vivo anti-inflammatory studies was obtained from Animal Ethics Committee of King Saud University, Riyadh Saudi Arabia. Institutional guidelines were strictly followed in order to perform these studies. All the animals were given standard laboratory conditions of “temperature and relative humidity”. The rats were kept in plastic cages and provided free access to standard laboratory pellet diet and water ad libitum. The anti-inflammatory effects were investigated by the Carrageenan-induced hind paw edema method developed by Winter et al. 1965 [Based on physicochemical characterization and maximum drug release profiles, SDs MA/PEG and FFA/PEG were selected for al. 1965 . The ratal. 1965 , 35.The results of this study were analyzed statistically using one way analysis of variance (ANOVA) analysis followed by Dennett’s test using GraphpadInstat software . The P vale of <0.05 was considered as statistically significant.Before starting the MW process, the study of the drug-carrier physicochemical characteristics is important which would help to adjust and optimize the experiment parameters that will lead to save time and efforts. The selection of suitable excipients is highly recommended in order to obtain excellent results and perfect products. In this study, four different carriers were selected depending upon their melting point, elasticity and their ability to convert the crystalline form of drug to an amorphous form. The obtained physical mixtures (PM) were subjected to MW irradiation at 900 W at different time of interval. PM powders were fine powder and easy to handle. When the binary mixtures of drugs (MA or FFA) with different carriers such as PL, EPO, GLU and PEG were prepared and subjected to MW, fine powder (mill and sieve) were obtained except for one mixture i.e. FFA/GLU. It was because of the low melting point of the drug and carrier. This mixture was also sticky and difficult to handle. Therefore, the binary mixture of this drug with the carrier was placed at -80°C for about 1 h in order to allow solidification. The solidified mixture was then subjected to grinding and sieving in order to obtain powder with uniform size. MW conditions used in the preparation of SDs of MA and FFA with suitable carriers are presented in TGA spectra of pure MA, pure FFA and different carriers are presented in DSC analysis was performed to evaluate drug–carrier interactions in different SDs , 23. DSCThe FTIR spectra of pure MA and its MW SDs are presented in −1. The C-O-C vibrational stretching is assigned to the peak of 1112 cm−1. EPO had a vibrational stretching of C = O at 1734 cm−1. Vibrational stretching of O-H and C-O-C for PEG 4000 is clear at 3447 and 1112 cm−1, respectively. The characteristic peaks found at 1112, 1736, 2888, and 3448 cm−1 is assigned to C-O, C = O, C-H, and O-H, respectively [For PL, the FTIR spectrum of C-H stretching showed a distinct peak at around 2889 cmectively , 41–43.−1 wavenumbers. For the MW formulation between MA and PL, all the assigned peak showed no significant difference in comparison with pure drugs except for the ether vibrational which shifted to higher wave number of 2886 cm−1, indicating a probability of a hydrogen bonding with C = C in the MA. No clear shifts in the vibrational bands were found in the MA with either EPO, or PEG 4000 or GLU mixtures  are displayed in Figs 5) of 7.79% was observed after 5 min with a dissolution efficiency (%DE60) value of 42.48 after 60 min  but did not result in enhancing its overall dissolution rate. MA-GLU SD also exhibited a slow dissolution rate with initial burst dissolution of 3.46% (IDR) and DE% of 10.16. The slower dissolution rates of both FFA and MA in their GLU binary systems might be due to the partial hydrophobic nature of GLU whose HLB value is reported as 13.0 [Unfortunately, the SD of FFA acid and MA in EPO matrix showed enhanced initial dissolution  but a slow dissolution rate was depicted during the whole dissolution period (only 23% was dissolved within 60 min). This might be due to a fact that EPO is swellable and permeable above this pH but poorly soluble above this pH .The drugs dissolution rates showed a noticeable enhancement in case of SD in PL and PEG 4000 binary systems. The drug DE% values calculated were 44.89% and 59.40% in case of FFA SD in PL and PEG matrices, respectively. Also, the MA exhibited DE% 58.74 and 61.18 in its-binary systems in PL and PEG matrices, respectively. However, slow initial dissolution rate (IDR) of both FFA and MA were observed from their binary systems with PL. The increase in the dissolution rate of FFA in its binary systems in the matrices of PL and PEG 4000 could be attributed on the basis of the crystallization of drug particles simultaneously in very minute crystals surrounded by water-soluble molecules of the carrier, as revealed by DSC and PXRD data. This results in an increase in the specific surface area subjected to the dissolution medium which in turn enhances drug dissolution rate , 48.Moreover, the hydrophilic SD carrier surrounding the drug particles may reduce aggregation and agglomeration of these particles, resulting in their rapid contact with dissolution medium with wetting of their surfaces, and so increase its dissolution rate .In vivo anti-inflammatory effects of MA/PEG and FFA/PEG SDs after oral administration were compared with those of MA suspension and FFA suspension. The results of in vivo anti-inflammatory studies in male Wistar rats are shown in in vivo anti-inflammatory effects of MA/PEG SD and FFA/PEG SD were highly significant in comparison with their suspension formulations. The enhanced anti-inflammatory effects of MD/PEG SD and FFA/PEG SD were possible due to the rapid absorption of MA and FFA from their SDs due to the presence of solubilizer such as PEG 4000 in comparison with their suspension formulations. The anti-inflammatory effects of MA/PEG SD and FFA/PEG SD were possible due to inhibition of prostaglandins and proinflammatory cytokines inductions.in vitro drug release studies. Physicochemical characterization of prepared SDs indicated that SDs of MA and FFA were developed successfully. In vitro drug release studies showed that SDs of MA and FFA prepared using PEG 4000 as carrier presented maximum drug release profile in comparison with pure MA, pure FFA and other SDs. Based on the best physicochemical parameters and maximum drug release profile, MA/PEG SD and FFA/PEG SD were selected for in vivo anti-inflammatory studies in male Wistar rats. In vivo anti-inflammatory studies in male Wistar rats showed that MA and FFA in their SDs were highly efficacious than their suspension formulations. MW SDs of MA as well as FFA were proved to be an excellent carriers for their oral administration. Overall, the results of this study showed that the MW SDs could be successfully used in the enhancement of in vitro dissolution rate and anti-inflammatory efficacy of poorly soluble drugs such as MA and FFA.In this study, various SDs of two anti-inflammatory drugs MA and FFA were developed using different carriers including PL, EPO, GLU and PEG 400 by MW technology. Prepared SDs of MA and FFA were characterized physicochemically and evaluated for