Optimization of acoustic performance of EPDM-based foams using Taguchi design of experiments: Appropriate content of additives

Novel EPDM-based polymer foams were prepared using a combination of nanomaterials, namely nano silica, nano clay, and graphene nanoplatelets. In order to achieve optimal acoustic performance, the Taguchi design (TD) technique was applied to reduce the number of experiments and optimize the formulation. By employing an orthogonal array of L9(34), four controlled factors, including content of the three nanomaterials and the blowing agent (Unicell D200A), were chosen. In practice, the acoustic properties of the nine suggested experiments with TD were examined with an impedance tube, and the signal-to-noise ratio analysis revealed two more optimal formulations for foam composites. Further experiments for the last two formulations compared to the nine Taguchi tests, showed an improvement of 13.04 and 19.68%, respectively, for noise reduction coefficient (NRC) and average transmission loss (ATL). It seemed that the idea of using multiple nanomaterials simultaneously is to be an effective way. Besides, the SEM images of nine samples proved that the smaller cell size of the foam were achieved using the higher concentration of nanoparticles. These findings are in accordance with the acoustic results, as the sample with larger cell size and more open cells (C3) showed higher NRC and the sample with larger cell size and closed cells (B2) showed higher ATL values. To complete the study, some blank samples with zero level or only one type of the nanomaterial were also investigated. Interestingly, the obtained results indicated that the formula should contain more than one type of nanoparticle to achieve a better acoustic performance. Comparing the result obtained in this study for EPDM foam with the same EVA foam in our previous work, it can be seen that EPDM showed an increase of 15.56% in NRC and a slight decrease of 2.5% in ATL. This behavior could be due to the difference in their morphology, in which the EPDM has probably more open cells and thinner cell walls

Aquaporins - from ion channels to human cancers

Despite advances in diagnostic techniques and cancer care management, cancer continues to be one of the leading causes of death worldwide. This thesis used two different approaches to advance cancer treatments: i) In the first approach, we used natural compounds as promising sources of new agents for controlling cancer proliferation and metastasis, and ii) second, we developed a mass-throughput technique for exploring the role and significance of aquaporins in cancer development. In phase 1A (chapter 2), using computational and experimental biology approaches, we identified candidate mechanisms of action of a traditional Chinese medicine, Compound Kushen Injection (CKI), in a breast cancer cell line. CKI disrupts the cell cycle and induces apoptosis in breast cancer cells; however, the exact mechanism of its single compounds and their effects on cancer proliferation, migration and invasion remained unknown. High-performance liquid chromatography (HPLC) fractionation and molecular biology techniques were used to define chemical fractions required for CKI to induce apoptosis. Bioinformatic analysis of RNA-seq data revealed correlations between different compounds and gene expression and phenotype. In phase 1B (chapter 3), CKI, fractionated mixtures, and isolated components were tested on migration assays with colon (HT-29, SW-480, DLD-1), brain (U87-MG, U251- MG), and breast (MDA-MB-231) cancer cell lines. Human embryonic kidney (HEK-293) and human foreskin fibroblast (HFF) served as non-cancerous controls. Wound closure, transwell invasion, and live cell imaging showed CKI reduced motility in all eight lines. Fractionation and reconstitution of CKI study on cancer cell lines demonstrated that combinations of compounds were required for activity. Live cell imaging confirmed that whole CKI strongly reduced migration of HT-29 and MDA-MB-231 cells, moderately slowed brain cancer cells, and had no effect on HEK-293. CKI uniformly blocked invasiveness through extracellular matrix. Apoptosis was increased by CKI in breast cancer but not in non-cancerous lines. Cell viability was not affected by CKI in all cell lines. Transcriptomic analyses of MDA-MB-231 indicated down-regulation of actin cytoskeletal and focal adhesion genes with CKI, consistent with the observed impairment of cell migration. As a result, we found the pharmacological complexity of CKI is important for effective blockade of cancer proliferation, cell migration and invasion. In phase 2 (chapter 4-5), our aim was to investigate the primary dogma that aquaporins (AQP) are only permeable to water and glycerol. Aquaporins are of interest internationally as therapeutic targets for treatment strategies in diverse classes of cancers, but understanding of their full range of substrate permeabilities remains incomplete. Our primarily aim was to discover new classes of aquaporins that serve as dual water and ion channels and then provide better insights into the novel function of aquaporins, their mechanism of gating and signaling networks in human-related diseases such cancer. Using a combination of molecular biology, electrophysiology, and computational biology, we introduced the first unbiased screening method for ion channel activity across all 13 classes of human aquaporins, addressing a major gap in knowledge. Using known AQP ion channel, hAQP1, we optimized an assay which, unlike traditional electrophysiology methods, provides 1- an unbiased high-throughput screen of ion channel functionality of diverse phyla, 2- screening a large number of intracellular signals that might govern their activity and function, 3- mass-screening of drugs, and 4- a broad range of mutagenesis study of AQP ion channels shorter time frame. Strikingly, we found all hAQPs appear to have cation permeability, though to some different degree. Moreover, we noted that ion functionality of hAQPs, unlike most of other ion channels, is active from acidic to neutral pH values ( pH 5.0-7.4). Finally, following our study in chapter 4, we used a combination of wet and dry lab approaches to investigate the potential significance of hAQPs in cancer development. Using transcriptome analysis, we identified an association between AQP mRNA expression and cancer severity and their translational importance in patient tissue samples. As a result, we found AQP9, -7, -5 and -3 as the most promising prognostic marker among other hAQPs in common cancers. This was followed by unrevealing these four AQP ion permeability modulatory mechanism using our optimised yeast screening. In summary, this work augmented our understanding of the fundamental properties of natural compounds for cancer treatment and introduced a novel approach to dissecting their downstream targets in different hallmarks of cancer. Moreover, we further discovered a new set of AQP ion channels and revealed their potential prognostic values in cancer. Outcomes from this dissertation are likely to serve as a strong foundation for the future basic research and clinical innovation and shed more light on the significance of ion channels in cancer development and paved the way for developing an AQP-based therapy.Thesis (Ph.D.) -- University of Adelaide, School of Biomedicine, 202

Identification of Loop D Domain Amino Acids in the Human Aquaporin-1 Channel Involved in Activation of the Ionic Conductance and Inhibition by AqB011

Aquaporins are integral proteins that facilitate the transmembrane transport of water and small solutes. In addition to enabling water flux, mammalian Aquaporin-1 (AQP1) channels activated by cyclic GMP can carry non-selective monovalent cation currents, selectively blocked by arylsulfonamide compounds AqB007 (IC50 170 μM) and AqB011 (IC50 14 μM). In silico models suggested that ligand docking might involve the cytoplasmic loop D (between AQP1 transmembrane domains 4 and 5), but the predicted site of interaction remained to be tested. Work here shows that mutagenesis of two conserved arginine residues in loop D slowed the activation of the AQP1 ion conductance and impaired the sensitivity of the channel to block by AqB011. Substitution of residues in loop D with proline showed effects on ion conductance amplitude that varied with position, suggesting that the structural conformation of loop D is important for AQP1 channel gating. Human AQP1 wild type, AQP1 mutant channels with alanines substituted for two arginines (R159A+R160A), and mutants with proline substituted for single residues threonine (T157P), aspartate (D158P), arginine (R159P, R160P), or glycine (G165P) were expressed in Xenopus laevis oocytes. Conductance responses were analyzed by two-electrode voltage clamp. Optical osmotic swelling assays and confocal microscopy were used to confirm mutant and wild type AQP1-expressing oocytes were expressed in the plasma membrane. After application of membrane-permeable cGMP, R159A+R160A channels had a significantly slower rate of activation as compared with wild type, consistent with impaired gating. AQP1 R159A+R160A channels showed no significant block by AqB011 at 50 μM, in contrast to the wild type channel which was blocked effectively. T157P, D158P, and R160P mutations had impaired activation compared to wild type; R159P showed no significant effect; and G165P appeared to augment the conductance amplitude. These findings provide evidence for the role of the loop D as a gating domain for AQP1 ion channels, and identify the likely site of interaction of AqB011 in the proximal loop D sequence

Divalent Cations Regulate the Ion Conductance Properties of Diverse Classes of Aquaporins

Aquaporins (AQPs) are known to facilitate water and solute fluxes across barrier membranes. An increasing number of AQPs are being found to serve as ion channels. Ion and water permeability of selected plant and animal AQPs (plant Arabidopsis thaliana AtPIP2;1, AtPIP2;2, AtPIP2;7, human Homo sapiens HsAQP1, rat Rattus norvegicus RnAQP4, RnAQP5, and fly Drosophila melanogaster DmBIB) were expressed in Xenopus oocytes and examined in chelator-buffered salines to evaluate the effects of divalent cations (Ca2+, Mg2+, Ba2+ and Cd2+) on ionic conductances. AtPIP2;1, AtPIP2;2, HsAQP1 and DmBIB expressing oocytes had ionic conductances, and showed differential sensitivity to block by external Ca2+. The order of potency of inhibition by Ca2+ was AtPIP2;2 > AtPIP2;1 > DmBIB > HsAQP1. Blockage of the AQP cation channels by Ba2+ and Cd2+ caused voltage-sensitive outward rectification. The channels with the highest sensitivity to Ca2+ (AtPIP2;1 and AtPIP2;2) showed a distinctive relief of the Ca2+ block by co-application of excess Ba2+, suggesting that divalent ions act at the same site. Recognizing the regulatory role of divalent cations may enable the discovery of other classes of AQP ion channels, and facilitate the development of tools for modulating AQP ion channels. Modulators of AQPs have potential value for diverse applications including improving salinity tolerance in plants, controlling vector-borne diseases, and intervening in serious clinical conditions involving AQPs, such as cancer metastasis, cardiovascular or renal dysfunction