Search results for: knock-out drums

Authors: Sung-Hee Hwang, Michael Lee

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Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas mediated genome editing. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence specific double-stranded DNA (dsDNA) cleavage. Interestingly, ATG5 KO cells clearly showed a greater proliferation rate than WT NIH 3T3 cells, implying that autophagy induction is cytotoxic. Also, the clonogenic survival of ATG5 KO cells was greater than WT cells. The MTT assay revealed that the cytotoxic effect of PP2 was weaker on ATG5 knockout cells than that WT cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. Furthermore, Cyto-ID autophagy assay also revealed that PP2 failed to induce autophagy in ATG5 knockout cells. Together, our findings suggest that the resistance to PP2 in ATG5 knockout cells is associated with defective autophagy.

Keywords: ATG5 knockout, Autophagy, CRISPR/Cas9, PP2

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Authors: Onwumere O. J.

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The teaching of stress at any level of education could be a daunting task for the second language teacher because most times, they are bereft of the right approach to use in teaching it even at the fact is that, teaching it. But the fact is that teaching stress even at the middle basic could be interesting if the right approach is employed. To this end, the researcher was of the view that grouping could be a very good strategy to employ in order to sustain the interest of the learner and that the use at drums would be a good way to concretise the teaching of stress at this level. He was able to do this by discussing stress, grouping as a good technique, and the use of drums in teaching stress. To establish the fact that the use of drums would be very effective, four research questions contained in a questionnaire were structured. Three hundred (300) teachers of English in four tertiary institutions, three secondary schools and three primary schools in Nigeria were used. Based on the data analysis and findings, suggestions were given on how teachers and learners could use drums to make the teaching and learning of stress enjoyable for both teachers and learners at the middle basic of education.

Keywords: concretise, grouping, right approach, second language

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Authors: Mona Heydari, Ehsan Motamedian, Seyed Abbas Shojaosadati

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Prediction of perturbations after genetic manipulation (especially gene knockout) is one of the important challenges in systems biology. In this paper, a new algorithm is introduced that integrates microarray data into the metabolic model. The algorithm was used to study the change in the cell phenotype after knockout of Gss gene in Escherichia coli BW25113. Algorithm implementation indicated that gene deletion resulted in more activation of the metabolic network. Growth yield was more and less regulating gene were identified for mutant in comparison with the wild-type strain.

Keywords: metabolic network, gene knockout, flux balance analysis, microarray data, integration

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Authors: Qiuhua Miao, Peng Huang, Yifei Ding

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Particles with different properties tend to be unevenly distributed along an axial direction of the rotating drum, which is usually ignored. Therefore, it is important to study the relationship between axial segregation characteristics and particle crushing efficiency in longer drums. In this paper, a relative area total energy (RRTE) index is proposed, which aims to evaluate the overall crushing energy distribution characteristics. Based on numerical simulation verification, the proposed RRTE index can reflect the overall grinding effect more comprehensively, clearly representing crushing energy distribution in different drum areas. Furthermore, the proposed method is applied to the relation between axial segregation and crushing energy in drums. Compared with the radial section, the collision loss energy of the axial section can better reflect the overall crushing effect in long drums. The axial segregation characteristics directly affect the total energy distribution between medium and abrasive, reducing overall crushing efficiency. Therefore, the axial segregation characteristics should be avoided as much as possible in the crushing of the long rotary drum.

Keywords: relative regional total energy, crushing energy, axial segregation characteristics, rotary drum

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Authors: Roberto Coppo, Francesca Orso, Daniela Dettori, Elena Quaglino, Lei Nie, Mehran M. Sadeghi, Daniela Taverna

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Malignant melanoma is currently the fifth most common cancer in the white population and it is fatal in its metastatic stage. Several research studies in recent years have provided evidence that cancer initiation and progression are driven by genetic alterations of the tumor and paracrine interactions between tumor and microenvironment. Scattered data show that the Endothelial and Smooth muscle cell-Derived Neuropilin-like molecule (ESDN) controls cell proliferation and movement of stroma and tumor cells. To investigate the role of ESDN in the tumor microenvironment during melanoma progression, murine melanoma cells (B16 or B16-F10) were injected in ESDN knockout mice in order to evaluate how the absence of ESDN in stromal cells could influence melanoma progression. While no effect was found on primary tumor growth, increased cell extravasation and lung metastasis formation was observed in ESDN knockout mice compared to wild type controls. In order to understand how cancer cells cross the endothelial barrier during metastatic dissemination in an ESDN-null microenvironment, structure, and permeability of lung blood vessels were analyzed. Interestingly, ESDN knockout mice showed structurally altered and more permeable vessels compared to wild type animals. Since cell surface molecules mediate the process of tumor cell extravasation, the expression of a panel of extravasation-related ligands and receptors was analyzed. Importantly, modulations of N-cadherin, E-selectin, ICAM-1 and VAP-1 were observed in ESDN knockout endothelial cells, suggesting the presence of a favorable tumor microenvironment which facilitates melanoma cell extravasation and metastasis formation in the absence of ESDN. Furthermore, a potential contribution of immune cells in tumor dissemination was investigated. An increased recruitment of macrophages in the lungs of ESDN knockout mice carrying subcutaneous B16-F10 tumors was found. In conclusion, our data suggest a functional role of ESDN in the tumor microenvironment during melanoma progression and the identification of the mechanisms that regulate tumor cell extravasation could lead to the development of new therapies to reduce metastasis formation.

Keywords: melanoma, tumor microenvironment, extravasation, cell surface molecules

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Authors: Tianqi Yu, Yingnan Ding, Yina Zhang, Yulan Liu, Yahui Li, Jing Lei, Jiyong Zhou, Suquan Song, Boli Hu

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Circular RNAs (circRNAs) play critical roles in various diseases. However, whether and how circular RNA regulates influenza A virus (IAV) infection is unknown. Here, we studied the role of circular RNA GATA Zinc Finger Domain Containing 2A (circ-GATAD2A) in the replication of IAV H1N1 in A549 cells. Circ-GATAD2A was formed upon H1N1 infection. Knockdown of circ-GATAD2A in A549 cells enhanced autophagy and inhibited H1N1 replication. By contrast, overexpression of circ-GATAD2A impaired autophagy and promoted H1N1 replication. Similarly, knockout of vacuolar protein sorting 34 (VPS34) blocked autophagy and increased H1N1 replication. However, the expression of circ-GATAD2A could not further enhance H1N1 replication in VPS34 knockout cells. Collectively, these data indicated that circ-GATAD2A promotes the replication of H1N1 by inhibiting autophagy.

Keywords: autophagy, circ-GATAD2A, H1N1, replication

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Authors: Kinnsley Travis, Camerron M. Crowder

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Mapk8ip3 (Mitogen-Activated Protein Kinase 8 Interacting Protein 3) is a gene that codes for the JIP3 protein, which is a part of the JIP scaffolding protein family. This protein is involved in axonal vesicle transport, elongation and regeneration. Variants in the Mapk8ip3 gene are associated with a rare-genetic condition that results in a neurodevelopmental disorder that can cause a range of phenotypes including global developmental delay and intellectual disability. Currently, there are 18 known individuals diagnosed to have sequenced confirmed Mapk8ip3 genetic disorders. This project focuses on examining the impact of a subset of missense patient variants on the Jip3 protein function by overexpressing the mRNA of these variants in a zebrafish knockout model for Jip3. Plasmids containing cDNA with individual missense variants were reverse transcribed, purified, and injected into single-cell zebrafish embryos (Wild Type, Jip3 -/+, and Jip3 -/-). At 6-days post mRNA microinjection, morphological, behavioral, and microscopic phenotypes were examined in zebrafish larvae. Morphologically, we compared the size and shape of the zebrafish during their development over a 5-day period. Total locomotive activity was assessed using the Microtracker assay and patterns of movement over time were examined using the DanioVision assay. Lastly, we used confocal microscopy to examine sensory axons for swelling and shortened length, which are phenotypes observed in the loss-of-function knockout Jip3 zebrafish model. Using these assays during embryonic development, we determined the impact of various missense variants on Jip3 protein function, compared to knockout and wild-type zebrafish embryo models. Variants in the gene Mapk8ip3 cause rare-neurodevelopmental disorders due to an essential role in axonal vesicle transport, elongation and regeneration. A subset of missense variants was examined by overexpressing the mRNA of these variants in a Jip3 knock-out zebrafish. Morphological, behavioral, and microscopic phenotypes were examined in zebrafish larvae. Using these assays, the spectrum of disorders can be phenotypically determined and the impact of variant location can be compared to knockout and wild-type zebrafish embryo models.

Keywords: rare disease, neurodevelopmental disorders, mrna overexpression, zebrafish research

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Authors: Hassan Hussin Zwida

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The liquified LPG (Liquefied Petroleum Gas) drying system at the Complex is designed to remove water and mercaptans from the LPG stream. Upon saturation of the desiccant beds, a regeneration cycle becomes necessary. The original design routed the regeneration gas, produced during the LPG dryer heating cycle, to the sulfur recovery unit to the incineration. However, concerns regarding high temperatures and potential unit disruptions led to a modification where the gas is currently vented to the acid flare for the initial hour before being diverted to the LP network fuel gas system. While this addresses the temperature concerns, it generates significant smoke due to the presence of liquid hydrocarbons. This paper proposes an approach to recover the regeneration gas and redirect it back to the gas plant's (off-gas compressors) instead of sending it to the AC (Acid Flare), by utilizing the existing pipe 6” and connected to off gas compressor KO (Knock-Out ) Drums . This option is simple to operate, flexible, environment-friendly solution as long-term solution, lower in capital expenditure and increase the company's profitability. The feasibility of this proposal is supported by dynamic simulations. The simulations suggest the possibility of operating two out of the three off-gas compressors and LPG (Liquefied petroleum gas) as a liquid phase, is foreseen to be carried over and gathered at the bottom level of the KO (Knock-Out) Drum.

Keywords: thermal incinerator, off-gas compressors, environment, knock-out drums, acid flare

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Authors: Lívia Pimentel Sant'ana Dourado, Raquel Das Neves Almeida, Luís Henrique Costa Corrêa Neto, Nayara Soares, Kelly Grace Magalhães

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Introduction: Obesity and high-fat diet intake have a crucial impact on immune cells and inflammatory profile, highlighting an emerging realization that obesity is an inflammatory disease. In the present work, we aimed to characterize the role of caspase-1/11 and NLRP3 inflammasome in the establishment of mice obesity and modulation of inflammatory lipid metabolism induced by high fat diet intake. Methods and results: Wild type, caspase-1/11 and NLRP3 knockout mice were fed with standard fat diet (SFD) or high fat diet (HFD) for 90 days. The weight of animals was measured weekly to monitor the weight gain. After 90 days, the blood, peritoneal lavage cells, heart and liver were collected from mice studied here. Cytokines were measured in serum by ELISA and analyzed in spectrophotometry. Lipid antigen presentation molecule CD1d expression, reactive oxygen species (ROS) generation and lipid droplets biogenesis were analyzed in cells from mice peritoneal cavity by flow cytometry. Liver histopathology was performed for morphological evaluation of the organ. The absence of caspase-1/11, but not NLRP3, in mice fed with HFD favored the mice weight gain, increased liver size, induced development of hepatic steatosis and IL-12 secretion in mice compared to mice fed with SFD. In addition, caspase-1/11 knockout mice fed with HFD presented an increased CD1d molecule expression, as well as higher levels of lipid droplets biogenesis and ROS generation compared to wild type mice also fed with HFD. Conclusion: Our data suggest that caspase-1/11 knockout mice have greater susceptibility to obesity as well as increased activation of lipid metabolism and inflammatory markers.

Keywords: caspase 1, caspase 11, inflamassome, obesity, lipids

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Authors: Li-Jun Zheng

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The use of drums as an accompanying instrument is a common phenomenon in traditional Chinese folk rituals and musical culture. Especially, some folk rituals construct ritual-related sounds and give them ritual-specific symbolic and meaningful systems through the combination and use of multiple percussion instruments. The Yue drum(岳鼓), an asymmetrically shaped thin waist drum, is currently used in Han Chinese Shigong（师公）rituals in Guangxi, China, and is an important ritual instrument in Shigong rituals. This paper examines the use of the Yue drum and other percussion instruments in Han Chinese Shigong rituals in Guangxi, China, and shows the current status of combining instrumental accompaniment forms with human voices in Shigong rituals. Through further analysis of the musical and dance forms of Han Chinese Shigong rituals, this paper shows how Han Chinese Shigong ritual performers construct the ritual field through "sound-human-dance" and further explains the relationship between the existing and fictitious performance fields in the rituals. In addition, this paper demonstrates the relationship between Han Chinese Shigong rituals and the religious beliefs they involve, such as Taoism and Buddhism. And it further explores how performers in Han Chinese Shigong rituals use Yue drums for the dual purpose of "entertaining the gods" and "entertaining the people".

Keywords: sound research, the Han Chinese Shigong ritual, the thin waist drum, folk beliefs, ritual music

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Authors: Qiang Xu, Xingxin Wu, Wenjie Guo, Xingqi Wang, Yang Sun, Renxiang Tan

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The phosphatase SHP-2 is known to exert regulatory activities on cytokine receptor signaling and the dysregulation of SHP-2 has been implicated in the pathogenesis of a variety of diseases. Here we report several small molecule regulators of SHP-2 for the treatment of T cell-mediated diseases. The new cyclodepsipeptide trichomides A, isolated from the fermentation products of Trichothecium roseum, increased the phosphorylation of SHP-2 in activated T cells, and ameliorated contact dermatitis in mice. The trichomides A’s effects were significantly reversed by using the SHP-2-specific inhibitor PHPS1 or T cell-conditional SHP-2 knockout mice. Another compound is a cerebroside Fusaruside isolated from the endophytic fungus Fusarium sp. IFB-121. Fusaruside also triggered the tyrosine phosphorylation of SHP-2, which provided a possible mean of selectively targeting STAT1 for the treatment of Th1 cell-mediated inflammation and led to the discovery of the non-phosphatase-like function of SHP-2. Namely, the Fusaruside-activated pY-SHP-2 selectively sequestrated the cytosolic STAT1 to prevent its recruitment to IFN-R, which contributed to the improvement of experimental colitis in mice. Blocking the pY-SHP-2-STAT1 interaction, with SHP-2 inhibitor NSC-87877 or using T cells from conditional SHP-2 knockout mice, reversed the effects of fusaruside. Furthermore, the fusaruside’s effect is independent of the phosphatase activity of SHP-2, demonstrating a novel role for SHP-2 in regulating STAT1 signaling and Th1-type immune responses.

Keywords: SHP-2, small molecules, T cell, T cell-mediated diseases

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Authors: Ludmila A. Gavriliuc, Jerry McLarty, Heather E. Kleiner, J. Michael Mathis

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Abstract -- Background: The citrus coumarine Auraptene (Aur) is an effective chemopreventive agent, as manifested in many models of diseases and cancer. Nuclear factor erythroid 2-related factor (Nrf2) is an important regulator of genes induced by oxidative stress, such as glutathione S-transferases, heme oxygenase-1, and peroxiredoxin 1, by activating the antioxidant response element (ARE). Genetic and biochemical evidence has demonstrated that glutathione (GSH) and glutathione-dependent enzymes, glutathione reductase (GR), glutathione peroxidases (GPs), glutathione S-transferases (GSTs) are responsible for the control of intracellular reduction-oxidation status and participate in cellular adaptation to oxidative stress. The effect of Aur on the activity of GR, GPs (Se-GP and Se-iGP), and content of GSH in the liver, kidney, and spleen is insufficiently explored. Aim: Our goal was the examination of the Aur influence on the redox-system of GSH in Nrf2 wild type and Nrf2 knockout mice via activation of Nrf2 and ARE. Methods: Twenty female mice, 10 Nrf2 wild-type (WT) and 10 Nrf2 (-/-) knockout (KO), were bred and genotyped for our study. The activity of GR, Se-GP, Se-iGP, GST, G6PD, CytP450 reductase, catalase (Cat), and content of GSH were analyzed in the liver, kidney, and spleen using Spectrophotometry methods. The results of the specific activity of enzymes and the amount of GSH were analyzed with ANOVA and Spearman statistical methods. Results: Aur (200 mg/kg) treatment induced hepatic GST, GR, Se-GP activity and inhibited their activity in the spleen of mice, most likely via activation of the ARE through Nrf2. Activation in kidney Se-GP and G6PD by Aur is also controlled, apparently through Nrf2. Results of the non-parametric Spearman correlation analysis indicated the strong positive correlation between GR and G6PD only in the liver in WT control mice (r=+0.972; p < 0.005) and in the kidney KO control mice (r=+0.958; p < 0.005). The observed low content of GSH in the liver of KO mice indicated an increase in its participation in the neutralization of toxic substances with the absence of induction of GSH-dependent enzymes, such as GST, GR, Se-GP, and Se-iGP. Activation of CytP450 in kidney and spleen and Cat in the liver in KO mice probably revealed another regulatory mechanism for these enzymes. Conclusion: Thereby, obtained results testify that Aur can modulate the activity of genes and antioxidant enzymatic redox-system of GSH, responsible for the control of intracellular reduction-oxidation status.

Keywords: auraptene, glutathione, GST, Nrf2

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Authors: Juthamath Komvongsa, Bancha Mahong, Kannika Phasai, Sukanya Luang, Jong-Seong Jeon, James Ketudat-Cairns

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Os9BGlu31 is a rice transglucosidase that transfers glucosyl moieties to various acceptors such as carboxylic acids and alcohols, including phenolic acids and flavonoids, in vitro. The role of Os9BGlu31 transglucosidase in rice plant metabolism has not been reported to date. Methanolic extracts of rice bran and flag leaves were found to contain substrates to which Os9BGlu31 could transfer glucose from 4-nitrophenyl β -D-glucopyranoside donor. The semi-purified substrate from rice bran was found to contain oleic acid and linoleic acid and the pure fatty acids were found to act as acceptor substrates for Os9BGlu31 transglucosidase to form 1-O-acyl glucose esters. Os9BGlu31 showed higher activity with oleic acid (18:1) and linoleic acid (18:2) than stearic acid (18:0), and had both higher kcat and higher Km for linoleic than oleic acid in the presence of 8 mM 4NPGlc donor. This transglucosidase reaction is reversible, Os9bglu31 knockout rice lines of flag leaves were found to have higher amounts of fatty acid glucose esters than wild type control lines, these data conclude that fatty acid glucose esters act as glucosyl donor substrates for Os9BGlu31 transglucosidase in rice.

Keywords: fatty acid, fatty acid glucose ester, transglucosidase, rice flag leaf, homologous knockout lines, tandam mass spectrometry

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Authors: Rueyhung Weng, Chia-En Huang, Jung-Chi-Liao

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The transition zone (TZ) serves as a diffusion barrier to regulate the ins and outs of the proteins recruited to the primary cilia. TCTN2 is one of the TZ proteins and its mutation causes Joubert syndrome, a serious multi-organ disease. Despite its important medical relevance, the functions of TCTN2 remain elusive. Here we created a TCTN2 gene deleted retinal pigment epithelial cells (RPE1) using CRISPR/Cas9-based genome editing technique and used this knockout line to reveal roles of TCTN2. TCTN2 knockout RPE1 cells displayed a significantly reduced ciliogenesis or a shortened primary cilium length in the cilium-remaining population. Intraflagellar transport protein IFT88 aberrantly accumulated at the tip of TCTN2 deficient cells. Guanine nucleotide exchange factor Arl13B was mostly absent from the ciliary compartment, with a small population localizing at the ciliary tip. The deficient TZ was corroborated with the mislocalization of two other TZ proteins TMEM67 and MKS1. In addition, TCTN2 deficiency induced TZ impairment led to the suppression of Sonic hedgehog signaling in response to Smoothened (Smo) agonist. Together, depletion of TCTN2 destabilizes other TZ proteins and considerably alters the localization of key transport and signaling-associated proteins, including IFT88, Arl13B, and Smo.

Keywords: CRISPR/Cas9, primary cilia, Sonic hedgehog signaling, transition zone

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Authors: Summer Hassan, David Crossman

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Myocardial fibrosis (MF) has been loosely defined as the process occurring in the pathological remodeling of the myocardium due to excessive production and deposition of extracellular matrix (ECM) proteins, including collagen. This reduces tissue compliance and accelerates progression to heart failure, as well as affecting the electrical properties of the myocytes resulting in arrhythmias. Microscopic interrogation of MF is key to understanding the molecular orchestrators of disease. It is well-established that recruitment and stimulation of myofibroblasts result in Collagen deposition and the resulting expansion in the ECM. Many types of Collagens have been identified and implicated in scarring of tissue. In a series of experiments conducted at our lab, we aim to elucidate the role collagen VI plays in the development of myocardial fibrosis and its direct impact on myocardial function. This was investigated through an animal experiment in Rats with Collagen VI knockout diseased and healthy animals as well as Collagen VI wild diseased and healthy rats. Echocardiogram assessments of these rats ensued at four-time points, followed by microscopic interrogation of the myocardium aiming to correlate the role collagen VI plays in myocardial function. Our results demonstrate a deterioration in cardiac function as represented by the ejection fraction in the knockout healthy and diseased rats. This elucidates a potential protective role that collagen-VI plays following a myocardial insult. Current work is dedicated to the microscopic characterisation of the fibrotic process in all rat groups, with the results to follow.

Keywords: heart failure, myocardial fibrosis, collagen, echocardiogram, confocal microscopy

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Authors: Meiling Yu, Nadia Rouatbi, Khuloud T. Al-Jamal

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Background: Treatment of neurological disorders with modern medical and surgical approaches remains difficult. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. The treatment of brain diseases with gene therapy requires the gene-editing tool to be delivered efficiently to the central nervous system. In this study, we explored the efficiency of different delivery routes, namely intravenous (i.v.), intra-cranial (i.c.), and intra-nasal (i.n.), to deliver stable nucleic acid-lipid particles (SNALPs) containing gene-editing tools namely Cas9 mRNA and sgRNA encoding for GFP as a reporter protein. We hypothesise that SNALPs can reach the brain and perform gene-editing to different extents depending on the administration route. Intranasal administration (i.n.) offers an attractive and non-invasive way to access the brain circumventing the blood–brain barrier. Successful delivery of gene-editing tools to the brain offers a great opportunity for therapeutic target validation and nucleic acids therapeutics delivery to improve treatment options for a range of neurodegenerative diseases. In this study, we utilised Rosa26-Cas9 knock-in mice, expressing GFP, to study brain distribution and gene-editing efficiency of SNALPs after i.v.; i.c. and i.n. routes of administration. Methods: Single guide RNA (sgRNA) against GFP has been designed and validated by in vitro nuclease assay. SNALPs were formulated and characterised using dynamic light scattering. The encapsulation efficiency of nucleic acids (NA) was measured by RiboGreen™ assay. SNALPs were incubated in serum to assess their ability to protect NA from degradation. Rosa26-Cas9 knock-in mice were i.v., i.n., or i.c. administered with SNALPs to test in vivo gene-editing (GFP knockout) efficiency. SNALPs were given as three doses of 0.64 mg/kg sgGFP following i.v. and i.n. or a single dose of 0.25 mg/kg sgGFP following i.c.. knockout efficiency was assessed after seven days using Sanger Sequencing and Inference of CRISPR Edits (ICE) analysis. In vivo, the biodistribution of DiR labelled SNALPs (SNALPs-DiR) was assessed at 24h post-administration using IVIS Lumina Series III. Results: Serum-stable SNALPs produced were 130-140 nm in diameter with ~90% nucleic acid loading efficiency. SNALPs could reach and stay in the brain for up to 24h following i.v.; i.n. and i.c. administration. Decreasing GFP expression (around 50% after i.v. and i.c. and 20% following i.n.) was confirmed by optical imaging. Despite the small number of mice used, ICE analysis confirmed GFP knockout in mice brains. Additional studies are currently taking place to increase mice numbers. Conclusion: Results confirmed efficient gene knockout achieved by SNALPs in Rosa26-Cas9 knock-in mice expressing GFP following different routes of administrations in the following order i.v.= i.c.> i.n. Each of the administration routes has its pros and cons. The next stages of the project involve assessing gene-editing efficiency in wild-type mice and replacing GFP as a model target with therapeutic target genes implicated in Motor Neuron Disease pathology.

Keywords: CRISPR, nanoparticles, brain diseases, administration routes

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Authors: Trenten Theis, Trevor Daubert, Kennedy Kluthe, Austin Nuxoll

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Staphylococcus aureus is a gram-positive bacterium responsible for an estimated 23,000 deaths in the United States and 25,000 deaths in the European Union annually. Recurring S. aureus bacteremia is associated with biofilm-mediated infections and can occur in 5 - 20% of cases, even with the use of antibiotics. Despite these infections being caused by drug-susceptible pathogens, they are surprisingly difficult to eradicate. One potential explanation for this is the presence of persister cells—a dormant type of cell that shows a high tolerance to antibiotic treatment. Recent studies have shown a connection between low intracellular ATP and persister cell formation. Specifically, this decrease in ATP, and therefore increase in persister cell formation, is due to an interrupted tricarboxylic acid (TCA) cycle. However, S. aureus persister cells’ role in pathogenesis remains unclear. Initial studies have shown that a fumC (TCA cycle gene) knockout survives challenge from aspects of the innate immune system better than wild-type S. aureus. Specifically, challenges from two antimicrobial peptides--LL-37 and hBD-3—show a log increase in survival of the fumC::N∑ strain compared to wild type S. aureus after 18 hours. Furthermore, preliminary studies show that the fumC knockout has a log more survival within a macrophage. These data lead us to hypothesize that the fumC knockout is better suited to other aspects of the innate immune system compared to wild-type S. aureus. To further investigate the mechanism for increased survival of fumC::N∑ within a macrophage, we tested bacterial growth in the presence of reactive oxygen species (ROS), reactive nitrogen species (RNS), and a low pH. Preliminary results suggest that the fumC knockout has increased growth compared to wild-type S. aureus in the presence of all three antimicrobial factors; however, no difference was observed in any single factor alone. To investigate survival within a host, a nine-day biofilm-associated catheter infection was performed on 6–8-week-old male and female C57Bl/6 mice. Although both sexes struggled to clear the infection, female mice were trending toward more frequently clearing the HG003 wild-type infection compared to the fumC::N∑ infection. One possible reason for the inability to reduce the bacterial burden is that biofilms are largely composed of persister cells. To test this hypothesis further, flow cytometry in conjunction with a persister cell marker was used to measure persister cells within a biofilm. Cap5A (a known persister cell marker) expression was found to be increased in a maturing biofilm, with the lowest levels of expression seen in immature biofilms and the highest expression exhibited by the 48-hour biofilm. Additionally, bacterial cells in a biofilm state closely resemble persister cells and exhibit reduced membrane potential compared to cells in planktonic culture, further suggesting biofilms are largely made up of persister cells. These data may provide an explanation as to why infections caused by antibiotic-susceptible strains remain difficult to treat.

Keywords: antibiotic tolerance, Staphylococcus aureus, host-pathogen interactions, microbial pathogenesis

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Authors: Josephine Hai, Jie Jiang, Karen M. Lyons

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Of the musculoskeletal tissues, tendon is least understood in terms of biological development. The current study examines Cysteine-rich angiogenic inducer 61, or CCN1, a member of the CCN family of secreted matricellular proteins that regulate cell behavior via intercellular signaling. Though CCN1 is notable in limiting fibrosis by inducing senescence in fibroblasts, little is known about its role in normal fibrous tissue, where it may be essential to the development of ECM-rich structures like tendons. We found that CCN1 knockout mice (using limb-specific Prx1-Cre) exhibited clubfoot and waddling gaits, a unique phenotype not described in any other mutant to date. Histological analysis showed that the Achilles and patellar tendons, where we previously found high CCN1 expression in adult reporter mice, were thicker and denser in the Prx1-Cre knockouts than in their wildtype littermates. We then hypothesized that CCN1 is required directly in tendon progenitor cells for normal tendon development and generated tendon-specific CCN1 knockout mice using Scx-Cre. We observed similar Achilles/patellar tendon morphology among the Scx-Cre and Prx1-Cre mutants, indicating that the phenotype is a direct result of CCN1’s loss in tendon. To further study phenotype onset and progression, we will histologically characterize these tendons across different developmental time-points. We will also perform RNA-seq and qPCR to analyze tenocyte gene expression and expect fibrotic marker upregulation in the Scx-Cre mutants if CCN1 is required to maintain a normal tendon phenotype. Thus, our study aims to elucidate the molecular mechanisms underlying tendon formation and maintenance. Understanding tendons at the most basic level invites novel approaches to tendon repair.

Keywords: development, matricellular, musculoskeletal, tendon

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Authors: Jin Young Kim, Kwon Kyoo Kang

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The SGR1 (STAYGREEN1) protein is a critical regulator of plant leaves in chlorophyll degradation and senescence. The functions and mechanisms of tomato SGR1 action are poorly understood and worthy of further investigation. To investigate the function of the SGR1 gene, we generated a SGR1-knockout (KO) null line via clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene editing and conducted RNA sequencing and gas chromatography tandem mass spectrometry (GC-MS/MS) analysis to identify the differentially expressed genes. The SlSGR1 (Solanum lycopersicum SGR1) knockout null line clearly showed a turbid brown color with significantly higher chlorophyll and carotenoid content compared to wild-type (WT) fruit. Differential gene expression analysis revealed 728 differentially expressed genes (DEGs) between WT and sgr1 #1-6 line, including 263 and 465 downregulated and upregulated genes, respectively, for which fold change was >2, and the adjusted p-value was <0.05. Most of the DEGs were related to photosynthesis and chloroplast function. In addition, the pigment, carotenoid changes in sgr1 #1-6 line was accumulated of key primary metabolites such as sucrose and its derivatives (fructose, galactinol, raffinose), glycolytic intermediates (glucose, G6P, Fru6P) and tricarboxylic acid cycle (TCA) intermediates (malate and fumarate). Taken together, the transcriptome and metabolite profiles of SGR1-KO lines presented here provide evidence for the mechanisms underlying the effects of SGR1 and molecular pathways involved in chlorophyll degradation and carotenoid biosynthesis.

Keywords: tomato, CRISPR/Cas9, null line, RNA-sequencing, metabolite profiling

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Authors: Muhammad Awais Afzal, Lorena Tuchscherr De Hauschopp, Christian Hübner

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Introduction: Autophagy is an evolutionarily conserved lysosome-dependent degradation pathway, which can be induced by extrinsic and intrinsic stressors in living systems to adapt to fluctuating environmental conditions. In the context of inflammatory stress, autophagy contributes to the elimination of invading pathogens, the regulation of innate and adaptive immune mechanisms, and regulation of inflammasome activity as well as tissue damage repair. Lysosomes can be recycled from autolysosomes by the process of autophagic lysosome reformation (ALR), which depends on the presence of several proteins including Spatacsin. Thus ALR contributes to the replenishment of lysosomes that are available for fusion with autophagosomes in situations of increased autophagic turnover, e.g., during bacterial infections, inflammatory stress or sepsis. Objectives: We aimed to assess whether ALR plays a role for cell survival in an in-vitro bacterial infection model. Methods: Mouse embryonic fibroblasts (MEFs) were isolated from wild-type mice and Spatacsin (Spg11-/-) knockout mice. Wild-type MEFs and Spg11-/- MEFs were infected with Staphylococcus aureus (multiplication of infection (MOI) used was 10). After 8 and 16 hours of infection, cell viability was assessed on BD flow cytometer through propidium iodide intake. Bacterial intake by cells was also calculated by plating cell lysates on blood agar plates. Results: in-vitro infection of MEFs with Staphylococcus aureus showed a marked decrease of cell viability in ALR deficient Spatacsin knockout (Spg11-/-) MEFs after 16 hours of infection as compared to wild-type MEFs (n=3 independent experiments; p < 0.0001) although no difference was observed for bacterial intake by both genotypes. Conclusion: Suggesting that ALR is important for the defense of invading pathogens e.g. S. aureus, we observed a marked increase of cell death in an in-vitro infection model in cells with compromised ALR.

Keywords: autophagy, autophagic lysosome reformation, bacterial infections, Staphylococcus aureus

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Authors: Ozden Saygili, Eser Cakti

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Mosques and minarets can be severely damaged as a result of earthquakes. Non-linear behavior of minarets of Mihrimah Sultan and Süleymaniye Mosques and the minaret of St. Sophia are analyzed to investigate seismic response, damage and failure mechanisms of minarets during earthquake. Selected minarets have different height and diameter. Discrete elements method was used to create the numerical minaret models. Analyses were performed using sine waves. Two parameters were used for evaluating the results: the maximum relative dislocation of adjacent drums and the maximum displacement at the top of the minaret. Both parameters were normalized by the drum diameter. The effects of minaret geometry on seismic behavior were evaluated by comparing the results of analyses.

Keywords: discrete element method, earthquake safety, nonlinear analysis, masonry structures

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Authors: Sunil Bandaru, Suresh Yv, Srinivas Vanapalli

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Heavy machinery especially compactors has more vibrations induced from the compactor mechanism than the engines. Since the operator’s comfort is most important in any of the machines, this paper shows interest in studying the vibrations on the steering wheel for a double drum compactor. As there are no standard procedures available for testing vibrations on the steering wheel of double drum compactors, this paper tries to evaluate the vibrations on the steering wheel by considering most of the possibilities. In addition to the feasibility for the operator to adjust the steering wheel tilt as in the case of automotive, there is an option for the operator to change the orientation of the operator platform for the complete view of the road’s edge on both the ends of the front and rear drums. When the orientation is either +/-180°, the operator will be closer to the compactor mechanism; also there is a possibility for the shuffle in the modes with respect to the operator. Hence it is mandatory to evaluate the vibrations levels in both cases. This paper attempts to evaluate the vibrations on the steering wheel by considering the two operator platform positions and three steering wheel tilting angles.

Keywords: FEA, CAE, steering column, steering column orientation position

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Authors: Ahmad Al Aboudi

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The Department of Archaeology at King Saud University conduct a long Term excavations since 2004 at the archaeological site of (Qurh) in Al-Ula area. The history of the site goes back to the eighth century AD. The main aim of the excavations is the training of the students on the archaeological field work associated with the scientific skills of exploring, surveying, classifying, documentations and other necessary in the field archaeology. During the 12th Season of Excavations, an area of 20 × 40 m2 of the site was excavated. The depth of the excavating the site was reached to 2-3 m. Many of the architectural features of a residential area in the northern part of the site were excavated this season. Circular walls made of mud-brick and a brick column drums and tiles made of clay were revealed this season. Additionally, lots of findings such as Gemstones, jars, ceramic plates, metal, glass, and fabric, as well as some jewelers and coins were discovered. This paper will deal with the main results of this field project including the architectural features and phenomena and their interpretations, the classification of excavated material culture remains and stratigraphy.

Keywords: Islamic architecture, Islamic art, excavations, early Islamic city

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Authors: Somboon Sarasit

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This research aims to develop a model small ceramic kiln using burner from a high-pressure household gas stove. The efficiency of the kiln and community technology transfer. The study of history shows that this area used to be a source of pottery on the old capital of Ayutthaya. There is evidence from pottery kilns unearthed many types of wood kiln since 2535 and was assumed that the production will end when the war with Burma in the Ayutthaya period. The result of the research design and performance testing of ceramic kiln using burners by gas cooker and outside from 200-liter steel drums inside with ceramic fiber. It was found that the Graze Firing of the products to be at a temperature of 1230°C. The duration of the burn approximately 5-6 hours and uses only 3-4 kg of LPG products, a coffee can burn up to 40-50 pieces. It is an energy-efficient Kiln. Use safe and appropriate opportunities for entrepreneurs, small ceramic and entrepreneurs with new investments or those who want to produce ceramic products as a hobby. The community interest in the pottery to create a new one to continue the product development and manufacturing in the harshest existence forever.

Keywords: ceramics kiln design and development, ceramic gas kiln, burners application, high-pressure of household gas stove

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Authors: H. A. J. Nguyen, V. A. Pham

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RE:SOUNDING is an ongoing research project and artwork seeking to return the sound and knowledge of Dong Son bronze drums back to contemporary musicians. Colonial collections of ethnographic instruments are problematic in how they commit acts of conceptual, cultural, and acoustic silencing. The collection (or more honestly), the plagiarism, and pillaging of these instruments have systemically separated them from living and breathing cultures. This includes diasporic communities, who have come to resettle in close proximity - but still have little access - to the museums and galleries that display their cultural objects. Despite recent attempts to 'open up' and 'recognise' the tensions and violence of these ethnographic collections, many museums continue to structurally organize and reproduce knowledge with the same procedural distance and limitations of imperial condescension. Impatient with the slowness of these museums, our diaspora led collaborations participated in the opaque economy of the auction market to gain access and begin the process of digitally recording and archiving the actual sounds of the ancient Dong Son drum. This self-directed, self-initiated artwork not only acoustically reinvigorated an ancient instrument but redistributed these sonic materials back to contemporary musicians, composers, and their diasporic communities throughout Vietnam, South East Asia, and Australia. Our methodologies not only highlight the persistent inflexibility of museum infrastructures but demand that museums refrain from their paternalistic practice of risk-averse ownership, to seriously engage with new technologies and political formations that require all public institutions to be held accountable for the ethical and intellectual viability of their colonial collections. The integrated and practical resolve of diasporic artists and their communities are more than capable of working with new technologies to reclaim and reinvigorate what is culturally and spiritually theirs. The motivation to rematriate – as opposed to merely repatriate – the acoustic legacies of these instruments to contemporary musicians and artists is a new model for decolonial and restorative practices. Exposing the inadequacies of western scholarship that continues to treat these instruments as discreet, disembodied, and detached artifacts, these collaborative strategies have thus far produced a wealth of new knowledge – new to the west perhaps – but not that new to these, our own communities. This includes the little-acknowledged fact that the Dong Son drum were political instruments of war and technology, rather than their simplistic description in the museum and western academia as agrarian instruments of fertility and harvest. Through the collective and continued sharing of knowledge and sound materials produced from this research, these drums are gaining a contemporary relevance beyond the cultural silencing of the museum display cabinet. Acknowledgement: We acknowledge the Wurundjeri and Boon Wurrung of the Kulin Nation and the Gadigal of the Eora Nation where we began this project. We pay our respects to the Peoples, Lands, Traditional Custodians, Practices, and Creator Ancestors of these Great Nations, as well as those First Nations peoples throughout Australia, Vietnam, and Indonesia, where this research continues, and upon whose stolen lands and waterways were never ceded.

Keywords: acoustic archaeology, decolonisation, museum collections, rematriation, repatriation, Dong Son, experimental music, digital recording

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Authors: Eun Gyeong Lee, Ji Young Park, Hyun Ae Woo

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Reactive oxygen species (ROS) production is involved in ischemia/reperfusion (I/R) injury in kidney of mice. Oxidative stress develops from an imbalance between ROS production and reduced antioxidant defenses. Many enzymatic and nonenzymatic antioxidant systems including peroxiredoxins (Prxs) are present in kidney to maintain an appropriate level of ROS and prevent oxidative damage. Prxs are a family of peroxidases that reduce peroxides, with a conserved cysteine residue serving as the site of oxidation by peroxides. In this study, we examined the protective role of Prx V against I/R-induced acute kidney injury (AKI) using Prx V wild type (WT) and knockout (KO) mice. We compared the response of Prx V WT and KO mice in mice model of I/R injury. Renal structure, functions, oxidative stress markers, protein levels of oxidative damage marker were worse in Prx V KO mice. Ablation of Prx V enhanced susceptibility to I/R-induced oxidative stress. Prx V KO mice were seen to have more severe renal damage than Prx V WT mice in mice model of I/R injury. Our results demonstrate that Prx V is protective against I/R-induced AKI.

Keywords: peroxiredoxin, ischemia/reperfusion, kidney, oxidative stress

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Authors: Rezwana Rahman

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Various simulations have been conducted to understand the particle's macroscopic behavior in the solid-gas multiphase flow in rotating drums in the past. In these studies, the particle-wall no-slip boundary condition was usually adopted. However, the non-slip boundary condition is rarely encountered in real systems. A little effort has been made to investigate the particle behavior at slip boundary conditions. The paper represents a study of the gas-solid flow in a horizontal rotating drum at a slip boundary wall condition. Two different sizes of particles with the same density have been considered. The Eulerian–Eulerian multiphase model with the kinetic theory of granular flow was used in the simulations. The granular pressure at the rolling flow regime with specularity coefficient 1 was examined and compared with that obtained based on the no-slip boundary condition. The results reveal that the profiles of granular pressure distribution on the transverse plane of the drum are similar for both boundary conditions. But, overall, compared with those for the no-slip boundary condition, the values of granular pressure for specularity coefficient 1 are larger for the larger particle and smaller for the smaller particle.

Keywords: boundary condition, eulerian–eulerian, multiphase, specularity coefficient, transverse plane

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Authors: Nafisa Komilova, Plamena Angelova, Andrey Abramov, Ulugbek Mirkhodjaev

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Parkinson’s disease (PD) is a progressive neurodegenerative disorder which is induced by the loss of dopaminergic neurons in the midbrain. The mechanism of neurodegeneration is associated with the aggregation of misfolded proteins, oxidative stress, and mitochondrial disfunction. Considering this, the process of removal of unwanted organelles or proteins by autophagy is vitally important in neurons, and activation of these processes could be protective in PD. Short-time acidification of cytosol can activate mitophagy and autophagy, and here we used sodium pyruvate and sodium lactate in human fibroblasts with PD mutations (Pink1, Pink1/Park2, α-syn triplication, A53T) to induce changes in intracellular pH. We have found that both lactate and pyruvate in millimolar concentrations can induce short-time acidification of cytosol in these cells. It induced activation of mitophagy and autophagy in control and PD fibroblasts and protected against cell death. Importantly, the application of lactate to acute brain slices of control and Pink1 knockout mice also induced a reduction of pH in neurons and astrocytes that increase the level of mitophagy. Thus, acidification of cytosol by compounds which play important role in cell metabolism also can activate mitophagy and autophagy and protect cells in the familial form of PD.

Keywords: Parkinson's disease, mutations, mitophagy, autophagy

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Authors: Manal Alosaimi

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Many rehabilitation strategies exist to aid persons with neurological disorders relearn motor skills through intensive training. Evidence supporting the theory that cortical areas involved in motor execution can be triggered by observing actions performed by others is attributed to the function of the mirror neuron system (MNS) indicates that activation of the MNS is associated with improvements in physical action and motor learning. Therefore, it is important to investigate the relationship between motor training (in this case, playing the drums) and the activation of the MNS. To achieve this, 15 healthy right-handed participants received drum-kit training for 21 weeks, during which time blood oxygen level-dependent (BOLD) signals were monitored in the brain using functional magnetic resonance imaging (fMRI). Participants were required to perform action–observation and action–execution fMRI tasks. The main results are that BOLD signals in classical regions of the MNS such as supramarginal gyri, inferior parietal lobule, and supplementary motor area increase significantly over the training period. Activation of these areas indicates that passive-observation of others performing these same skills may facilitate recovery of persons suffering from neurological disorders, and complement conventional rehabilitation programs that focus on action execution or intense training.

Keywords: fMRI, mirror neuron system, magnetic resonance imaging, neuroplasticity, drumming, learning, music, action observation, action execution

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Authors: M. J. Kim, D. B. Lee

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Centrifugal casting is a metal casting method that uses forces make by centripetal acceleration to distribute molten material in mold. Centrifugal cast parts manufactured in industry contain gas pipes and water supply lines, moreover rings, turbocharger, bushings, brake drums. Turbochargers were exposed to exhaust temperatures of 900-1050°C require a material for the corrosion resistance that will withstand such high component temperatures during the entire service life of the vehicle. Hence, the study of corrosion resistance for turbocharger is important for practical application. DIN1.4091 steels were used widely. The DIN1.4091 steels whose compositions were Fe-34.4Cr-14.5Ni-2.5Mo-0.4W-0.4Mn-0.5Si-(0.009 or 0.35)S (wt.%) were centrifugally cast, and oxidized at 900°C for 50-200 h in order to find the effect of sulfur on the high-temperature oxidation of Fe-34.4Cr-14.5Ni-2.5Mo-0.4W-0.4Mn-0.5Si-(0.009 or 0.35)S (wt.%) alloys. These alloys formed oxide scales that consisted primarily of Cr₂O₃ as the major oxide and Cr₂MnO₄ as the minor one through preferential oxidation of Cr and Mn. Cr formed a thin CrOx oxide film on the surface to prevent further oxidation, and when it is added more than 20%, the sulphide decreased corrosion rate. The high affinity of Mn with S, led to the formation of scattered MnS inclusions, particularly in the 0.35S-containing cast alloy. Sulfur was harmful to the oxidation resistance because it deteriorated the scale/alloy adherence so as to accelerate the adherence and compactness of the formed scales. Acknowledgement: This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1A2B1013169).

Keywords: centrifugal casting, turbocharger, sulfur, oxidation, Fe-34.4Cr-14.5Ni alloy

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