Prevention of Pseudomonas aeruginosa adhesion by electric currents.

Shim, Soojin,Hong, Seok Hoon,Tak, Yongsug,Yoon, Jeyong Harwood Academic Publishers 2011 Biofouling Vol.27 No.2

<P>The process of controlling bacterial adhesion using an electric current deserves attention because of its ease of automation and environmentally friendly nature. This study investigated the role of electric currents (negative, positive, alternating) for preventing adhesion of Pseudomonas aeruginosa and achieving bacterial inactivation. Indium tin oxide (ITO) film was used as a working electrode to observe adhesion and inactivation under electric polarization. Electric current types were classified into negative, positive, and alternating current. The working electrode acted as a cathode or anode by applying a negative or positive current, and an alternating current indicates that the negative current was combined sequentially with the positive current. The numbers of adhered cells were compared under a flow condition, and the in situ behavior of the bacterial cells and the extent of their inactivation were also investigated using time-lapse recording and live/dead staining, respectively. The application of a negative current prevented bacterial adhesion significantly (??1% at 15.0 관A cm(-2)). The positive current did not significantly inhibit adhesion (<20% at 15.0 관A cm(-2)), compared to the nonpolarized case. The alternating current had a similar effect as the negative current on preventing bacterial adhesion, but it also exhibited bactericidal effects, making it the most suitable method for bacterial adhesion control.</P>

Analysis of RNA expression patterns in THP-1 cells after B. abortus mutant infection.

( Soojin Shim ),( Young Bin Im ),( Woo Bin Park ),( Han Sang Yoo ) 대한인수공통전염병학회 2017 창립총회 및 학술대회 초록집 Vol.2017 No.1

Introduction: Brucella abortus (B. abortus) is a zoonotic pathogen that mainly causes abortion in domestic animals and serious symptoms in human. Early detection and removal of the intracellular bacteria have been received attention because these intracellular bacteria invade and survive in macrophages through their ability to modulate host cell function. Therefore, identification of interaction between B. abortus and host cells could be crucial to control the bacteria. In this study, B. abortus mutant strains were generated using transposon mutagenesis. The disrupted genes of the mutant strains, C1, C10, C27 and C32, were analyzed through genome sequencing and THP-1 cell, the human macrophage cell line was infected by these mutant strains to profile transcriptome. Methods: Firstly, genome sequence was performed using Illumina Hi-seq 2500 to identify the insertion site of transposon by NICEM. Then, THP-1 cells were each grown in 2 x 10⁵ cells per ml in 6-well plates and infected with the B. abortus wild type and the mutant strains at a MOI of 1: 100. The cells infected by B. abortus were lysed and total RNA was extracted from the samples. Then, microarray was conducted. Statistical significance of the expression data was determined using LPE test and fold change and the microarray result was verified through real-time PCR. Gene-Enrichment and Functional Annotation analysis for significant probe list was performed using Gene Ontology (http://geneontology.org) and KEGG pathway(http://kegg.jp). Microarray using obtained total RNA was conducted. Statistical significance of the expression data was determined using LPE test and fold change and the microarray result was verified through real-time PCR. Results: As a result, the insertion sites of transposon of C1, C10, C27 and C32 mutants were BRUAB_RS13225, BRUAB_RS00455, BRUAB_RS03675, BRUAB_RS08885. Also microarray data showed 391, 428, 459 and 210 of genes expression changes as compared with the cells infected to the B. abortus wild type strain. Genes with significant changes in expression were associated with the cellular process such as expression of interferon by infection (IFI44L, ISG20) and promoting M1 differentiation of macrophages (CCL2, CCL8, CXCL10, CXCL13). Gene network analysis suggest that B. abortus wild type, mutant strain C27 and C32 was activated inflammatory response by expression of CCL3, APOL3 and IFIH1. Also, it implies that mutant strain C1 and C10 was inactivated inflammatory compared to wild type infected cell, suppressing IL8, TNFAIP3 and CCL3. Conclusion: B. abortus mutants strains infected to THP-1 macrophage cell and microarray data showed that genes related with cellular immune response were differently expressed according to strains. Also, expression patterns were divided into two groups: C1 and C10, C27 and C32 showed similar expression, respectively. Although genes of C1 and C10 infected cell down regulated the expression of CCL3 or TNFAIP3 inducing inhibition of chemokines and cell apoptosis, genes of C27 and C32 infected cell up regulated the expression of APOL3 or IFIH1 activating cytokine production, Interferon. Our result suggests that these expression pattern in the cells might be closely related with the disrupted genes of mutants and these loci are associated to pathogenicity of B. abortus.S Acknowledgment: This work was supported by KHIDI (No. HI16C2130), the BK21 PLUS program for Creative Veterinary Science Research and the Research Institute of Veterinary Science, Seoul National University, Republic of Korea.

Different invasion efficiencies of Brucella abortus wild-type and mutants in RAW 264.7 and THP-1 phagocytic cells and HeLa non-phagocytic cells

Shim, Soojin,Im, Young Bin,Jung, Myunghwan,Park, Woo Bin,Yoo, Han Sang The Korean Society of Veterinary Science 2018 大韓獸醫學會誌 Vol.58 No.2

Brucellosis is one of the common zoonoses caused by Brucella abortus (B. abortus). However, little has been reported on factors affecting invasion of B. abortus into host cells. To investigate cell-type dependent invasion of B. abortus, phagocytic RAW 264.7 and THP-1 cells and non-phagocytic HeLa cells were infected with wild-type and mutant B. abortus, and their invasion efficiencies were compared. The invasion efficiencies of the strains were cell-type dependent. Wild-type B. abortus invasion efficiency was greater in phagocytic cells than in epithelial cells. The results also indicated that there are different factors involved in the invasion of B. abortus into phagocytic cells.

Induction of pro-inflammatory cytokines in THP-1 cells by chitosan microspheres loaded with Brucella abortus antigen, malate dehydrogenase

( Soojin Shim ),( Sang Hee Soh ),( Young Bin Im ),( Han Sang Yoo ) 대한인수공통전염병학회 2018 창립총회 및 학술대회 초록집 Vol.2018 No.1

Introduction: Brucellosis is one of most important zoonosis caused by Brucella abortus (B. abortus). Over the years, great efforts have been made to identify effective vaccine targets for the bacteria. In our previous study, among the four B. abortus recombinant protein, malate dehydrogenase (MDH) induced strong pro-inflammatory cytokines of tumor necrosis factor-alpha (TNF-□□, interleukin 1-beta (IL-1□) and IL-6 in human macrophage, THP-1 cells. These cytokines have been shown to be significant in host defenses and elimination of Brucella infection. To attempts to develop effective B. abortus antigen for mucosal immunity, the MDH was loaded with chitosan microspheres (CMs) which is biocompatible, nontoxic, and immune-enhancing adjuvant. In the study, as a first step to evaluate the effect of antigen loaded chitosan, the MDH and MDH loaded chitosan were stimulated to THP-1 cells, and cell-mediated immune responses and cytotoxicity of these antigens were measured. Methods: The CMs were provided by another student in our lab. THP-1 cells were each grown in 1 x 106 cells per ml in 6-well plates and stimulated with the TF, MDH, CMs, CMs+TF, and CMs+MDH. The culture supernatants were collected from THP-1 cells after stimulation with the antigens at different times post-infection (p.i.): 6 hrs, 12 hrs and 24 hrs. Amounts of the cytokines such as IL-1β, IL-6, IFN-γ, and TNF-α were quantified with ELISA. Also, MTT based cytotoxicity assay of THP-1 cells was conducted after stimulation with the antigens at p.i. 6 hr, 12 hr and 24 hr. Results: Both of MDH and MDH-loaded CMs induced TNF-□, IL-1□□ and IL-6. Of the cytokines, the production of IL-6 showed increase in time-dependent manner after stimulation with the antigens until 24hrs. Conversely, the production of TNF-□□ showed slight decrease in time-dependent manner. The production of IL-1□□ showed increase until at p.i. 12 hrs, and it showed slight decrease at p.i. 24 hrs. The production of IFN-□□ was not detected after stimulation. The Mdh-loaded CMs induced higher production of IL-6 and IL-1□□ compared to MDH and TF. The CMs induced no production of TNF-□□ and IL-6 while it induced production of IL-1□. The THP-1 cytotoxicity showed no significant difference after stimulation with these antigens until 12 hrs. However, cell viability of TF-loaded CMs and MDH-loaded CMs stimulated cells showed gradually decrease until p.i. 24 hrs whereas CMs were similar with non-treated cells. The results suggest that MDH-loaded CMs induced effective production of pro-inflammatory cytokines without cytotoxicity but it could inhibit cell proliferation. Conclusion: Pro-inflammatory cytokines of TNF-□, IL-1□□and IL-6 were produced in THP-1 cells after stimulation with MDH and MDH-loaded CMs. Also, the production level of IL-1□□and IL-6 induced by MDH-loaded CMs was higher than TF, TF-loaded CMs, and MDH. Also, although no cytotoxicity was detected after stimulation with MDH-loaded CMs until p.i. 12 hrs, cell viability was gradually decreased compared to non-treated cells. Therefore, MDH-loaded CMs is considered as an effective antigen candidate and in vivo experiment should be investigated as further study. Acknowledgment: This work was supported by KHIDI (No. HI16C2130), the BK21 PLUS program for Creative Veterinary Science Research and the Research Institute of Veterinary Science, Seoul National University, Republic of Korea.

Comprehensive Evaluation of Immune Responses Induced by Nasal-associated Lymphoid Tissue (NALT) of Mice Intranasally Immunized with Brucella abortus malate dehydrogenase Loaded with Chitosan Nanoparticles

( Soojin Shim ),( Sang Hee Soh ),( Young Bin Im ),( Han Sang Yoo ) 대한인수공통전염병학회 2019 창립총회 및 학술대회 초록집 Vol.2019 No.1

Introduction: Brucella abortus malate dehydrogenase (Mdh) is promising vaccine candidate. To develop subunit vaccine, Mdh was loaded to mucoadhesive chitosan nanoparticles (CNs) and it was intranasally immunized to mice. The NALT plays an important role in inducing a first immune response against pathogens. We evaluated immunogenicity of Mdh and elucidated adjuvant ability of CNs, by studying the comprehensive innate immune responses in NALT after intranasal immunization of mice. Methods: CNs were generated with chitosan dissolved in D.W (0.5 w/v %) and mixed with 150ul of 8% Tween 80. 6-week-old BALB/c mice were intranasally immunized with of 30 μg of CNs loaded with the Mdh. NALT was isolated from immunized mice and transcriptomic analysis was conducted using mRNA from the NALT. The production of IgG and IgA from serum, nasal washes, genital secretions, and fecal excretions were detected. Results: As an adjuvant activity, CNs triggered activation of PRRs and chemotaxis of innate immune cells. The pathway analysis revealed that intranasal immunization of Mdh induced maturation of APCs such as dendritic cells and T lymphocytes eliciting Th17 cell differentiation. Nasal vaccination of CNs-Mdh in mice also resulted in production of specific IgG and higher levels of mucosal secretory IgA in digestive and genital mucosa. Conclusion: These results suggest that CNs-Mdh could be promising delivery system and nasal vaccine candidate that can trigger activation of APCs, Th17 cell response and production of IgG and secretory IgA through NALT. The study will be beneficial for further understanding of immune responses against Mdh, promising antigen candidate for B. abortus, within NALT and the rational design of vaccination strategies. Acknowledgment: This work was supported by Korea Health Industry Development Institute (No. HI16C2130) and the Brain Korea 21 PLUS program for Creative Veterinary Science Research and the Research Institute of Veterinary Science, SNU, ROK

Genes Related to Intracellular Survival of Brucella abortus in THP-1 Macrophage Cells

( Soojin Shim ),( Young Bin Im ),( Myunghwan Jung ),( Woo Bin Park ),( Han Sang Yoo ) 한국미생물생명공학회(구 한국산업미생물학회) 2018 Journal of microbiology and biotechnology Vol.28 No.10

Brucella abortus can survive and replicate within host macrophages, and great efforts have been made to demonstrate the genes involved in pathogenicity, such as internalization, in Brucella research. Here, intracellular responses were compared between THP-1 macrophage cells stimulated with B. abortus wild-type and four mutants (C1, C10, C27, and C32) using microarray to demonstrate the role of genes related to intracellular survival and replication. These mutants were generated by deleting genes encoding BAB_RS13225 (4-hydrobenzoate 3- monooxygenase, PHBH), BAB_RS00455 (heme exporter protein cytochrome C, CcmC), BAB_RS03675 (exopolyphosphatase, PPX), and BAB_RS13225 (peptidase M24). The results showed that mutants C1 and C10 induced significant suppression of survival levels and cytokine expression relative to wild-type in the THP-1 macrophage cells. These findings suggest that the BAB_RS13225 and BAB_RS00455 genes play important roles in survival within human macrophages. Conversely, mutants C27 and C32 induced significantly higher survival level than wild-type in the cells inhibiting cellular signal transduction. It is assumed that the BAB_RS03675 and BAB_RS13225 genes play a role in cellular resistance to B. abortus. Therefore, the disrupted genes are involved in B. abortus intracellular growth, and especially in its survival, and they could be effective targets for understanding the intracellular bacterium, B. abortus.

이산화납 전극 제조 시 전기화학적 증착인자가 수산화라디칼 발생에 미치는 영향

심수진(Soojin Shim),윤제용(Jeyong Yoon) 대한환경공학회 2016 대한환경공학회지 Vol.38 No.12

Lead dioxide (PbO₂)는 전기화학적 고도산화공정(electrochemical advanced oxidation process, EAOP)에서 hydroxyl radical (˙OH) 발생에 기반한 유기오염물 분해에 효과적인 전극물질이다. PbO₂ 전극의 대표적인 제조방법인 전기화학적 증착법(electrodeposition)의 주요 인자로는 전류/전압세기, 온도, 반응시간, Pb(II)의 농도, 전해질 종류 및 농도가 있다. 본 연구에서는 Ti/PbO₂ 산화전극을 전기화학적 증착법을 통해 전류인가 시간, 전류밀도, 온도, HNO₃3 전해질 농도를 각각 조절하여 제조하였고, ˙OH 검출물질인 p-Nitrosodimethylaniline (RNO)의 전기화학적 탈색 측면에서 ˙OH 발생에 대한 PbO₂ 증착인자의 영향을 조사하였다. 주요 결과로, PbO₂의 ˙OH 발생 성능은 PbO₂ 증착과정에서 대체로 전류인가 시간이 길어질수록(1-90 min), 전류밀도가 감소할수록(0.5-50 ㎃/㎠), 증착온도가 증가할수록(5-65℃), HNO3 전해질 농도(0.01-1.0 M)가 감소할수록 향상되었다. 특히, 0.01 M의 낮은 HNO₃3 농도 상에서 20 ㎃/㎠ 전류를 10분 이상 인가하여 증착시킨 PbO₂에서 ˙OH 발생이 가장 촉진되었다. RNO 탈색속도 측면에서 가장 성능이 좋은 PbO₂와 저조한 PbO₂ 사이에 최대 41% 정도 차이가 나타났다. PbO의 ˙OH 발생 성능을 결정짓는 특성으로 PbO₂ 층 전도도, Ti 기판 산화, PbO₂ 결정크기를 고려한 결과, PbO₂ 층의 전도도 및 Ti 기판의 산화가 ˙OH 발생에 주요하게 영향을 미치는 것으로 확인되었다. PbO₂ 층의 전도도 향상과 Ti 표면 산화 억제로 인한 Ti/PbO₂ 계면에서 전도도 향상이 ˙OH 발생을 촉진시키는 효과를 가져왔다. 그리고 일부 전극에서는 표면에서 PbO₂ 결정 크기 증가가 ˙OH 발생을 저감시키는 역할을 하였다. Lead dioxide (PbO₂) is an electrode material that is effective for organic pollutant degradation based on hydroxyl radical (˙OH) attack. Representative parameters for PbO₂ electrodeposition are summarized to current, temperature, reaction time, concentration of Pb(II) and electrolyte agent. In this study, Ti/PbO₂ electrodes were fabricated by electrodeposition method under controlled reaction time, current density, temperature, concentration of HNO₃ electrolyte. Effects of deposition parameters on ˙OH evolution were investigated in terms of electrochemical bleaching of p-Nitrosodimethylaniline (RNO). As major results, the ˙OH evolution was promoted at the PbO₂ that was deposited in longer reaction time (1-90 min), lower current density (0.5-50 ㎃/㎠), higher temperature (5-65℃) and lower HNO₃ concentration (0.01-1.0 M). Especially, the PbO₂ which was deposited in 0.01 M of lowest HNO₃ concentration by applying 20 ㎃/㎠ for above 10 min was most effective on ˙OH evolution. The performance gap between PbO₂s that was best and worst in ˙OH evolution was about 41%. Among the properties of PbO₂ related on ˙OH evolution performance, conductivity of Ti/PbO₂ significantly influenced on ˙OH evolution. The increase in conductivity promoted ˙OH evolution. In addition, the increase in crystal size of PbO₂ interfered ˙OH evolution at surface of some PbO₂ deposits.

Effects of Newly Synthesized Recombinant Human Amyloid-β Complexes and Poly-Amyloid-β Fibers on Cell Apoptosis and Cognitive Decline

( Soojin Park ),( Jae-won Huh ),( Taekil Eom ),( Naeun Park ),( Youngjeon Lee ),( Ju-sung Kim ),( Sun-uk Kim ),( Insop Shim ),( Sang-rae Lee ),( Ekyune Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.11

The main pathological hallmark of Alzheimer’s disease is the deposition of amyloid-beta (Aβ) peptides in the brain. Aβ has been widely used to mimic several aspects of Alzheimer’s disease. However, several characteristics of amyloid-induced Alzheimer’s disease pathology are not well established, especially in mice. The present study aimed to develop a new Alzheimer’s disease model by investigating how Aβ can be effectively aggregated using prokaryotes and eukaryotes. To express the Aβ42 complex in HEK293 cells, we cloned the Aβ42 region in a tandem repeat and incorporated the resulting construct into a eukaryotic expression vector. Following transfection into HEK293 cells via lipofection, cell viability assay and western blotting analysis revealed that exogenous Aβ42 can induce cell death and apoptosis. In addition, recombinant His-tagged Aβ42 was successfully expressed in Escherichia coli BL21 (DE3) and not only readily formed Aβ complexes, but also inhibited the proliferation of SH-SY5Y cells and E. coli. For in vivo testing, recombinant His-tagged Aβ42 solution (3 μg/ μl in 1× PBS containing 1 mM Ni²⁺) was injected stereotaxically into the left and right lateral ventricles of the brains of C57BL/6J mice (n = 8). Control mice were injected with 1× PBS containing 1 mM Ni²⁺ following the same procedure. Ten days after the sample injection, the Morris water maze test confirmed that exogenous Aβ caused an increase in memory loss. These findings demonstrated that Ni²⁺ is capable of complexing the 50-kDa amyloid and that intracerebroventricular injection of Aβ42 can lead to cognitive impairment, thereby providing improved Alzheimer’s disease models.

Inductions of Caspase-, MAPK- and ROS-dependent Apoptosis and Chemotherapeutic Effects Caused by an Ethanol Extract of Scutellaria barbata D. Don in Human Gastric Adenocarcinoma Cells

Shim, Ji Hwan,Gim, Huijin,Lee, Soojin,Kim, Byung Joo KOREAN PHARMACOPUNCTURE INSTITUTE 2016 Journal of pharmacopuncture Vol.19 No.2

Objectives: The crude extracts of Scutellaria barbata D. Don (SB) have traditionally demonstrated inhibitory effects on numerous human cancers both in vitro and in vivo. Gastric cancer is one of the most common types of cancer on world. The authors investigated the effects of an ethanol extract of Scutellaria barbata D. Don (ESB) on the growth and survival of MKN-45 cells (a human gastric adenocarcinoma cell line). Methods: The MKN-45 cells were treated with different concentrations of ESB, and cell death was examined using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Analyses of sub-G1 peaks, caspase-3 and -9 activities, and mitochondrial membrane depolarizations were conducted to determine the anti-cancer effects of SB on MKN-45 cells. Also, intracellular reactive oxygen species (ROS) generation was investigated. Results: ESB inhibited the growth of MKN-45 cells, caused cell cycle arrest, and increased the sub-G1 population. In addition, ESB markedly increased mitochondrial membrane depolarization and the activities of caspase-3 and -9. ESB exerted anti-proliferative effects on MKN-45 cells by modulating the mitogen-activated protein kinase (MAPK) signaling pathway and by increasing the generation of ROS. Furthermore, combinations of anti-cancer drugs plus ESB suppressed cell growth more than treatments with an agent or ESB, and this was especially true for cisplatin, etoposide, and doxorubicin. Conclusion: ESB has a dose-dependent cytotoxic effect on MKN-45 cells and this is closely associated with the induction of apoptosis. ESB-induced apoptosis is mediated by mitochondria-, caspase- and MAPK dependent pathways. In addition, ESB enhances ROS generation and increases the chemosensitivity of MKN-45 cells. These results suggest that treatment with ESB can inhibit the proliferation and promote the apoptosis of human gastric adenocarcinoma cells by modulating the caspase-, MAPK- and ROS-dependent pathway.

외전형 BLDC 모터의 열유동 해석

강수진(Soojin Kang),이관수(Kwan-Soo Lee),왕세명(Semyung Wang),심호경(Hokyung Shim) 대한기계학회 2007 대한기계학회 춘추학술대회 Vol.2007 No.5

In this paper, thermo-flow characteristics of an outer-rotor type of a BLDC motor are numerically analyzed using three-dimensional turbulence modeling. In an advance design of BLDC motor, cooling blades and holes are preferred for the enhanced cooling performances. Rotating the blades and holes generates axial air flow passing through stator slots, which cools down stator by forced convection. For the present study, a new design of the BLDC motor has been developed and major design parameters such as the arrangement of cooling holes, the area of cooling holes, and cooling blades and the cooling blade angle, are analyzed for the enhanced convective heat transfer rate. It is found that the convective heat transfer rate of the new BLDC motor model is increased by about 8.1%, compared to that of the reference model.