• Title/Summary/Keyword: Single-Cell Analysis

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Gene Expression Profiling of the Habenula in Rats Exposed to Chronic Restraint Stress

  • Yoo, Hyeijung;Kim, Hyun Jung;Yang, Soo Hyun;Son, Gi Hoon;Gim, Jeong-An;Lee, Hyun Woo;Kim, Hyun
    • Molecules and Cells
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    • v.45 no.5
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    • pp.306-316
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    • 2022
  • Chronic stress contributes to the risk of developing depression; the habenula, a nucleus in epithalamus, is associated with many neuropsychiatric disorders. Using genome-wide gene expression analysis, we analyzed the transcriptome of the habenula in rats exposed to chronic restraint stress for 14 days. We identified 379 differentially expressed genes (DEGs) that were affected by chronic stress. These genes were enriched in neuroactive ligand-receptor interaction, the cAMP (cyclic adenosine monophosphate) signaling pathway, circadian entrainment, and synaptic signaling from the Kyoto Encyclopedia of Genes and Genomes pathway analysis and responded to corticosteroids, positive regulation of lipid transport, anterograde trans-synaptic signaling, and chemical synapse transmission from the Gene Ontology analysis. Based on protein-protein interaction network analysis of the DEGs, we identified neuroactive ligand-receptor interactions, circadian entrainment, and cholinergic synapse-related subclusters. Additionally, cell type and habenular regional expression of DEGs, evaluated using a recently published single-cell RNA sequencing study (GSE137478), strongly suggest that DEGs related to neuroactive ligand-receptor interaction and trans-synaptic signaling are highly enriched in medial habenular neurons. Taken together, our findings provide a valuable set of molecular targets that may play important roles in mediating the habenular response to stress and the onset of chronic stress-induced depressive behaviors.

VIP IMMUNOREACTIVITY IN THE RAT TRIGEMINAL GANGLION AFTER INFERIOR ALVEOLAR NERVE AXOTOMY (하치조신경 절단 후 흰쥐 삼차신경절에서 VIP-IR의 변화)

  • Kim, Heung-Joong;Park, Joo-Cheol;Kim, Hyun-Sub;Moon, Joo-Hoon
    • Restorative Dentistry and Endodontics
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    • v.25 no.2
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    • pp.225-234
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    • 2000
  • The purpose of this study was to investigate the distribution and fluorescene intensity of vasoactive intestinal polypeptide(VIP) immunoreactive cells in rat trigeminal ganglion after inferior alveolar nerve axotomy. The animals were divided into normal and two experimental groups. The experimental animals were sacrificed at 14th and 28th day after inferior alveolar nerve axotomy. The trigeminal ganglion was removed and immersed in the 4% paraformaldehyde-0.2% picric acid in 0.1M phosphate buffer. Serial frozon sections about $16{\mu}m$ in thickness were cut with a cryostat. The immunofluorescence staining was performed. The rabbit anti-VIP(1 : 8,000) was used as primary antibody and fluorescene isothiocynate(FITC)-conjugated anti-rabbit IgG(1 : 80) as secondary antibody. The slides were observed under confocal laser scanning microscope. Three-dimensional images were constructed from 9 serial images(each $1{\mu}m$ in thickness) made by automatic optical sectioning. Unprocessed optical sections were obtained and stored on a optical disk. Color picture were printed by a video copy processor. The results were as follows; 1. The appearance of VIP immunoreactive cells in the mandibular part of trigeminal ganglion was 8.79${\pm}$1.99% in normal group and 39.16${\pm}$5.62% in 14 days, 16.25${\pm}$2.39% in 28 days after inferior alveolar nerve axotomy groups. 2. The relative fluorescence intensity of VIP immunoreactive cell bodies in the mandibular part of trigeminal ganglion was 134.40${\pm}$10.39 in normal group and 192.88${\pm}$14.06 in 14 days, 143.10${\pm}$5.02 in 28 days after nerve axotomy groups. Therefore, the relative fluorescence intensity of 14 days after nerve axotomy group was 43.3% higher than intensity of normal group. 3. In optical single section analysis of VIP immunoreactive cell bodies, white cell bodies(moderate fluorescence intensity) were the most abundant in normal and 28 days after nerve axotomy groups. Whereas, in 14 days after nerve axotomy group, red cell bodies(high fluorescence intensity) were the most abundant. 4. In optical serial section analysis of VIP immunoreactive cell bodies, red cell bodies(high fluorescence intensity) were observed in a part of the 9 sections of normal and 24 days after nerve axotomy groups. Whereas, red cell bodies were observed in all of the 9 sections of 14 days after nerve axotomy group. 5. The results indicates that number and fluorescence intensity of VIP immunoreactive cells were increased in the mandibular part of trigeminal ganglion following inferior alveolar nerve axotomy.

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Epigenetic Regulation of Human Riboflavin Transporter 2(hRFT2) in Cervical Cancers from Uighur Women

  • Ma, Jun-Qi;Kurban, Shajidai;Zhao, Jun-Da;Li, Qiao-Zhi;Hasimu, Ayshamgul
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.6
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    • pp.2485-2489
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    • 2014
  • In the present study, we studied the hypermethylation of the human riboflavin transporter 2 (hRFT2) gene and regulation of protein expression in biopsies from resected tissues from Uighur cervical squamous cell carcinoma (CSCC) patients and their neighboring normal tissues. hRFT2 gene promoter region methylation sequences were mapped in cervical cancer cell line SiHa by bisulfite-sequencing PCR and quantitative detection of methylated DNA from 30 pairs of Uighur's CSCCs and adjacent normal tissues by MassARRAY (Sequenom, San Diego, CA, USA) and hRFT2 protein expression was analyzed by immunohistochemistry. In SiHa, we identified 2 CG sites methylated from all of 12CpG sites of the hRFT2 gene. Analysis of the data from quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform showed that the methylation level between two CpG sites (CpG 2 and CpG 3) from CpG 1~12 showed significant differences between CSCC and neighboring normal tissues. However, the methylation level of whole target CpG fragments demonstrated no significant variation between CSCC ($0.476{\pm}0.020$) and neighboring normal tissues ($0.401{\pm}0.019$, p>0.05). There was a tendency for translocation the hRFT2 proteins from cytoplasm/membrane to nucleus in CSCC with increase in methylation of CpG 2 and CpG 3 in hRFT2gene promoter regions, which may relate to the genesis of CSCC. Our results suggested that epigenetic modifications are responsible for aberrant expression of the hRFT2 gene, and may help to understand mechanisms of cervical carcinogenesis.

Mechanism study on DNA damage and Apoptosis induced by heak shock using Comet Assay

  • Seo, Young-Rok;Han, Sung-Sik;Kim, L. O′Neill;Ryu, Jae-Chun
    • Proceedings of the Korea Society of Environmental Toocicology Conference
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    • 1997.12a
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    • pp.101-101
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    • 1997
  • Comet assay, single cell gel electrophoresis has been known as useful, rapid, simple, visual, and sensitive technique for measuring the DNA breakage in mammalian ce1ls. For evaluation of DNA damage using comet assay, early studies reported a change in comet length and intensity with DNA damage using simple visual technique, such as fluorescence microscopy with eyespiece. In recent, some workers are observing and analyzing nucleotide of comets using quantitative fluorescence image analysis system to estimate 'tail moment', which is defined as the product of the tail length and the fraction of total DNA in tail. Our laboratory also adopted the image analysis software for qualification. In addition, many of the practical features of comet assay render it potentially attractive as useful tool for molecular toxicology and carcinogenesis, because the system is already showing considerable promise as rapid predictor in both in vitro and in vivo experimental designs. Recently, the comet assay becomes a attractive technique to study of apoptosis, because apoptotic fragmentation of nuclear DNA into nucleosomal sizes can be evaluated by the comet assay. So, we attempted to apply the comet assay to studying the effect of various stress on the apoptosis-sensitive cell lines. Particularly, focusing on the hyperthermic apoptosis, we could find that heat shock(44˚C for 60 minutes) was sufficient to induced apoptosis in these cell lines. But using the highly sensitive comet assay, we could not detect DNA breaks immediately after heat shock.

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Comparative proteomics and global genome-wide expression data implicate role of ARMC8 in lung cancer

  • Amin, Asif;Bukhari, Shoiab;Mokhdomi, Taseem A;Anjum, Naveed;Wafai, Asrar H;Wani, Zubair;Manzoor, Saima;Koul, Aabid M;Amin, Basit;Qurat-ul-Ain, Qurat-ul-Ain;Qazi, Hilal;Tyub, Sumira;Lone, Ghulam Nabi;Qadri, Raies A
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.9
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    • pp.3691-3696
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    • 2015
  • Background: Cancer loci comprise heterogeneous cell populations with diverse cellular secretions. Therefore, disseminating cancer-specific or cancer-associated protein antigens from tissue lysates could only be marginally correct, if otherwise not validated against precise standards. Materials and Methods: In this study, 2DE proteomic profiles were examined from lysates of 13 lung-adenocarcinoma tissue samples and matched against the A549 cell line proteome. A549 matched-cancer-specific hits were analyzed and characterized by MALDI-TOF/MS. Results: Comparative analysis identified a total of 13 protein spots with differential expression. These proteins were found to be involved in critical cellular functions regulating pyrimidine metabolism, pentose phosphate pathway and integrin signaling. Gene ontology based analysis classified majority of protein hits responsible for metabolic processes. Among these, only a single non-predictive protein spot was found to be a cancer cell specific hit, identified as Armadillo repeat-containing protein 8 (ARMC8). Pathway reconstruction studies showed that ARMC8 lies at the centre of cancer metabolic pathways. Conclusions: The findings in this report are suggestive of a regulatory role of ARMC8 in control of proliferation and differentiation in lung adenocarcinomas.

Inhibitory effects of ginsenosides on basic fibroblast growth factor-induced melanocyte proliferation

  • Lee, Ji Eun;Park, Jong Il;Myung, Cheol Hwan;Hwang, Jae Sung
    • Journal of Ginseng Research
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    • v.41 no.3
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    • pp.268-276
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    • 2017
  • Background: UV-B-exposed keratinocytes secrete various paracrine factors. Among these factors, basic fibroblast growth factor (bFGF) stimulates the proliferation of melanocytes. Ginsenosides, the major active compounds of ginseng, are known to have broad pharmacological effects. In this study, we examined the antiproliferative effects of ginsenosides on bFGF-induced melanocyte proliferation. Methods: We investigated the inhibitory effects of Korean Red Ginseng and ginsenosides from Panax ginseng on bFGF-induced proliferation of melan-a melanocytes. Results: When melan-a melanocytes were treated with UV-B-irradiated SP-1 keratinocytes media, cell proliferation increased. This increased proliferation of melanocytes decreased with a neutralizing anti-bFGF antibody. To elucidate the effects of ginsenosides on melanocyte proliferation induced by bFGF, we tested 15 types of ginsenoside compounds. Among them, Rh3, Rh1, F1, and CK demonstrated antiproliferative effects on bFGF-induced melanocyte proliferation after 72 h of treatment. bFGF stimulated cell proliferation via extracellular signal-regulated kinase (ERK) activation in various cell types. Western blot analysis found bFGF-induced ERK phosphorylation in melan-a. Treatment with Rh3 inhibited bFGF-induced maximum ERK phosphorylation and F1-delayed maximum ERK phosphorylation, whereas Rh1 and CK had no detectable effects. In addition, cotreatment with Rh3 and F1 significantly suppressed bFGF-induced ERK phosphorylation. Western blot analysis found that bFGF increased microphthalmia-associated transcription factor (MITF) protein levels in melan-a. Treatment with Rh3 or F1 had no detectable effects, whereas cotreatment with Rh3 and F1 inhibited bFGF-induced MITF expression levels more strongly than a single treatment. Conclusion: In summary, we found that ginsenosides Rh3 and F1 have a synergistic antiproliferative effect on bFGF-induced melan-a melanocyte proliferation via the inhibition of ERK-mediated upregulation of MITF.

Prognostic Analysis of Stage I Non-Small Cell Lung Cancer Abutting Adjacent Structures on Preoperative Computed Tomography

  • Soohwan Choi;Sun Kyun Ro;Seok Whan Moon
    • Journal of Chest Surgery
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    • v.57 no.2
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    • pp.136-144
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    • 2024
  • Background: Early non-small cell lung cancer (NSCLC) that abuts adjacent structures requires careful evaluation due to its potential impact on postoperative outcomes and prognosis. We examined stage I NSCLC with invasion into adjacent structures, focusing on the prognostic implications after curative surgical resection. Methods: We retrospectively analyzed the records of 796 patients who underwent curative surgical resection for pathologic stage IA/IB NSCLC (i.e., visceral pleural invasion only) at a single center from 2008 to 2017. Patients were classified based on tumor abutment and then reclassified by the presence of visceral pleural invasion. Clinical characteristics, pathological features, and survival rates were compared. Results: The study included 181 patients with abutting NSCLC (22.7% of all participants) and 615 with non-abutting tumors (77.3%). Those with tumor abutment exhibited higher rates of non-adenocarcinoma (26.5% vs. 9.9%, p<0.01) and visceral/lymphatic/vascular invasion (30.4%/33.1%/12.7% vs. 8.5%/22.4%/5.7%, respectively; p<0.01) compared to those without abutment. Multivariable analysis identified lymphatic invasion and male sex as risk factors for overall survival (OS) and disease-free survival (DFS) in stage I NSCLC measuring 3 cm or smaller. Age, smoking history, vascular invasion, and recurrence emerged as risk factors for OS, whereas the presence of non-pure ground-glass opacity was a risk factor for DFS. Conclusion: NSCLC lesions 3 cm or smaller that abut adjacent structures present higher rates of various risk factors than non-abutting lesions, necessitating evaluation of tumor invasion into adjacent structures and lymph node metastasis. In isolation, however, the presence of tumor abutment without visceral pleural invasion does not constitute a risk factor.

Performance Analysis of Multi-Carrier DS-CDMA System in Multipath Rician Fading Channel (다중경로 라이시안 페이딩 채널에서 Multi-Carrier DS-CDMA 시스템의 성능 해석)

  • 김영철;노재성;오창헌;조성준
    • The Journal of Korean Institute of Electromagnetic Engineering and Science
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    • v.12 no.3
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    • pp.378-390
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    • 2001
  • In this paper, it is analyzed that the error performance of a Multi-Carrier DS-CDMA system in a single cell with multipath Rician fading and multiple access interference (MAI) and the error performance of the system is compared with that of a Sing1e-Carrier DS-CDMA system. Moreover, the convolutional coding techniques with code rate of 1/2, 1/3, and 1/4 are adopted in order to improve the error performance degraded by the multipath fading and MAI and performance improvement through the coding techniques is analyzed. As a result, it is shown that the number of users in each system can be determined by the number of branches of the rake receiver in a Single-Carrier DS-CDMA system and the number of carriers in a Multi-Carrier DS-CDMA system. Furthermore, the convolutional coding should be chosen with considering the trade-off between coding gain and a power limitation in a Multi-Carrier DS-CDMA system. In case of increasing the number of carriers, the processing gain is decreased but the error performance is improved through the effect of frequency diversity and the system can be possibility implemented due to the low chip rate.

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Studies on Multi-step Addition of NMP in (LiNi0.80Co0.15Al0.05) (NCA) Cathode Slurry Preparation and its Rheological, Mechanical Strength and Electrochemical Properties for Li-ion Cells

  • Vasudevarao Pasala;Satyanarayana Maddukuri;V. Sethuraman;Rekha Lankipalli;Devi Gajula;Venkateswarlu Manne
    • Journal of Electrochemical Science and Technology
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    • v.14 no.3
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    • pp.262-271
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    • 2023
  • For electrode stability and the electrochemical performance of the Li-ion cell, it is essential that the active ingredients and unique additives in the polymer binder be well dispersed with the solvent-based slurry. The efficient procedure used to create the slurry affects the rheological characteristics of the electrode slurry. When successively adding different steps of Nmethyl-2-pyrrolidone (NMP) solvent to the cathode composition, it is evenly disseminated. The electrochemical performance of the Li-ion cells and the electrodes made with slurry formed by single step and multiple steps of addition of NMP solvent are examined. To preform rheological properties of cathode electrode slurry on Ni-rich Lithium Nickel-Cobalt-Aluminum Oxide (LiNi0.80Co0.15Al0.05) (NCA). Also, we investigate different step addition of electrode formation and mechanical strength characterization like peel strength. According to the EIS study, a multi-step electrode slurry has lower internal resistance than a single-step electrode slurry, which results in better electrical characteristics and efficiency. Further, microstructure of electrodes is obtained electrochemical performance in the 18650 cylindrical cells with targeted capacity of 1.5 Ah. The slurry of electrodes prepared by single step and multiple steps of addition of NMP solvent and its effect on the fabrication of 1.5 Ah cells. A three-step solvent addition on slurry has been found to be a lower internal resistance than a single-step electrode slurry as confirmed by the EIS analysis, yielding improved electrical properties and efficiency.

Synthesis of Enzyme-Containing PEG Hydrogel Nanospheres for Optical Biosensors (광바이오센서용 효소를 함유한 PEG 수화젤 나노입자의 합성)

  • Kim, Bum-Sang
    • Polymer(Korea)
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    • v.29 no.6
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    • pp.613-616
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    • 2005
  • In this word as the first step to develop optical biosensors for a single cell level analysis, the preparation method of nano-scale polymer hydrogel spheres containing an enzyme was set up and the feasibility of the spheres as optical biosensors was investigated. The horseradish peroxidase (HRP) was encapsulated in the PEG hydrogel spheres by suspension photopolymerization, yielding spheres of the average size of 305 nm. After the polymerization, the incorporation and activity of HRP within the spheres were determined by the production of fluorescence resulted from the enzymatic reaction between HRP and $\H_{2}O_{2}$. The fluorescence emission response of the HRP-loaded PEG hydrogel spheres increased by nearly 300$\%$ as hydrogen peroxide concentration was changed from 0 to 11 nM in the presence of Amplex Red. The results suggest that the method to prepare the PEG hydrogel nanospheres containing an enzyme could be used for developing optical biosensors to measure various analytes in the very small samples like a single cell.