Potential role of HIF-1-responsive microRNA210/HIF3 axis on gemcitabine resistance in cholangiocarcinoma cells

MicroRNA-210 (miR-210) is a robust target for hypoxia-inducible factor, and its overexpression has been detected in a variety of solid tumors. However, the role of miR-210 in the development, progression and response to therapy in cholangiocarcinoma (CCA) remains undefined. We report here that high miR-210 expression was significantly correlated with the shorter survival of CCA patients. Overexpression of miR-210 inhibited CCA cell proliferation at the G2/M phase and reduced the gemcitabine sensitivity in CCA cells under CoCl2-induced pseudohypoxia. Concomitantly, inhibition of endogenous miR-210 activity using miRNA sponges increased cell proliferation under CoCl2-induced pseudohypoxia, resulting in an increase in gemcitabine sensitivity in CCA cells. We showed that HIF-3alpha, a negative controller of HIF-1alpha, was a target of miR-210 constituting a feed-forward hypoxic regulatory loop. Our data suggest an important role of miR-210 in sustaining HIF-1alpha activity via the suppression of HIF-3alpha, regulating cell growth and chemotherapeutic drug resistance in CCA.ope

Anti-helminthic niclosamide inhibits Ras-driven oncogenic transformation via activation of GSK-3

Despite the importance of Ras oncogenes as a therapeutic target in human cancer, their 'undruggable' tertiary structures limit the effectiveness of anti-Ras drugs. Canonical Wnt signaling contributes to Ras activity by glycogen synthase kinase 3 (GSK-3)-dependent phosphorylation at the C-terminus and subsequent degradation. In the accompanying report, we show that the anti-helminthic niclosamide directly binds to GSK-3 and inhibits Axin functions in colon cancer cells, with reversion of Snail-mediated epithelial-mesenchymal transition. In this study, we report that niclosamide effectively suppresses Ras and nuclear NFAT activities regardless of the mutational status of Ras at nM levels. Mechanistically, niclosamide increased endogenous GSK-3 activity, shortening the half-life of mutant Ras. Further, niclosamide activates Raf-1 kinase inhibitory protein, a downstream target of Snail repressor. Niclosamide treatment attenuates Ras-induced oncogenic potential in vitro and in vivo. These findings provide a clinically available repositioned Ras inhibitor as well as a novel strategy for inhibiting the Ras via GSK-3.ope

Dishevelled has a YAP nuclear export function in a tumor suppressor context-dependent manner

Phosphorylation-dependent YAP translocation is a well-known intracellular mechanism of the Hippo pathway; however, the molecular effectors governing YAP cytoplasmic translocation remains undefined. Recent findings indicate that oncogenic YAP paradoxically suppresses Wnt activity. Here, we show that Wnt scaffolding protein Dishevelled (DVL) is responsible for cytosolic translocation of phosphorylated YAP. Mutational inactivation of the nuclear export signal embedded in DVL leads to nuclear YAP retention, with an increase in TEAD transcriptional activity. DVL is also required for YAP subcellular localization induced by E-cadherin, alpha-catenin, or AMPK activation. Importantly, the nuclear-cytoplasmic trafficking is dependent on the p53-Lats2 or LKB1-AMPK tumor suppressor axes, which determine YAP phosphorylation status. In vivo and clinical data support that the loss of p53 or LKB1 relieves DVL-linked reciprocal inhibition between the Wnt and nuclear YAP activity. Our observations provide mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer.ope

Niclosamide is a potential therapeutic for familial adenomatosis polyposis by disrupting Axin-GSK3 interaction

The epithelial-mesenchymal transition (EMT) is implicated in tumorigenesis and cancer progression, and canonical Wnt signaling tightly controls Snail, a key transcriptional repressor of EMT. While the suppression of canonical Wnt signaling and EMT comprises an attractive therapeutic strategy, molecular targets for small molecules reverting Wnt and EMT have not been widely studied. Meanwhile, the anti-helminthic niclosamide has been identified as a potent inhibitor of many oncogenic signaling pathways although its molecular targets have not yet been clearly identified. In this study, we show that niclosamide directly targets Axin-GSK3 interaction, at least in part, resulting in suppression of Wnt/Snail-mediated EMT. In vitro and in vivo, disruption of Axin-GSK3 complex by niclosamide induces mesenchymal to epithelial reversion at nM concentrations, accompanied with suppression of the tumorigenic potential of colon cancer. Niclosamide treatment successfully attenuates Snail abundance while increasing E-cadherin abundance in xenograft tumor. Notably, oral administration of niclosamide significantly suppressed adenoma formation in an APC-MIN mice model, indicating that niclosamide is an effective therapeutic for familial adenomatosis polyposis (FAP) patients. In this study, we identified a novel target to control the canonical Wnt pathway and Snail-mediated EMT program, and discovered a repositioned therapeutics for FAP patients.ope

Snail reprograms glucose metabolism by repressing phosphofructokinase PFKP allowing cancer cell survival under metabolic stress

Dynamic regulation of glucose flux between aerobic glycolysis and the pentose phosphate pathway (PPP) during epithelial-mesenchymal transition (EMT) is not well-understood. Here we show that Snail (SNAI1), a key transcriptional repressor of EMT, regulates glucose flux toward PPP, allowing cancer cell survival under metabolic stress. Mechanistically, Snail regulates glycolytic activity via repression of phosphofructokinase, platelet (PFKP), a major isoform of cancer-specific phosphofructokinase-1 (PFK-1), an enzyme involving the first rate-limiting step of glycolysis. The suppression of PFKP switches the glucose flux towards PPP, generating NADPH with increased metabolites of oxidative PPP. Functionally, dynamic regulation of PFKP significantly potentiates cancer cell survival under metabolic stress and increases metastatic capacities in vivo. Further, knockdown of PFKP rescues metabolic reprogramming and cell death induced by loss of Snail. Thus, the Snail-PFKP axis plays an important role in cancer cell survival via regulation of glucose flux between glycolysis and PPP.ope

Bacillus circulans xylanase 의 최적 pH 이동을 위한 단백질 공학

Thesis (master`s)--서울대학교 대학원 :응용화학부,2003.Maste

Detection of nosocomial pneumonia pathogens using a fluorescence-based device

Background: Early detection of nosocomial pneumonia pathogens is a significant factor in hospital-acquired pneumonia care. This study aimed to determine the autofluorescence properties of five nosocomial pneumonia pathogens using a fluorescence-based device and to establish evidence for clinical guidelines. Methods: The following bacterial strains were assessed: Acinetobacter baumannii (AB), Escherichia coli (EC), Enterococcus faecalis (EF), Klebsiella pneumoniae (KP), and Staphylococcus aureus (SA). The bacteria were cultured separately on tryptic soy agar at 37 °C under aerobic conditions for 168 h. Fluorescence photographs of each species were captured every 24 h using a fluorescence-based device with fixed camera settings. The images were analyzed by measuring the red and green values (R/G ratio) at a central point in each colony, and the R/G ratios were analyzed using the Kruskal-Wallis non-parametric test. Results: KP and SA showed red fluorescence with their R/G values, which were significantly higher than those of the other strains (p < 0.001). In particular, the R/G ratio of KP increased steadily until 72 h of incubation, peaking at 3.65. In addition, AB and EC showed orange fluorescence with higher red ratios than green ratios. EF and SA showed green fluorescence all through 168 h of incubation, with R/G ratio less than 1.0. Conclusions: Nosocomial pneumonia pathogens can be identified and classified via bacterial autofluorescence emission. It is possible to develop a rapid and easy-to-use identification technology based on bacterial autofluorescence for clinical applications.restrictio

The influence of professional oral hygiene care on reducing ventilator-associated pneumonia in trauma intensive care unit patients

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전사체 분석을 통한 살모넬라-박테리오파지 상호작용 연구 및 이를 이용한 식품에서 파지의 병원균 제어능 향상

학위논문(석사)--서울대학교 대학원 :농업생명과학대학 농생명공학부,2020. 2. 유상열.Understanding the bacterial response to bacteriophage infection can provide more comprehensive solutions to control pathogens using phages. In this study, we used total RNA sequencing analysis to investigate global gene expression change during phage infection and find new functional genes involved in phage-host interaction. When Salmonella Typhimurium LT2 was infected by virulent phage SPC32N, several genes in Fep system, required to ferric iron uptake, were significantly up-regulated. Among these up-regulated genes, we speculated that the gene cluster fepDGC, encoding an inner membrane transporter complex, is thought be an important factor for phage infection because experiments using knockout strain ∆fepDGC showed that the plaque formed by SPC32N became significantly turbid while at the same time this deletion mutant seemed resistant to SPC32N in challenge assay. In addition, the adsorption rate of SPC32N against the ∆fepDGC was significantly reduced compared to the wild-type strain and for mimicking the RNA sequencing data of this system, ferric iron chelators were used and we can identify depletion of ferric irons induces delay of host cells resistance acquisition. From this understanding, to overcome the limit of phage as biocontrol agent at foods, deferiprone, one of ferric iron chelating agents which is allowed as edible agent for human by FDA was selected as a synergetic resource. By treating deferiprone to food, it was shown that bacterial resistance to phage was significantly postponed.박테리오파지의 감염에 대한 숙주 박테리아의 반응 체계를 연구, 이해하는 것은 박테리오파지를 이용하여 식중독균을 제어하는 데에 있어 보다 심화되고 통찰력 있는 해결책을 제시하게 한다. 본 연구에서는 숙주 박테리아가 박테리오파지에 감염되었을 때의 전사체를 추출, 분석을 통하여 해당 조건에서 숙주의 전반적인 유전자 발현 변화를 이해하고자 하였으며 이를 통해 박테리오파지와 숙주 사이의 상호작용에 연관되어 있는 새로운 기능적인 유전자를 찾고자 하였다. 살모넬라 타이피뮤리움 LT2를 숙주로 하는 용균성 박테리오파지 SPC32N을 해당 균에 감염시킨 결과 숙주 내 존재하는 Fep 체계와 연관 되어 있는 유전자들이 유의미하게 발현량이 증가하였다. 해당 체계는 3가 철 이온을 외부로부터 유입하는데 역할을 하며 분석을 통해 걸러진 유전자 중 fepDGC 유전자가 다른 유전자에 비해 큰 발현량 변화폭을 보였으므로 해당 유전자를 타겟유전자로 선정하였다. 해당 유전자는 Fep 체계에 속해 있는 세포 내막운송체로 이 유전자를 숙주로부터 제거 (ΔfepDGC)한 뒤, SPC32N을 감염시켜 감염성 표현형을 확인하였다. 그 결과, ΔfepDGC 는 해당 박테리오파지로부터 확연하게 저항성이 나타났으며 이는 흡착 과정을 확인해본 결과 ΔfepDGC에서 SPC32N의 흡착률이 유의미하게 감소하는 것과 연관이 있다고 판단된다. 한편 Fep 체계와 관련된 유전자의 발현량을 전사체분석 결과와 유사하게 임의로 모방하고자 3가 철 이온을 킬레이트하는 화학물질을 배양하는 과정에서 처리, 해당 물질이 숙주세포가 박테리오파지에 대해 저항성을 갖지 못하게 한다는 것을 결과로 얻었으며 이러한 현상을 임의로 오염시킨 식품에서 관찰하였을 때, 마찬가지로 박테리오파지의 제어능을 향상시키는 것을 확인하였다. 결과적으로 전사체분석을 통해 Fep 체계를 이용하면 박테리오파지의 균제어능을 향상시킬 수 있다는 것을 밝힐 수 있었다.1 INTRODUCTION ７ 1.1 Salmonella Typhimurium and its Fe3+ uptake systems ７ 1.2 Bacteriophages and their biocontrol １１ 1.3 Purpose of this study １３ 2 MATERIALS AND METHODS １５ 2.1 Bacterial strains, bacteriophage and growth conditions １５ 2.2 Construction of Salmonella mutant strains １６ 2.3 Plasmid construction １７ 2.4 RNA extraction and total RNA sequencing ２２ 2.5 Heat map generation ２４ 2.6 Quantitative real-time PCR (qRT-PCR) analysis ２４ 2.7 Bacterial challenge assay ２６ 2.8 Phage adsorption assay ２６ 2.9 One-step growth curve analysis ２７ 2.10 LPS extraction of bacterial strains ２８ 2.11 Antimicrobial activity of SPC32N with deferiprone in food samples ２９ 2.12 Statistical analysis. ３１ 3 RESULTS ３２ 3.1 Analysis of transcriptome after phage infection and Fe3+ uptake system ３２ 3.2 Validation of the expression value of DEGs belonging to Fe3+ uptake systems by qRT-PCR ３５ 3.3 Effects of various mutants on infectivity ３８ 3.4 Adsorption assay and one-step growth curve of ΔfepDGC ４０ 3.5 LPS analysis of ΔfepDGC ４２ 3.6 Challenge assay under Fe3+-chelated condition ４４ 3.7 Challenge assay of ΔfepDGC and ΔfhuB under Fe3+-chelated condition ４７ 3.8 The effect of deferiprone to phage biocontrol at foods ４９ 4 DISCUSSION ５１ 5 REFERENCES ５５ 국문 초록 ６１Maste