Publications by authors named "Keun Pil Kim"

17 Publications

  • Page 1 of 1

Molecular characterization of Hsf1 as a master regulator of heat shock response in the thermotolerant methylotrophic yeast Ogataea parapolymorpha.

J Microbiol 2021 Feb 1;59(2):151-163. Epub 2021 Feb 1.

Department of Life Science, College of Natural Science, Chung-Ang University, Seoul, 06974, Republic of Korea.

Ogataea parapolymorpha (Hansenula polymorpha DL-1) is a thermotolerant methylotrophic yeast with biotechnological applications. Here, O. parapolymorpha genes whose expression is induced in response to heat shock were identified by transcriptome analysis and shown to possess heat shock elements (HSEs) in their promoters. The function of O. parapolymorpha HSF1 encoding a putative heat shock transcription factor 1 (OpHsf1) was characterized in the context of heat stress response. Despite exhibiting low sequence identity (26%) to its Saccharomyces cerevisiae homolog, OpHsf1 harbors conserved domains including a DNA binding domain (DBD), domains involved in trimerization (TRI), transcriptional activation (AR1, AR2), transcriptional repression (CE2), and a C-terminal modulator (CTM) domain. OpHSF1 could complement the temperature sensitive (Ts) phenotype of a S. cerevisiae hsf1 mutant. An O. parapolymorpha strain with an H221R mutation in the DBD domain of OpHsf1 exhibited significantly retarded growth and a Ts phenotype. Intriguingly, the expression of heat-shock-protein-coding genes harboring HSEs was significantly decreased in the H221R mutant strain, even under non-stress conditions, indicating the importance of the DBD for the basal growth of O. parapolymorpha. Notably, even though the deletion of C-terminal domains (ΔCE2, ΔAR2, ΔCTM) of OpHsf1 destroyed complementation of the growth defect of the S. cerevisiae hsf1 strain, the C-terminal domains were shown to be dispensable in O. parapolymorpha. Overexpression of OpHsf1 in S. cerevisiae increased resistance to transient heat shock, supporting the idea that OpHsf1 could be useful in the development of heat-shock-resistant yeast host strains.
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http://dx.doi.org/10.1007/s12275-021-0646-2DOI Listing
February 2021

Maintenance of genome integrity and active homologous recombination in embryonic stem cells.

Exp Mol Med 2020 08 7;52(8):1220-1229. Epub 2020 Aug 7.

Department of Life Sciences, Chung-Ang University, Seoul, 06974, South Korea.

Embryonic stem cells (ESCs) possess specific gene expression patterns that confer the ability to proliferate indefinitely and enable pluripotency, which allows ESCs to differentiate into diverse cell types in response to developmental signals. Compared to differentiated cells, ESCs harbor an elevated level of homologous recombination (HR)-related proteins and exhibit exceptional cell cycle control, characterized by a high proliferation rate and a prolonged S phase. HR is involved in several aspects of chromosome maintenance. For instance, HR repairs impaired chromosomes and prevents the collapse of DNA replication forks during cell proliferation. Thus, HR is essential for the maintenance of genomic integrity and prevents cellular dysregulation and lethal events. In addition, abundant HR proteins in the prolonged S phase can efficiently protect ESCs from external damages and protect against genomic instability caused by DNA breaks, facilitating rapid and accurate DNA break repair following chromosome duplication. The maintenance of genome integrity is key to preserving the functions of ESCs and reducing the risks of cancer development, cell cycle arrest, and abnormal replication. Here, we review the fundamental links between the stem cell-specific HR process and DNA damage response as well as the different strategies employed by ESCs to maintain genomic integrity.
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http://dx.doi.org/10.1038/s12276-020-0481-2DOI Listing
August 2020

FOXL2 directs DNA double-strand break repair pathways by differentially interacting with Ku.

Nat Commun 2020 04 24;11(1):2010. Epub 2020 Apr 24.

School of Pharmacy, Chung-Ang University, Seoul, 06974, Korea.

The balance between major DNA double-strand break (DSB) repair pathways is influenced by binding of the Ku complex, a XRCC5/6 heterodimer, to DSB ends, initiating non-homologous end joining (NHEJ) but preventing additional DSB end resection and homologous recombination (HR). However, the key molecular cue for Ku recruitment to DSB sites is unknown. Here, we report that FOXL2, a forkhead family transcriptional factor, directs DSB repair pathway choice by acetylation-dependent binding to Ku. Upon DSB induction, SIRT1 translocates to the nucleus and deacetylates FOXL2 at lysine 124, leading to liberation of XRCC5 and XRCC6 from FOXL2 and formation of the Ku complex. FOXL2 ablation enhances Ku recruitment to DSB sites, imbalances DSB repair kinetics by accelerating NHEJ and inhibiting HR, and thus leads to catastrophic genomic events. Our study unveils the SIRT1-(de)acetylated FOXL2-Ku axis that governs the balance of DSB repair pathways to maintain genome integrity.
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http://dx.doi.org/10.1038/s41467-020-15748-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181608PMC
April 2020

E2F1 facilitates DNA break repair by localizing to break sites and enhancing the expression of homologous recombination factors.

Exp Mol Med 2019 09 18;51(9):1-12. Epub 2019 Sep 18.

Department of Life Sciences, Chung-Ang University, Seoul, 06974, South Korea.

The human genome is constantly exposed to both endogenous and exogenous stresses, which can lead to errors in DNA replication and the accumulation of DNA mutations, thereby increasing the risk of cancer development. The transcription factor E2F1 is a key regulator of DNA repair. E2F1 also has defined roles in the replication of many cell cycle-related genes and is highly expressed in cancer cells, and its abundance is strongly associated with poor prognosis in cancers. Studies on colon cancer have demonstrated that the depletion of E2F1 leads to reduced levels of homologous recombination (HR), resulting in interrupted DNA replication and the subsequent accumulation of DNA lesions. Our results demonstrate that the depletion of E2F1 also causes reduced RAD51-mediated DNA repair and diminished cell viability resulting from DNA damage. Furthermore, the extent of RAD51 and RPA colocalization is reduced in response to DNA damage; however, RPA single-stranded DNA (ssDNA) nucleofilament formation is not affected following the depletion of E2F1, implying that ssDNA gaps accumulate when RAD51-mediated DNA gap filling or repair is diminished. Surprisingly, we also demonstrate that E2F1 forms foci with RAD51 or RPA at DNA break sites on damaged DNA. These findings provide evidence of a molecular mechanism underlying the E2F1-mediated regulation of HR activity and predict a fundamental shift in the function of E2F1 from regulating cell division to accelerating tumor development.
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http://dx.doi.org/10.1038/s12276-019-0307-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802646PMC
September 2019

Yeast-based assays for characterization of the functional effects of single nucleotide polymorphisms in human DNA repair genes.

PLoS One 2018 9;13(3):e0193823. Epub 2018 Mar 9.

Department of Biomedical Science, College of Life Science, CHA University, Seongnam-si, Gyeonggi-do, Republic of Korea.

DNA repair mechanisms maintain genomic integrity upon exposure to various types of DNA damage, which cause either single- or double-strand breaks in the DNA. Here, we propose a strategy for the functional study of single nucleotide polymorphisms (SNPs) in the human DNA repair genes XPD/ERCC2, RAD18, and KU70/XRCC6 and the checkpoint activation gene ATR that are essentially involved in the cell cycle and DNA damage repair. We analyzed the mutational effects of the DNA repair genes under DNA-damaging conditions, including ultraviolet irradiation and treatment with genotoxic reagents, using a Saccharomyces cerevisiae system to overcome the limitations of the human cell-based assay. We identified causal variants from selected SNPs in the present analyses. (i) R594C SNP in RAD3 (human XPD/ERCC2) caused severe reductions in the growth rate of mutant cells upon short-wavelength UV irradiation or chemical reagent treatment. (ii) The growth rates of the selected variants in RAD18, YKU70, and MEC1 were similar to those of wild-type cells on methyl methanesulfonate and hydroxyurea treated media. (iii) We also assessed the structural impact of the SNPs by analyzing differences in the structural conformation and calculating the root mean square deviation, which is a measure of the discordance of the Cα atoms between protein structures. Based on the above results, we propose that these analytical approaches serve as efficient methods for the identification of causal variants of human disease-causing genes and elucidation of yeast-cell based molecular mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193823PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844570PMC
June 2018

Hed1 Promotes Meiotic Crossover Formation in .

J Microbiol Biotechnol 2017 Feb;27(2):405-411

Department of Life Sciences, Chung-Ang University, Seoul 06974, Republic of Korea.

Homologous recombination occurs between homologous chromosomes and is significantly involved in programmed double-strand break (DSB) repair. Activation of two recombinases, Rad51 and Dmc1, is essential for an interhomolog bias during meiosis. Rad51 participates in both mitotic and meiotic recombination, and its strand exchange activity is regulated by an inhibitory factor during meiosis. Thus, activities of Rad51 and Dmc1 are coordinated to promote homolog bias. It has been reported that Hed1, a meiosis-specific protein in budding yeast, regulates Rad51-dependent recombination activity. Here, we investigated the role of Hed1 in meiotic recombination by ectopic expression of the protein after pre-meiotic replication in . DNA physical analysis revealed that the overexpression of Hed1 delays the DSB-to-joint molecule (JM) transition and promotes interhomolog JM formation. The study indicates a possible role of Hed1 in controlling the strand exchange activity of Rad51 and, eventually, meiotic crossover formation.
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http://dx.doi.org/10.4014/jmb.1610.10074DOI Listing
February 2017

Novel Function of Sprouty4 as a Regulator of Stemness and Differentiation of Embryonic Stem Cells.

Dev Reprod 2016 Jun;20(2):171-7

Dept. of Biomedical Science, College of Life Science, CHA University, Seoul 06135, Korea.

Sprouty (Spry) genes encode inhibitors of the receptor tyrosine kinase signaling cascade, which plays important roles in stem cells. However, the role of Spry4 in the stemness of embryonic stem cells has not been fully elucidated. Here, we used mouse embryonic stem cells (mESCs) as a model system to investigate the role of Spry4 in the stem cells. Suppression of Spry4 expression results in the decreases of cell proliferation, EB formation and stemness marker expression, suggesting that Spry4 activity is associated with stemness of mESCs. Teratoma assay showed that the cartilage maturation was facilitated in Spry4 knocked down mESCs. Our results suggest that Spry4 is an important regulator of the stemness and differentiation of mESCs.
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http://dx.doi.org/10.12717/DR.2016.20.2.171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027223PMC
June 2016

Rad51 Regulates Reprogramming Efficiency through DNA Repair Pathway.

Dev Reprod 2016 Jun;20(2):163-9

Dept. of Biomedical Science, College of Life Science, CHA University, Seoul 06135, Korea.

Rad51 is a key component of homologous recombination (HR) to repair DNA double-strand breaks and it forms Rad51 recombinase filaments of broken single-stranded DNA to promote HR. In addition to its role in DNA repair and cell cycle progression, Rad51 contributes to the reprogramming process during the generation of induced pluripotent stem cells. In light of this, we performed reprogramming experiments to examine the effect of co-expression of Rad51 and four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc, on the reprogramming efficiency. Co-expression of Rad51 significantly increased the numbers of alkaline phosphatase-positive colonies and embryonic stem cell-like colonies during the process of reprogramming. Co-expression ofRad51 significantly increased the expression of epithelial markers at an early stage of reprogramming compared with control cells. Phosphorylated histone H2AX (γH2AX), which initiates the DNA double-strand break repair system, was highly accumulated in reprogramming intermediates upon co-expression of Rad51. This study identified a novel role of Rad51 in enhancing the reprogramming efficiency, possibly by facilitating mesenchymal-to-epithelial transition and by regulating a DNA damage repair pathway during the early phase of the reprogramming process.
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http://dx.doi.org/10.12717/DR.2016.20.2.163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027222PMC
June 2016

The ELK3-GATA3 axis orchestrates invasion and metastasis of breast cancer cells in vitro and in vivo.

Oncotarget 2016 10;7(40):65137-65146

Department of Biomedical Science, College of Life Science, CHA University, Seoul, Korea.

Triple-negative breast cancer is a highly aggressive tumor subtype that lacks effective therapeutic targets. Here, we show that ELK3 is overexpressed in a subset of breast cancers, in particular basal-like and normal-like/claudin-low cell lines. Suppression of ELK3 in MDA-MB-231 cells led to transdifferentiation from an invasive mesenchymal phenotype to a non-invasive epithelial phenotype both in vitro and in vivo. Suppression of ELK3 resulted in extensive changes in genome expression profiles. Among these, GATA3, a master suppressor of metastasis, was epigenetically activated. Also, suppression of GATA3 led to the restoration of migration and invasion. These results suggest that the ELK3-GATA3 axis is a major pathway that promotes metastasis of MDA-MB-231 cells.
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http://dx.doi.org/10.18632/oncotarget.11427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323143PMC
October 2016

Hop2 and Sae3 Are Required for Dmc1-Mediated Double-Strand Break Repair via Homolog Bias during Meiosis.

Mol Cells 2016 Jul 21;39(7):550-6. Epub 2016 Jun 21.

Department of Life Sciences, Chung-Ang University, Seoul 06974, Korea.

During meiosis, exchange of DNA segments occurs between paired homologous chromosomes in order to produce recombinant chromosomes, helping to increase genetic diversity within a species. This genetic exchange process is tightly controlled by the eukaryotic RecA homologs Rad51 and Dmc1, which are involved in strand exchange of meiotic recombination, with Rad51 participating specifically in mitotic recombination. Meiotic recombination requires an interaction between homologous chromosomes to repair programmed double-strand breaks (DSBs). In this study, we investigated the budding yeast meiosis-specific proteins Hop2 and Sae3, which function in the Dmc1-dependent pathway. This pathway mediates the homology searching and strand invasion processes. Mek1 kinase participates in switching meiotic recombination from sister bias to homolog bias after DSB formation. In the absence of Hop2 and Sae3, DSBs were produced normally, but showed defects in the DSB-to-single-end invasion transition mediated by Dmc1 and auxiliary factors, and mutant strains failed to complete proper chromosome segregation. However, in the absence of Mek1 kinase activity, Rad51-dependent recombination progressed via sister bias in the hop2Δ or sae3Δ mutants, even in the presence of Dmc1. Thus, Hop2 and Sae3 actively modulate Dmc1-dependent recombination, effectively progressing homolog bias, a process requiring Mek1 kinase activation.
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http://dx.doi.org/10.14348/molcells.2016.0069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959020PMC
July 2016

PI3K/Akt/mTOR activation by suppression of ELK3 mediates chemosensitivity of MDA-MB-231 cells to doxorubicin by inhibiting autophagy.

Biochem Biophys Res Commun 2016 08 11;477(2):277-82. Epub 2016 Jun 11.

Department of Biomedical Science, College of Life Science, CHA University, Seoul, South Korea. Electronic address:

Drug resistance in breast cancer remains a major obstacle of clinical therapy. We found that suppression of ELK3 in the triple negative breast cancer cell line MDA-MB-231 impaired autophagy and led to a hypersensitive response to doxorubicin treatment. In ELK3-knockdown MDA-MB-231 cells (ELK3 KD), autophagy was not activated under starvation conditions, which is a major stimulus of autophagy activation. We revealed that activation of the PI3K/Akt pathway was the main cause of impaired autophagy in ELK3 KD. Our results suggest that targeting ELK3 may be a potential approach to overcome doxorubicin resistance in breast cancer therapeutics.
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http://dx.doi.org/10.1016/j.bbrc.2016.06.057DOI Listing
August 2016

Monitoring of chromosome dynamics of single yeast cells in a microfluidic platform with aperture cell traps.

Lab Chip 2016 Apr;16(8):1358-65

Department of Chemical Engineering, Chungnam National University, 99 Daehak-ro, Yuseong-Gu, Daejeon 305-764, Republic of Korea.

Chromosome movement plays important roles in DNA replication, repair, genetic recombination, and epigenetic phenomena during mitosis and meiosis. In particular, chromosome movement in the nuclear space is essential for the reorganization of the nucleus. However, conventional methods for analyzing the chromosome movements in vivo have been limited by technical constraints of cell trapping, cell cultivation, oxygenation, and in situ imaging. Here, we present a simple microfluidic platform with aperture-based cell trapping arrays to monitor the chromosome dynamics in single living cells for a desired period of time. Under the optimized conditions, our microfluidic platform shows a single-cell trapping efficiency of 57%. This microfluidic approach enables in situ imaging of intracellular dynamics in living cells responding to variable input stimuli under the well-controlled microenvironment. As a validation of this microfluidic platform, we investigate the fundamental features of the dynamic cellular response of the individual cells treated with different stimuli and drug. We prove the basis for dynamic chromosome movement in single yeast cells to be the telomere and nuclear envelope ensembles that attach to and move in concert with nuclear actin cables. Therefore, these results illustrate the monitoring of cellular functions and obtaining of dynamic information at a high spatiotemporal resolution through the integration of a simple microfluidic platform.
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http://dx.doi.org/10.1039/c5lc01422kDOI Listing
April 2016

Ycs4 is Required for Efficient Double-Strand Break Formation and Homologous Recombination During Meiosis.

J Microbiol Biotechnol 2015 Jul;25(7):1026-35

Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea.

Condensin is not only responsible for chromosome condensation, but is also involved in double-strand break (DSB) processing in the cell cycle. During meiosis, the condensin complex serves as a component of the meiotic chromosome axis, and mediates both proper assembly of the synaptonemal complex and DSB repair, in order to ensure proper homologous chromosome segregation. Here, we used the budding yeast Saccharomyces cerevisiae to show that condensin participates in a variety of chromosome organization processes and exhibits crucial molecular functions that contribute to meiotic recombination during meiotic prophase I. We demonstrate that Ycs4 is required for efficient DSB formation and establishing homolog bias at the early stage of meiotic prophase I, which allows efficient formation of interhomolog recombination products. In the Ycs4 meiosis-specific allele (ycs4S), interhomolog products were formed at substantial levels, but with the same reduction in crossovers and noncrossovers. We further show that, in prophase chromosomal events, ycs4S relieved the defects in the progression of recombination interactions induced as a result of the absence of Rec8. These results suggest that condensin is a crucial coordinator of the recombination process and chromosome organization during meiosis.
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http://dx.doi.org/10.4014/jmb.1504.04013DOI Listing
July 2015

Mitotic cohesin subunit Mcd1 regulates the progression of meiotic recombination in budding yeast.

J Microbiol Biotechnol 2015 May;25(5):598-605

Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea.

The cohesin complex holds sister chromatids together and prevents premature chromosome segregation until the onset of anaphase. Mcd1 (also known as Scc1), the α-kleisin subunit of cohesin, is a key regulatory subunit of the mitotic cohesin complex and is required for maintaining sister chromatid cohesion, chromosome organization, and DNA repair. We investigated the function of Mcd1 in meiosis by ectopically expressing Mcd1 during early meiotic prophase I in Saccharomyces cerevisiae. Mcd1 partially regulated the progression of meiotic recombination, sister chromatid separation, and nuclear division. DNA physical analysis during meiotic recombination showed that Mcd1 induced double-strand breaks (DSBs) but negatively regulated homologous recombination during DSB repair; Mcd1 expression delayed post-DSB stages, leading to inefficiencies in the DSB-to-joint molecule (JM) transition and subsequent crossover formation. These findings indicate that meiotic cells undergo Mcd1-mediated DSB formation during prophase I, and that residual Mcd1 could regulate the progression of JM formation during meiotic recombination.
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http://dx.doi.org/10.4014/jmb.1501.01081DOI Listing
May 2015

Rad51 regulates cell cycle progression by preserving G2/M transition in mouse embryonic stem cells.

Stem Cells Dev 2014 Nov 18;23(22):2700-11. Epub 2014 Aug 18.

1 Department of Life Science, Chung-Ang University , Seoul, Korea.

Homologous recombination (HR) maintains genomic integrity against DNA replication stress and deleterious lesions, such as double-strand breaks (DSBs). Rad51 recombinase is critical for HR events that mediate the exchange of genetic information between parental chromosomes in eukaryotes. Additionally, Rad51 and HR accessory factors may facilitate replication fork progression by preventing replication fork collapse and repair DSBs that spontaneously arise during the normal cell cycle. In this study, we demonstrated a novel role for Rad51 during the cell cycle in mouse embryonic stem cells (mESCs). In mESCs, Rad51 was constitutively expressed throughout the cell cycle, and the formation of Rad51 foci increased as the cells entered S phase. Suppression of Rad51 expression caused cells to accumulate at G2/M phase and activated the DNA damage checkpoint, but it did not affect the self-renewal or differentiation capacity of mESCs. Even though Rad51 suppression significantly inhibited the proliferation rate of mESCs, Rad51 suppression did not affect the replication fork progression and speed, indicating that Rad51 repaired DNA damage and promoted DNA replication in S phase through an independent mechanism. In conclusion, Rad51 may contribute to G2/M transition in mESCs, while preserving genomic integrity in global organization of DNA replication fork.
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http://dx.doi.org/10.1089/scd.2014.0129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4216517PMC
November 2014

Shu1 promotes homolog bias of meiotic recombination in Saccharomyces cerevisiae.

Mol Cells 2013 Nov 8;36(5):446-54. Epub 2013 Nov 8.

Department of Life Sciences, Chung-Ang University, Seoul, 156-756, Korea.

Homologous recombination occurs closely between homologous chromatids with highly ordered recombinosomes through RecA homologs and mediators. The present study demonstrates this relationship during the period of "partner choice" in yeast meiotic recombination. We have examined the formation of recombination intermediates in the absence or presence of Shu1, a member of the PCSS complex, which also includes Psy3, Csm2, and Shu2. DNA physical analysis indicates that Shu1 is essential for promoting the establishment of homolog bias during meiotic homologous recombination, and the partner choice is switched by Mek1 kinase activity. Furthermore, Shu1 promotes both crossover (CO) and non-crossover (NCO) pathways of meiotic recombination. The inactivation of Mek1 kinase allows for meiotic recombination to progress efficiently, but is lost in homolog bias where most doublestrand breaks (DSBs) are repaired via stable intersister joint molecules. Moreover, the Srs2 helicase deletion cells in the budding yeast show slightly reduced COs and NCOs, and Shu1 promotes homolog bias independent of Srs2. Our findings reveal that Shu1 and Mek1 kinase activity have biochemically distinct roles in partner choice, which in turn enhances the understanding of the mechanism associated with the precondition for homolog bias.
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http://dx.doi.org/10.1007/s10059-013-0215-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3887942PMC
November 2013

In situ monitoring of antibiotic susceptibility of bacterial biofilms in a microfluidic device.

Lab Chip 2010 Dec 11;10(23):3296-9. Epub 2010 Oct 11.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

Antibiotic resistance of biofilms is a growing public health concern due to overuse and improper use of antibiotics. Thus, determining an effective minimal concentration of antibiotics to eradicate bacterial biofilms is crucial. Here we present a simple, novel one-pot assay for the analysis of antibiotic susceptibility of bacterial biofilms using a microfluidics system where continuous concentration gradients of antibiotics are generated. The results of minimal biofilm eradication concentration (MBEC) clearly confirm that the concentration required to eradicate biofilm-grown Pseudomonas aeruginosa is higher than the minimal inhibitory concentration (MIC) that has been widely used to determine the lowest concentration of antibiotics against planktonically grown bacteria.
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http://dx.doi.org/10.1039/c0lc00154fDOI Listing
December 2010