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Search Results: 1 - 10 of 28519 matches for " Sanghyun Lee equal contributor "
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Human Cytomegalovirus Clinical Strain-Specific microRNA miR-UL148D Targets the Human Chemokine RANTES during Infection
Youngkyun Kim equal contributor,Sanghyun Lee equal contributor,Sungchul Kim,Donghyun Kim,Jin-Hyun Ahn,Kwangseog Ahn
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002577
Abstract: The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a 15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b' sequences of the Toledo genome, targeted the 3′-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV.
Cryptococcus neoformans Overcomes Stress of Azole Drugs by Formation of Disomy in Specific Multiple Chromosomes
Edward Sionov equal contributor,Hyeseung Lee equal contributor,Yun C. Chang equal contributor,Kyung J. Kwon-Chung
PLOS Pathogens , 2010, DOI: 10.1371/journal.ppat.1000848
Abstract: Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.
Novel Low Abundance and Transient RNAs in Yeast Revealed by Tiling Microarrays and Ultra High–Throughput Sequencing Are Not Conserved Across Closely Related Yeast Species
Albert Lee equal contributor,Kasper Daniel Hansen equal contributor,James Bullard equal contributor,Sandrine Dudoit,Gavin Sherlock
PLOS Genetics , 2008, DOI: 10.1371/journal.pgen.1000299
Abstract: A complete description of the transcriptome of an organism is crucial for a comprehensive understanding of how it functions and how its transcriptional networks are controlled, and may provide insights into the organism's evolution. Despite the status of Saccharomyces cerevisiae as arguably the most well-studied model eukaryote, we still do not have a full catalog or understanding of all its genes. In order to interrogate the transcriptome of S. cerevisiae for low abundance or rapidly turned over transcripts, we deleted elements of the RNA degradation machinery with the goal of preferentially increasing the relative abundance of such transcripts. We then used high-resolution tiling microarrays and ultra high–throughput sequencing (UHTS) to identify, map, and validate unannotated transcripts that are more abundant in the RNA degradation mutants relative to wild-type cells. We identified 365 currently unannotated transcripts, the majority presumably representing low abundance or short-lived RNAs, of which 185 are previously unknown and unique to this study. It is likely that many of these are cryptic unstable transcripts (CUTs), which are rapidly degraded and whose function(s) within the cell are still unclear, while others may be novel functional transcripts. Of the 185 transcripts we identified as novel to our study, greater than 80 percent come from regions of the genome that have lower conservation scores amongst closely related yeast species than 85 percent of the verified ORFs in S. cerevisiae. Such regions of the genome have typically been less well-studied, and by definition transcripts from these regions will distinguish S. cerevisiae from these closely related species.
CPAF: A Chlamydial Protease in Search of an Authentic Substrate
Allan L. Chen equal contributor,Kirsten A. Johnson equal contributor,Jennifer K. Lee equal contributor,Christine Sütterlin ,Ming Tan
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002842
Abstract: Bacteria in the genus Chlamydia are major human pathogens that cause an intracellular infection. A chlamydial protease, CPAF, has been proposed as an important virulence factor that cleaves or degrades at least 16 host proteins, thereby altering multiple cellular processes. We examined 11 published CPAF substrates and found that there was no detectable proteolysis when CPAF activity was inhibited during cell processing. We show that the reported proteolysis of these putative CPAF substrates was due to enzymatic activity in cell lysates rather than in intact cells. Nevertheless, Chlamydia-infected cells displayed Chlamydia-host interactions, such as Golgi reorganization, apoptosis resistance, and host cytoskeletal remodeling, that have been attributed to CPAF-dependent proteolysis of host proteins. Our findings suggest that other mechanisms may be responsible for these Chlamydia-host interactions, and raise concerns about all published CPAF substrates and the proposed roles of CPAF in chlamydial pathogenesis.
The Roles of APC and Axin Derived from Experimental and Theoretical Analysis of the Wnt Pathway
Ethan Lee equal contributor,Adrian Salic equal contributor,Roland Krüger equal contributor,Reinhart Heinrich ,Marc W Kirschner
PLOS Biology , 2003, DOI: 10.1371/journal.pbio.0000010
Abstract: Wnt signaling plays an important role in both oncogenesis and development. Activation of the Wnt pathway results in stabilization of the transcriptional coactivator β-catenin. Recent studies have demonstrated that axin, which coordinates β-catenin degradation, is itself degraded. Although the key molecules required for transducing a Wnt signal have been identified, a quantitative understanding of this pathway has been lacking. We have developed a mathematical model for the canonical Wnt pathway that describes the interactions among the core components: Wnt, Frizzled, Dishevelled, GSK3β, APC, axin, β-catenin, and TCF. Using a system of differential equations, the model incorporates the kinetics of protein–protein interactions, protein synthesis/degradation, and phosphorylation/dephosphorylation. We initially defined a reference state of kinetic, thermodynamic, and flux data from experiments using Xenopus extracts. Predictions based on the analysis of the reference state were used iteratively to develop a more refined model from which we analyzed the effects of prolonged and transient Wnt stimulation on β-catenin and axin turnover. We predict several unusual features of the Wnt pathway, some of which we tested experimentally. An insight from our model, which we confirmed experimentally, is that the two scaffold proteins axin and APC promote the formation of degradation complexes in very different ways. We can also explain the importance of axin degradation in amplifying and sharpening the Wnt signal, and we show that the dependence of axin degradation on APC is an essential part of an unappreciated regulatory loop that prevents the accumulation of β-catenin at decreased APC concentrations. By applying control analysis to our mathematical model, we demonstrate the modular design, sensitivity, and robustness of the Wnt pathway and derive an explicit expression for tumor suppression and oncogenicity.
DNA Display III. Solid-Phase Organic Synthesis on Unprotected DNA
David R Halpin equal contributor,Juanghae A Lee equal contributor,S. Jarrett Wrenn equal contributor,Pehr B Harbury
PLOS Biology , 2004, DOI: 10.1371/journal.pbio.0020175
Abstract: DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl–based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide–DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation.
U87MG Decoded: The Genomic Sequence of a Cytogenetically Aberrant Human Cancer Cell Line
Michael James Clark equal contributor,Nils Homer equal contributor,Brian D. O'Connor equal contributor,Zugen Chen equal contributor,Ascia Eskin,Hane Lee,Barry Merriman,Stanley F. Nelson
PLOS Genetics , 2010, DOI: 10.1371/journal.pgen.1000832
Abstract: U87MG is a commonly studied grade IV glioma cell line that has been analyzed in at least 1,700 publications over four decades. In order to comprehensively characterize the genome of this cell line and to serve as a model of broad cancer genome sequencing, we have generated greater than 30× genomic sequence coverage using a novel 50-base mate paired strategy with a 1.4kb mean insert library. A total of 1,014,984,286 mate-end and 120,691,623 single-end two-base encoded reads were generated from five slides. All data were aligned using a custom designed tool called BFAST, allowing optimal color space read alignment and accurate identification of DNA variants. The aligned sequence reads and mate-pair information identified 35 interchromosomal translocation events, 1,315 structural variations (>100 bp), 191,743 small (<21 bp) insertions and deletions (indels), and 2,384,470 single nucleotide variations (SNVs). Among these observations, the known homozygous mutation in PTEN was robustly identified, and genes involved in cell adhesion were overrepresented in the mutated gene list. Data were compared to 219,187 heterozygous single nucleotide polymorphisms assayed by Illumina 1M Duo genotyping array to assess accuracy: 93.83% of all SNPs were reliably detected at filtering thresholds that yield greater than 99.99% sequence accuracy. Protein coding sequences were disrupted predominantly in this cancer cell line due to small indels, large deletions, and translocations. In total, 512 genes were homozygously mutated, including 154 by SNVs, 178 by small indels, 145 by large microdeletions, and 35 by interchromosomal translocations to reveal a highly mutated cell line genome. Of the small homozygously mutated variants, 8 SNVs and 99 indels were novel events not present in dbSNP. These data demonstrate that routine generation of broad cancer genome sequence is possible outside of genome centers. The sequence analysis of U87MG provides an unparalleled level of mutational resolution compared to any cell line to date.
Use of Saliva for Early Dengue Diagnosis
Grace Yap equal contributor,Bijon Kumar Sil equal contributor,Lee-Ching Ng
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0001046
Abstract: Background The necessity of a venous blood collection in all dengue diagnostic assays and the high cost of tests that are available for testing during the viraemic period hinder early detection of dengue cases and thus could delay cluster management. This study reports the utility of saliva in an assay that detects dengue virus (DENV)–specific immunoglobulin A (Ig A) early in the phase of a dengue infection. Methods and Findings Using an antigen capture anti-DENV IgA (ACA) ELISA technique, we tested saliva samples collected from dengue-confirmed patients. The sensitivity within 3 days from fever onset was over 36% in primary dengue infections. The performance is markedly better in secondary infections, with 100% sensitivity reported in saliva samples from day 1 after fever onset. Serum and salivary IgA levels showed good correlation (Pearson's r = 0.69, p<0.001). Specificity was found to be 97%. Conclusion Our findings suggest that this technique would be very useful in dengue endemic regions, where the majority of dengue cases are secondary. The ACA-ELISA is easy to perform, cost effective, and especially useful in laboratories without sophisticated equipment. Our findings established the usefulness and reliability of saliva for early dengue diagnosis.
Role for Sumoylation in Systemic Inflammation and Immune Homeostasis in Drosophila Larvae
Indira Paddibhatla,Mark J. Lee,Marta E. Kalamarz equal contributor,Roberto Ferrarese equal contributor,Shubha Govind
PLOS Pathogens , 2010, DOI: 10.1371/journal.ppat.1001234
Abstract: To counter systemic risk of infection by parasitic wasps, Drosophila larvae activate humoral immunity in the fat body and mount a robust cellular response resulting in encapsulation of the wasp egg. Innate immune reactions are tightly regulated and are resolved within hours. To understand the mechanisms underlying activation and resolution of the egg encapsulation response and examine if failure of the latter develops into systemic inflammatory disease, we correlated parasitic wasp-induced changes in the Drosophila larva with systemic chronic conditions in sumoylation-deficient mutants. We have previously reported that loss of either Cactus, the Drosophila (IκB) protein or Ubc9, the SUMO-conjugating enzyme, leads to constitutive activation of the humoral and cellular pathways, hematopoietic overproliferation and tumorogenesis. Here we report that parasite infection simultaneously activates NF-κB-dependent transcription of Sp?tzle processing enzyme (SPE) and cactus. Endogenous Sp?tzle protein (the Toll ligand) is expressed in immune cells and excessive SPE or Sp?tzle is pro-inflammatory. Consistent with this function, loss of Spz suppresses Ubc9? defects. In contrast to the pro-inflammatory roles of SPE and Sp?tzle, Cactus and Ubc9 exert an anti-inflammatory effect. We show that Ubc9 maintains steady state levels of Cactus protein. In a series of immuno-genetic experiments, we demonstrate the existence of a robust bidirectional interaction between blood cells and the fat body and propose that wasp infection activates Toll signaling in both compartments via extracellular activation of Sp?tzle. Within each organ, the IκB/Ubc9-dependent inhibitory feedback resolves immune signaling and restores homeostasis. The loss of this feedback leads to chronic inflammation. Our studies not only provide an integrated framework for understanding the molecular basis of the evolutionary arms race between insect hosts and their parasites, but also offer insights into developing novel strategies for medical and agricultural pest control.
Two Separate Interfaces between the Voltage Sensor and Pore Are Required for the Function of Voltage-Dependent K+ Channels
Seok-Yong Lee equal contributor,Anirban Banerjee equal contributor,Roderick MacKinnon
PLOS Biology , 2009, DOI: 10.1371/journal.pbio.1000047
Abstract: Voltage-dependent K+ (Kv) channels gate open in response to the membrane voltage. To further our understanding of how cell membrane voltage regulates the opening of a Kv channel, we have studied the protein interfaces that attach the voltage-sensor domains to the pore. In the crystal structure, three physical interfaces exist. Only two of these consist of amino acids that are co-evolved across the interface between voltage sensor and pore according to statistical coupling analysis of 360 Kv channel sequences. A first co-evolved interface is formed by the S4-S5 linkers (one from each of four voltage sensors), which form a cuff surrounding the S6-lined pore opening at the intracellular surface. The crystal structure and published mutational studies support the hypothesis that the S4-S5 linkers convert voltage-sensor motions directly into gate opening and closing. A second co-evolved interface forms a small contact surface between S1 of the voltage sensor and the pore helix near the extracellular surface. We demonstrate through mutagenesis that this interface is necessary for the function and/or structure of two different Kv channels. This second interface is well positioned to act as a second anchor point between the voltage sensor and the pore, thus allowing efficient transmission of conformational changes to the pore's gate.
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