Web-based design and analysis tools for CRISPR base editing

Background: As a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed. Results: We present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (< 1 GB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/be-designer/ and http://www.rgenome.net/be-analyzer /, respectively. Conclusion: We develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors

UNSUK CHIN’S PIANO MUSIC: 6 PIANO ETUDES (1995-2003) AND PIANO CONCERTO (1996-97)

Unsuk Chin (b. 1961) is the most internationally recognised living Korean-born composer. She has received numerous awards including the prestigious 2004 Grawemeyer Award for her Violin Concerto, the 2005 Arnold Schoenberg prize, the 2010 Prince Pierre Foundation Music Award, and the 2012 Ho-Am Prize, and her works are performed worldwide by major orchestras and contemporary ensembles. The 6 Piano Etudes (1995-2003) and Piano Concerto (1996-1997) are important pieces of Chin’s, because these are her only compositions involving solo piano up to this point. They include many of the elements of her musical world as well as her various compositional styles and approaches to writing for the piano. In Chapter 1 of this Doctoral dissertation I provide a brief introduction to my research. In Chapter 2, Chin’s biography is outlined and an overview is given of her 6 Piano Etudes and Piano Concerto. Following this are in-depth analyses of the 6 Piano Etudes and Piano Concerto in Chapters 3 and 4 respectively, examining form and structure, harmony, rhythm, and Chin’s unique compositional methods. Chapters 3 and 4 focus on the influences and models for Chin’s music: gamelan ensemble, Olivier Messiaen (1908-1992), and Chin’s teachers, György Ligeti (1923-2006) and Sukhi Kang (b. 1934) as well as aspects of Korean music. In examining the synthesis of these influences along with her mature compositional approaches, I hope to add to the growing body of knowledge and understanding of Chin’s significance and of her work, the piano works in particular

Determining the coordination structure of metalloproteins with mass spectrometry

Due to its versatility, mass spectrometry (MS) has been recognized as an excellent tool for the analysis of peptides and proteins. The structural information that this technique has provided for metalloproteins so far, however, has not extended much to coordination structure. Metal catalyzed oxidation (MCO) reactions in conjunction with MS analysis have been utilized to determine the metal-binding sites of proteins. This method involves first selectively oxidizing the protein by generating reactive oxygen species (ROS) in the immediate vicinity of the metal. These ROS can quickly react with the amino acids that are part of the metal-binding site. The oxidatively modified residues are then identified using a combination of proteolytic enzymes and the peptide sequencing ability of MS. The initial focus of the dissertation was on finding the most specific MCO reaction conditions to site-specifically oxidize the amino acids bound to Cu and Fe in several proteins with known binding sites to test the viability of this approach. Relatively general reaction conditions involving ascorbate as a reducing agent and O2 and/or H2O2 as oxidizing agents have been found. The utility of this MCO/MS approach was assessed by the application of the method to β-2-microglobulin (β2m), which has unknown Cu-binding sites. The MCO/MS approach allowed us to gather information on Cu coordination under conditions that would make such determinations very difficult by other techniques. Interestingly, our results show that different conformers of β2m have different Cu coordination environments, and these results may provide insight into the Cu-induced amyloidogenesis of this protein. The MCO/MS procedure relies on collision-induced dissociation (CID) of oxidized peptide fragments. Hence, a better understanding of the dissociation patterns of oxidatively modified peptides has also been sought to facilitate identification of the oxidized residues. In general, the dissociation patterns of oxidized peptide ions are found to depend on peptide composition, charge state, and type of modification. More specifically, oxidation of methionine and cysteine residues, but not histidine residues, has a dramatic effect on the dissociation of peptide ions when the charge state of the peptide is less than or equal to the number of basic residues in the peptide

Study of the removal difference in indoor particulate matter and volatile organic compounds through the application of plants

This study was conducted to evaluate the ability of plants to purify indoor air by observing the effective reduction rate among pollutant types of particulate matter (PM) and volatile organic compounds (VOCs). PM and four types of VOCs were measured in a new building that is less than three years old and under three different conditions: before applying the plant, after applying the plant, and a room without a plant. The removal rate of each pollutant type due to the plant was also compared and analyzed. In the case of indoor PM, the removal effect was negligible because of outdoor influence. However, 9% of benzene, 75% of ethylbenzene, 72% of xylene, 75% of styrene, 50% of formaldehyde, 36% of acetaldehyde, 35% of acrolein with acetone, and 85% of toluene were reduced. The purification of indoor air by natural ventilation is meaningless because the ambient PM concentration has recently been high. However, contamination by gaseous materials such as VOCs can effectively be removed through the application of plants

A Noise Model for Mass Spectrometry Based Proteomics

Motivation: Mass spectrometry data are subjected to considerable noise. Good noise models are required for proper detection and quantification of peptides. We have characterized noise in both quadrupole time-of-flight (Q-TOF) and ion trap data, and have constructed models for the noise. Results: We find that the noise in Q-TOF data from Applied Biosystems QSTAR fits well to a combination of multinomial and Poisson model with detector dead-time correction. In comparison, ion trap noise from Agilent MSD-Trap-SL is larger than the Q-TOF noise and is proportional to Poisson noise. We then demonstrate that the noise model can be used to improve deisotoping for peptide detection, by estimating appropriate cutoffs of the goodness of fit parameter at prescribed error rates. The noise models also have implications in noise reduction, retention time alignment and significance testing for biomarker discovery

Al2O3-Coated Ni/CeO2 nanoparticles as coke-resistant catalyst for dry reforming of methane

Nickel is considered an economically feasible catalyst for the dry reforming of methane (DRM) owing to its high activity. Because the highly endothermic DRM requires a high reaction temperature to activate both CH4 and CO2, deactivation of the Ni catalyst may be induced by sintering and carbon coking. To mitigate catalyst deactivation, Ni/CeO2 catalysts composed of monodisperse Ni nanoparticles supported on CeO2 nanorods are designed and coated with Al2O3 layers by atomic layer deposition (ALD). The performance of the catalyst in DRM and amount of carbon deposited are correlated with the thickness of the Al2O3 layer in the Ni/CeO2/Al2O3 catalysts. As the number of ALD cycles increases from 1 to 10, the conversion of CO2 and CH4 at 700 and 800 degrees C decreases, but the Ni/CeO2/Al2O3 catalysts remain coke-free as thermogravimetric analysis shows no weight loss up to 800 degrees C. The Al2O3 layer generated by ALD curtails the coking substantially, but the weakly metallic character of Ni and blocking of Ni sites by the Al2O3 layer are the major factors contributing to decreasing the catalytic conversion. The ALD technique provides an efficient way to fabricate atomically controlled oxide layers for improving the stability of catalysts against coke deposition and sintering

Comparison of Fucose-Specific Lectins to Improve Quantitative AFP-L3 Assay for Diagnosing Hepatocellular Carcinoma Using Mass Spectrometry

Glycoproteins have many important biological functions. In particular, aberrant glycosylation has been observed in various cancers, such as liver cancer. A well-known glycoprotein biomarker is alpha-fetoprotein (AFP), a surveillance biomarker for hepatocellular carcinoma (HCC) that contains a glycosylation site at asparagine 251. The low diagnostic sensitivity of AFP led researchers to focus on AFP-L3, which has the same sequence as conventional AFP but contains a fucosylated glycan. AFP-L3 has high affinity for Lens culinaris agglutinin (LCA) lectin, prompting many groups to use it for detecting AFP-L3. However, a few studies have identified more effective lectins for fractionating AFP-L3. In this study, we compared the amounts of enriched AFP-L3 with five fucose-specific lectins-LCA, Lotus tetragonolobus lectin (LTL), Ulex europaeus agglutinin I (UEA I), Aleuria aurantia lectin (AAL), and Aspergillus oryzae lectin (AOL)-to identify better lectins and improve HCC diagnostic assays using mass spectrometry (MS). Our results indicate that LTL was the most effective lectin for capturing AFP-L3 species, yielding approximately 3-fold more AFP-L3 than LCA from the same pool of HCC serum samples. Thus, we recommend the use of LTL for AFP-L3 assays, given its potential to improve the diagnostic sensitivity in patients having limited results by conventional LCA assay. The MS data have been deposited to the PeptideAtlas (PASS01752).N