Regulation of Histone Covalent Modifications During Yeast Apoptosis

Chromatin compaction is a hallmark property of apoptosis, a highly coordinated suicide mechanism generally believed to be confined to vertebrates. However, invertebrates such as the budding yeast, Saccharomyces cerevisiae, display an apoptotic-like phenotypes including chromatin condensation, although its functions and mechanism are unclear. One mechanism that alters chromatin structure is the covalent modification of histones, which associates with DNA to form the nucleosome, the fundamental unit of chromatin. Phosphorylation of histone H2B at serine 14 (H2BS14ph), catalyzed by Mst1 kinase, has been linked to chromatin compaction during mammalian apoptosis. I extended these results to yeast by demonstrating that Ste20 kinase, a yeast orthologue of Mst1, directly phosphorylates H2B at serine 10 (H2BS10ph) in a hydrogen peroxide-induced cell death pathway. Unlike Mst1, Ste20 translocates into the nucleus in a caspase-independent fashion to mediate phosphorylation of H2B. H2BS10ph is dependent on the removal of acetylation mark on adjacent lysine residue, (H2BK11ac), which exists in growing yeast. During yeast apoptosis, the HDAC Hos3 deacetylates K11, which in turn, mediates H2BS10ph by Ste20 kinase. My studies underscore a concerted series of enzyme reactions governing histone modifications that promote a switch from cell proliferation to cell death. Moreover, the conservation of targeted H2B phosphorylation and the enzyme system point to an ancient, late-stage chromatin remodeling event that likely governs cellular homeostasis in a wide range of organisms. H2B phosphorylation may mediate apoptotic chromatin compaction by 1) directly affecting internucleosomal contacts and histone DNA interaction (“cis mechanismâ€), or 2) recruiting binding partners that then induce and direct downstream functions (“trans†mechanisms). Peptides corresponding to the phosphorylated form of yeast H2B and human H2B have the intrinsic ability to form “aggregates†in SDS polyacrylamide gel electrophoresis. In addition, nucleosome array containing yeast S10E or human S14E H2B fold into compacted conformation as measured by analytical ultracentrifugation. Moreover, an interaction between the forkhead homology-associated domain 1 (FHA1) of Rad53 and H2BS10ph was uncovered. This interaction inactivates the DNA damage checkpoint pathway and promotes apoptotic chromatin condensation. Thus, both mechanisms may contribute to chromatin remodeling event that govern apoptotic chromatin compaction in a pathway conserved from yeast to humans

재현성있는 말디스펙트럼의 형성과 분석물의 정량분석에 응용

학위논문 (박사)-- 서울대학교 대학원 : 화학부 물리화학 전공, 2017. 2. 이성훈.Matrix-assisted laser desorption ionization (MALDI) is a useful ionization technique for the mass spectrometry of biomolecules. One of the requirements for generating reproducible MALDI spectra is to prepare samples with good homogeneity. Until now, we have not been successful in producing homogeneous solid samples from popular matrixes apart from α-cyano-4-hydroxycinnamic acid (CHCA). In chapter 1, we showed the production of an outwardly homogeneous solid sample by loading a methanol solution of a matrix into a shallow reservoir on a coated MALDI sample plate and then vacuum-drying it. Out of ten popular matrixes tested, seven yielded homogeneous samples. These were 9-aminoacridine, 6-aza-2-thiothymine, CHCA, 2,5-dihydroxybenzoic acid (DHB), ferulic acid, sinapinic acid, and 2,4,6-trihydroxyacetophenone. For MALDI with these matrixes, linear calibration curves plotted in the form of I(A+H+)/I(M+H+) versus analyte concentration were acquired. Features of these matrixes in various aspects of analyte quantification have been examined. In our previous MALDI studies of peptides, we also found that their mass spectra were practically determined by the effective temperature in the early matrix plume, Tearly, when samples were homogeneous. But calculating Tearly was complicated. In chapter 2, we explained another empirical rule that the total number of particles hitting the detector (TIC) was a good measure of the spectral temperature. We also succeeded in generating reproducible spectra throughout a measurement by controlling TIC near a preset value through feedback adjustment of laser pulse energy. TIC control substantially reduced the shot-to-shot spectral variation in a spot, spot-to-spot variation in a sample, and even sample-to-sample variation in MALDI using CHCA or DHB as matrix. This technique produced calibration curves with excellent linearity, suggesting their utility in quantification of peptides. In chapter 3, we proposed to divide matrix suppression in MALDI into two parts, normal and anomalous. In quantification of peptides, the normal effect can be accounted for by constructing the calibration curve in the form of peptide-to-matrix ion abundance ratio versus concentration. The anomalous effect forbids reliable quantification and is noticeable when matrix suppression is larger than 70% for CHCA matrix. With this 70% rule, matrix suppression becomes a guideline for reliable quantification, rather than a nuisance. A peptide in a complex mixture can be quantified even in the presence of large amounts of contaminants, as long as matrix suppression is below 70%. Lastly, we attempted to quantify various molecules. In chapter 4, we quantified proteins by quantifying their tryptic peptides with the aforementioned method. We modified the digestion processe.g. disulfide bonds were not cleaved, so that hardly any reagent other than trypsin remained after the digestion process. This allowed the preparation of a sample by the direct mixing of a digestion mixture with a matrix solution. We also observed that the efficiency of the matrix-to-peptide proton transfer, as measured by its reaction quotient was similar for peptides with arginine at the C-terminus. With the reaction quotient averaged over many such peptides, we could rapidly quantify proteins. Most importantly, no peptide standard, not to mention its isotopically labeled analog, was needed in this method. In chapter 5, the utility of sodium ion adducts produced by MALDI for the quantification of analytes with multiple oxygen atoms was evaluated. The method resulted in a direct proportionality between the ion abundance ratio I([A + Na]+)/I([M + Na]+) versus analyte concentration, which could be used as a calibration curve. This was showed for carbohydrates, glycans, and polyether diols with dynamic range exceeding three orders of magnitude.1. Preparation of Homogenous Solid Samples 1 1.1 Introduction 1 1.2 Experimental 2 1.3 Results and Discussion 7 1.3.1 Analyte distribution in a solid sample 7 1.3.2 Analyte ion abundances 9 1.3.3 Calibration curves 9 1.3.4 Temperature of the early plume 13 1.4 Conclusion 15 2. TIC (Total Ion Count) control technique 16 2.1 Introduction 16 2.2 Experimental 18 2.3 Results and Discussion 19 2.3.1 Finding a measure of spectral temperature 19 2.3.2 Quantitative reproducibility of TIC selected spectra 22 2.3.3 Acquisition of reproducible spectra by TIC control 26 2.4 Conclusion 38 3. Matrix Suppression 40 3.1 Introduction 40 3.2 Experimental 44 3.3 Results and Discussion 44 3.3.1 Calibration curves 44 3.3.2 Peptide mixtures 46 3.4 Conclusion 50 4. Quick Quantification of Proteins 51 4.1 Introduction 51 4.2 Experimental 55 4.3 Results and Discussion 56 4.3.1 Myoglobin 58 4.3.2 Lysozyme 62 4.3.3 Human growth hormone 64 4.3.4 Quick absolute quantification of proteins without using peptide standards 65 4.4 Conclusion 71 5. Quantification of Carbohydrates and Related Materials 74 5.1 Introduction 74 5.2 Experimental 76 5.3 Results and Discussion 77 5.4 Conclusion 82 References 83 Publication lists 95Docto

Top-down and bottom-up neurodynamic evidence in patients with tinnitus

AbstractAlthough a peripheral auditory (bottom-up) deficit is an essential prerequisite for the generation of tinnitus, central cognitive (top-down) impairment has also been shown to be an inherent neuropathological mechanism. Using an auditory oddball paradigm (for top-down analyses) and a passive listening paradigm (for bottom-up analyses) while recording electroencephalograms (EEGs), we investigated whether top-down or bottom-up components were more critical in the neuropathology of tinnitus, independent of peripheral hearing loss. We observed significantly reduced P300 amplitudes (reflecting fundamental cognitive processes such as attention) and evoked theta power (reflecting top-down regulation in memory systems) for target stimuli at the tinnitus frequency of patients with tinnitus but without hearing loss. The contingent negative variation (reflecting top-down expectation of a subsequent event prior to stimulation) and N100 (reflecting auditory bottom-up selective attention) were different between the healthy and patient groups. Interestingly, when tinnitus patients were divided into two subgroups based on their P300 amplitudes, their P170 and N200 components, and annoyance and distress indices to their tinnitus sound were different. EEG theta-band power and its Granger causal neurodynamic results consistently support a double dissociation of these two groups in both top-down and bottom-up tasks. Directed cortical connectivity corroborates that the tinnitus network involves the anterior cingulate and the parahippocampal areas, where higher-order top-down control is generated. Together, our observations provide neurophysiological and neurodynamic evidence revealing a differential engagement of top-down impairment along with deficits in bottom-up processing in patients with tinnitus but without hearing loss

SOLiDzipper: A High Speed Encoding Method for the Next-Generation Sequencing Data

Background Next-generation sequencing (NGS) methods pose computational challenges of handling large volumes of data. Although cloud computing offers a potential solution to these challenges, transferring a large data set across the internet is the biggest obstacle, which may be overcome by efficient encoding methods. When encoding is used to facilitate data transfer to the cloud, the time factor is equally as important as the encoding efficiency. Moreover, to take advantage of parallel processing in cloud computing, a parallel technique to decode and split compressed data in the cloud is essential. Hence in this review, we present SOLiDzipper, a new encoding method for NGS data. Methods The basic strategy of SOLiDzipper is to divide and encode. NGS data files contain both the sequence and non-sequence information whose encoding efficiencies are different. In SOLiDzipper, encoded data are stored in binary data block that does not contain the characteristic information of a specific sequence platform, which means that data can be decoded according to a desired platform even in cases of Illumina, Solexa or Roche 454 data. Results The main calculation time using Crossbow was 173 minutes when 40 EC2 nodes were involved. In that case, an analysis preparation time of 464 minutes is required to encode data in the latest DNA compression method like G-SQZ and transmit it on a 183 Mbit/s bandwidth. However, it takes 194 minutes to encode and transmit data with SOLiDzipper under the same bandwidth conditions. These results indicate that the entire processing time can be reduced according to the encoding methods used, under the same network bandwidth conditions. Considering the limited network bandwidth, high-speed, high-efficiency encoding methods such as SOLiDzipper can make a significant contribution to higher productivity in labs seeking to take advantage of the cloud as an alternative to local computing. Availability http://szipper.dinfree.com . Academic/non-profit: Binary available for direct download at no cost. For-profit: Submit request for for-profit license from the web-site

Chiroptical signal enhancement in quasi-null-polarization-detection geometry: Intrinsic limitations

Despite its unique capability of distinguishing molecular handedness, chiroptical spectroscopy suffers from the weak-signal problem, which has restricted more extensive applications. The quasi-null-polarization-detection (QNPD) method has been shown to be useful for enhancing the chiroptical signal. Here, the underlying enhancement mechanism in the QNPD method combined with a heterodyne detection scheme is elucidated. It is experimentally demonstrated that the optical rotatory dispersion signal can be amplified by a factor of similar to 400, which is the maximum enhancement effect achievable with our femtosecond laser setup. The upper limit of the QNPD enhancement effect of chiroptical measurements could, in practice, be limited by imperfection of the polarizer and finite detection sensitivity. However, we show that there exists an intrinsic limit in the enhancement with the QNPD method due to the weak but finite contribution from the homodyne chiroptical signal. This is experimentally verified by measuring the optical rotation of linearly polarized light with the QNPD scheme. We further provide discussions on the connection between this intrinsic limitation in the QNPD scheme for enhanced detection of weak chiroptical signals and those in optical enantioselectivity and Raman optical activity with a structured chiral field. We anticipate that the present work could be useful in further developing time-resolved nonlinear chiroptical spectroscopy.111Nsciescopu

An Outbreak of Caparinia tripilis in a Colony of African Pygmy Hedgehogs (Atelerix albiventris) from Korea

In February 2010, dermatitis characterized by scale and self-trauma due to puritis was recognized in a group of 22 four-toed hedgehogs (Atelerix albiventris Wagner, 1841) from a local pet shop in Gwangju, Korea. Microscopic examinations of skin scraping samples showed numerous mites of all developmental stages. Morphologically, pedicels of adult mites were short and unjointed. Tarsal caruncles were bell-shaped on all legs of males while they were absent on legs III and IV of females. Three long setae on the third pair of legs in both sexes were present. Adult males had posterior end of the abdomen with trilobate projection on each side, each lobe with a long seta. Based on these features, the mites were identified as Caparinia tripilis. This is the first report of caparinic mite infestation in hedgehogs from Korea. Identification keys for the family Psoroptidae and the genus Caparinia are provided

Comparison of Clinical Outcomes Following Gefitinib and Erlotinib Treatment in Non–Small-Cell Lung Cancer Patients Harboring an Epidermal Growth Factor Receptor Mutation in Either Exon 19 or 21

Background:Gefitinib and erlotinib, small-molecule kinase inhibitors that block epidermal growth factor receptor (EGFR) signaling, have demonstrated a dramatic response rate and prolonged progression-free survival (PFS) in patients harboring an activating EGFR mutation. We compared the clinical outcomes in gefitinib- and erlotinib-treated patients harboring EGFR mutations who had recurrent or metastatic non–small-cell lung cancer (NSCLC).Methods:A total of 375 patients with recurrent or metastatic stage IIIB/IV NSCLC, who had either exon 19 deletion or the L858R mutation in exon 21, and had received either gefitinib (n = 228) or erlotinib (n = 147), were included in the study. A matched-pair case-control study design was implemented in the analysis, where 121 pairs of gefitinib-treated and erlotinib-treated patients were matched according to sex, smoking history, Eastern Cooperative Oncology Group performance status, and types of EGFR mutation.Results:The median age of all patients was 58 years (range, 30–84), and more than half of patients had never been smokers (63.6%). Most patients had adenocarcinoma (98.3%) and good Eastern Cooperative Oncology Group performance status (0, 1) (90.9%). The median number of cycles of EGFR tyrosine kinase inhibitor (TKI) treatment was 12.7 in the gefitinib group and 10.8 in the erlotinib group. Of the 242 patients, 63 (26%) received EGFR TKI as first-line therapy. The overall response rates and disease control rates in the gefitinib- or erlotinib-treated groups were 76.9% versus 74.4% (p = 0.575) and 90.1% versus 86.8%, respectively (p = 0.305). There was no statistically significant difference with regard to PFS (median, 11.7 versus 9.6; p = 0.056) between the gefitinib- and erlotinib-treated groups. For patients receiving EGFR TKI as the first-line treatment, there was no significant difference between the two treatment groups in overall response rates (76.7% and 90.0%) (p = 0.431) and median PFS (11.7 versus 14.5 months) (p = 0.507).Conclusion:In NSCLC patients harboring EGFR mutation, treatment with gefitinib and erlotinib resulted in similar effectiveness