Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

Synergistic Antibacterial Effects of Metallic Nanoparticle Combinations

© The Author(s) 2019.Metallic nanoparticles have unique antimicrobial properties that make them suitable for use within medical and pharmaceutical devices to prevent the spread of infection in healthcare. The use of nanoparticles in healthcare is on the increase with silver being used in many devices. However, not all metallic nanoparticles can target and kill all disease-causing bacteria. To overcome this, a combination of several different metallic nanoparticles were used in this study to compare effects of multiple metallic nanoparticles when in combination than when used singly, as single elemental nanoparticles (SENPs), against two common hospital acquired pathogens (Staphylococcus aureus and Pseudomonas. aeruginosa). Flow cytometry LIVE/DEAD assay was used to determine rates of cell death within a bacterial population when exposed to the nanoparticles. Results were analysed using linear models to compare effectiveness of three different metallic nanoparticles, tungsten carbide (WC), silver (Ag) and copper (Cu), in combination and separately. Results show that when the nanoparticles are placed in combination (NPCs), antimicrobial effects significantly increase than when compared with SENPs (P < 0.01). This study demonstrates that certain metallic nanoparticles can be used in combination to improve the antimicrobial efficiency in destroying morphologically distinct pathogens within the healthcare and pharmaceutical industry.Peer reviewe

Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution

August 1, 2010Bisulfite sequencing measures absolute levels of DNA methylation at single-nucleotide resolution, providing a robust platform for molecular diagnostics. Here, we optimize bisulfite sequencing for genome-scale analysis of clinical samples. Specifically, we outline how restriction digestion targets bisulfite sequencing to hotspots of epigenetic regulation; we show that 30ng of DNA are sufficient for genome-scale analysis; we demonstrate that our protocol works well on formalinfixed, paraffin-embedded (FFPE) samples; and we describe a statistical method for assessing significance of altered DNA methylation patterns.National Institutes of Health (U.S.) (Grant R01HG004401)National Institutes of Health (U.S.) (Grant U54HG03067)National Institutes of Health (U.S.) (Grant U01ES017155

Genome-wide DNA methylation analysis for diabetic nephropathy in type 1 diabetes mellitus

BACKGROUND: Diabetic nephropathy is a serious complication of diabetes mellitus and is associated with considerable morbidity and high mortality. There is increasing evidence to suggest that dysregulation of the epigenome is involved in diabetic nephropathy. We assessed whether epigenetic modification of DNA methylation is associated with diabetic nephropathy in a case-control study of 192 Irish patients with type 1 diabetes mellitus (T1D). Cases had T1D and nephropathy whereas controls had T1D but no evidence of renal disease. METHODS: We performed DNA methylation profiling in bisulphite converted DNA from cases and controls using the recently developed Illumina Infinium(R) HumanMethylation27 BeadChip, that enables the direct investigation of 27,578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14,495 genes. RESULTS: Singular Value Decomposition (SVD) analysis indicated that significant components of DNA methylation variation correlated with patient age, time to onset of diabetic nephropathy, and sex. Adjusting for confounding factors using multivariate Cox-regression analyses, and with a false discovery rate (FDR) of 0.05, we observed 19 CpG sites that demonstrated correlations with time to development of diabetic nephropathy. Of note, this included one CpG site located 18 bp upstream of the transcription start site of UNC13B, a gene in which the first intronic SNP rs13293564 has recently been reported to be associated with diabetic nephropathy. CONCLUSION: This high throughput platform was able to successfully interrogate the methylation state of individual cytosines and identified 19 prospective CpG sites associated with risk of diabetic nephropathy. These differences in DNA methylation are worthy of further follow-up in replication studies using larger cohorts of diabetic patients with and without nephropathy

Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP) assays in using in vitro methylated genomic DNA

<p>Abstract</p> <p>Background</p> <p>DNA methylation contributes to the regulation of gene expression during development and cellular differentiation. The recently developed Methylated DNA ImmunoPrecipitation (MeDIP) assay allows a comprehensive analysis of this epigenetic mark at the genomic level in normal and disease-derived cells. However, estimating the efficiency of the MeDIP technique is difficult without previous knowledge of the methylation status of a given cell population. Attempts to circumvent this problem have involved the use of <it>in vitro </it>methylated DNA in parallel to the investigated samples. Taking advantage of this stratagem, we sought to improve the sensitivity of the approach and to assess potential biases resulting from DNA amplification and hybridization procedures using MeDIP samples.</p> <p>Findings</p> <p>We performed MeDIP assays using <it>in vitro </it>methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using <it>in vitro </it>methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA), we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data.</p> <p>Conclusion</p> <p>We illustrate the use of <it>in vitro </it>methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.</p

High Resolution Detection and Analysis of CpG Dinucleotides Methylation Using MBD-Seq Technology

Methyl-CpG binding domain protein sequencing (MBD-seq) is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM) to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ∼100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution

EFEKTIVITAS DESENTRALISASI PENDIDIKAN DALAM MENINGKATKAN PROFESIONALISME GURU DI SMA NEGERI 1 LAKEA

Skripsi ini membahas tentang efektivitas desentralisasi pendidikan dalammeningkatkan profesionalisme guru di SMA Negeri 1 Lakea dengan pokok pembahasana bagaimana deskripsi desentralisasi pendidikan dalam meningkatkan profesionalisme guru di SMA Negeri 1 Lakea? dan bagaimana implikasi desentralisasi pendidikan dalam meningkatkan profesionalisme guru di SMA Negeri 1 Lakea. Tujuan penelitian ini untuk mengetahuideskripsi dan implikasi desentralisasi pendidikan dalam meningkatkan prodfesionalisme guru di sekolah tersebut.Untuk menjawab permsalahan tersebut, penelitian ini menggunakan metode kualitatif dengan teknik pengumpulan data melalui observasi, wawancara dan dokumentasi, serta menggunakan teknik analisis data melalui reduksi data, penyajian data, verifikasi data dan penarikan kesimpulan.Hasil penelitian skripsi ini yaitu: deskripsi Desentralisasi Pendidikan dalam Meningkatkan Profesionalisme Guru di SMA Negeri 1 Lakea: (1) Desentralisasi pendidikan merupakan pemberian kewenangan kepada daerah untuk mengelola pendidikannya, lalu daerah melimpahkan kepada masing-masing sekolah (2) Pemerintah pusat tetap mengontrol pendidikan yang diselenggarakan oleh pemerintah daerah dan masing-masing sekolah melalui akreditasi nasional yang dilaksanakan oleh BAN-SM (3) Aspek-aspek yang menjadi kewenangan sekolah dalam melaksanakan desentralisai pendidikan yakni: (a) Perencanaan dan evaluasi program sekolah dalam hal ini sekolah berupaya meningkatkan mutu pendidikan dengan cara merencanakan kegiatan-kegiatan yang dapat meningkatkan profesionalisme guru, misalnya mengutus guru untuk mengikuti Musyawarah Guru Mata Pelajaran (MGMP) dan merencanakan bimbingan teknis untuk meningkatkan keterampilan guru dan mengevaluasi berbagai perencaan kegiatan program sekolah (b) Aspek pengelolaan proses belajar, memberikan kewenangan kepada masing-masing guru untuk mengelola proses pembelajaran pada mata pelajaran yang dipegangnya dan kepala sekolah melukan supervisi kepada guru untuk mengevaluasi dan membimbing pelaksanaan proses pembelajaran guru tersebut (c) Aspek pengelolaan ketenagaan, SMA Negeri 1 Lakea mengelola 25 orang guru dan 3 orang tenaga administrasi, pengelolaan yang dilakukan misalnya memberikan guru mata pelajaran yang sesuai dengan keahliannya, dan mengutus guru sebagai perwakilannya dalam kegiatan seminar mapun workshop (d) Aspek pengelolaan keuangan, sekolah mengelola dana BOS dengan prosedur sesuai dengan aturan penggunaan dana tersebut yakni sebagai operasional sekolah, misalnya mengupayakan pembayaran honor guru honorer yang tidak pernah terlambat dibayarkan. Implikasi Desentralisasi Pendidikan dalam Meningkatkan Profesionalisme Guru di SMA Negeri 1 Lakea: (1) Program sekolah dalam meningkatkan profesionalisme dapat terlaksana dengan baik (2) Masing-masing guru berupaya meningkatkan pengelolaan proses belajarnya sebagai implikasi dari pelaksanaan supervisi (3) Sekolah memiliki tenaga pendidik sesuai dengan kebutuhan dan (4) Pengelolaan keuangan yang dilaksanakan oleh sekolah dapat memotivasi guru untuk melaksanakan tugasnya dengan baik.Kata Kunci        :    Desentralisasi pendidikan, profesionalisme gur

Analytic philosophy for biomedical research: the imperative of applying yesterday's timeless messages to today's impasses

The mantra that "the best way to predict the future is to invent it" (attributed to the computer scientist Alan Kay) exemplifies some of the expectations from the technical and innovative sides of biomedical research at present. However, for technical advancements to make real impacts both on patient health and genuine scientific understanding, quite a number of lingering challenges facing the entire spectrum from protein biology all the way to randomized controlled trials should start to be overcome. The proposal in this chapter is that philosophy is essential in this process. By reviewing select examples from the history of science and philosophy, disciplines which were indistinguishable until the mid-nineteenth century, I argue that progress toward the many impasses in biomedicine can be achieved by emphasizing theoretical work (in the true sense of the word 'theory') as a vital foundation for experimental biology. Furthermore, a philosophical biology program that could provide a framework for theoretical investigations is outlined