Project description:Here we examined whether histones H3 and H4 produced using native chemical ligation reaction affect protein binding to di-nucleosomes. To test this we performed a set of affinity purification pull-downs using di-nucleosomes containing the following histones H3 and H4: 1. unmodified ligated histone H3 and recombinant wild type histone H4 (unmod. H3), 2. recombinant wild type histone H3 and ligated unmodified histone H4 (unmod. H4) 3. ligated unmodified histones H3 and H4 (unmod. H3&H4). 4. recombinant wild type histones H3 and H4 (WT) Each pull-down was performed in three replicates. Co-purified proteins were quantified using label-free MS. Note that ligated histones H3 and H4 contain T32C or I29C single amino acid substitutions, respectively, that are introduced by the native chemical ligation procedure. In addition, ligated histone H4 is N-terminally acetylated to mimic its naturally blocked N-terminus.
Project description:Purpose: Analyze transcriptome changes in response to metal treatment in wildtype and sensitized genetic backgrounds. Methods: Zinc or manganese chloride supplementation of wildtype or mutant Drosophila S2R+ cultured cells, followed by RNA extraction. Results: Zinc chloride supplementation induces transcriptional responses in wildtype, ia-2 mutant, and ZnT63C mutant cells. Conclusions: Wildtype S2R+ cells have a transcriptional response to mild zinc chloride supplementation and the zinc response is greater (in degree and in number of genes) in genetically sensitized cell backgrounds that perturb zinc mobilization.
Project description:Only 59% of Alaska Native people have been adequately screened for colorectal cancer (CRC) despite having the highest reported incidence of CRC in the world. A new at-home multi-target stool DNA screening test (MT-sDNA; Cologuard) with high sensitivity for pre-cancerous polyps and CRC is now available. MT-sDNA has not been tested for feasibility or acceptability within the Alaska tribal health care delivery system, and it is unknown whether use of this new test will increase Alaska Native CRC screening rates. The long-term study goal is to improve screening and reduce CRC-attributable mortality. The objective of this application is to test the effectiveness of MT-sDNA for increasing CRC screening in Alaska Native communities using a mixed methods, community-based participatory research (CBPR) approach. The study will be conducted in collaboration with regional Tribal health organizations responsible for providing health care to geographically remote Alaska Native communities. Although the proposed implementation strategy is evidence-informed and promising, it is novel in that MT-sDNA has not been evaluated in the tribal health setting or among rural/remote populations. Using the Social Ecological Model, the research will be multi-level, examining influence on patients, providers, and tribal health organizations (THOs). This research study will pursue two specific aims: (1) Identify patient-, provider-, and system-level factors associated with CRC screening preferences, uptake, and follow-up; and (2) test the effectiveness of graded intensity MT-sDNA intervention in the Alaska Native community setting. For the first aim, focus groups with Alaska Native people who are not adherent to CRC screening guidelines and interviews with healthcare providers will be used to identify factors for future intervention. For the second aim, a three-arm cluster randomized controlled trial (high intensity with patient navigation, medium intensity with mailed reminders, usual care) will provide evidence on the MT-sDNA usefulness (MT-sDNA sample quality and neoplastic yield) as well as the first data on MT-sDNA follow up adherence rates in the Alaska Native population, which will inform plans to scale-up the intervention model. This research has the potential to sustainably improve public health by increasing CRC screening rates among a rural/remote tribal population as well as provide a model for other integrated health systems that provide care to high-risk or underserved populations in the U.S. and worldwide.
Project description:Here we examined whether histones H3 and H4 produced using native chemical ligation reaction affect protein binding to di-nucleosomes. To test this we performed a set of affinity purification pull-downs using di-nucleosomes containing the following histones H3 and H4: 1. ligated unmodified histone H3 and recombinant wild type histone H4 (unmod. H3), 2. recombinant wild type histone H3 and ligated unmodified histone H4 (unmod. H4) 3. ligated unmodified histones H3 and H4 (unmod. H3&H4). 4. recombinant wild type histones H3 and H4 (WT) Each pull-down was performed in three replicates. Co-purified proteins were quantified using label-free MS. Note that ligated histones H3 and H4 contain T32C or I29C single amino acid substitutions, respectively, that are introduced by the native chemical ligation procedure. In addition, ligated histone H4 is N-terminally acetylated to mimic its naturally blocked N-terminus.
Project description:The "Assay for Transposase Accessible Chromatin sequencing" (ATAC-seq) is an efficient and easy to implement protocol to measure chromatin accessibility that has been widely used in multiple applications studying gene regulation. While several modifications or variants of the protocol have been published since it was first described, there has not yet been an extensive evaluation of the effects of specific protocol choices head-to-head in a consistent experimental setting. In this study, we tested multiple protocol options for major ATAC-seq components (including three reaction buffers, two reaction temperatures, two enzyme sources, and the use of either native or fixed nuclei) in a well-characterized cell line. In addition, the native conditions were tested in a primary sample type (mouse lung tissue) with two different input amounts. In general, native samples yielded more peaks (particularly at loci not overlapping transcription start sites) than fixed samples, and the temperature at which the enzymatic reaction was carried out had a major impact on data quality metrics for both fixed and native nuclei. However, the effect of various conditions tested was not always consistent between the native and fixed samples. For example, the Nextera and Omni buffers were largely interchangeable across all other conditions, while the THS buffer resulted in markedly different profiles in native samples. In-house and commercial enzymes performed similarly. We found that the relationship between commonly used measures of library quality differed across temperature and fixation, and so evaluating multiple metrics in assessing the quality of a sample is recommended. Notably, we also found that these choices can bias the functional class of elements profiled and so we recommend evaluating several formulations in any new experiments. Finally, we hope the ATAC-seq workflow formulated in this study on crosslinked samples will help to profile archival clinical specimens.
Project description:To understand widespread differences in the DNA methylation patterns of Conyza canadensis leaf samples from its native and non-native ranges. Using Whole Genome Bisulfite Sequencing, we found average read coverages in high mapped reads across native and non-native samples of Conyza canadensis. Using R bioconductor package, we found enrichment score of methylated sites in both native and non-native samples. while analyzing CG, CHG and CHH methylation, we found relatively low CG and CHG methylation across transcriptional units in natives over non-natives. However, differentially methylated regions were found to be 53% hypomethylated and 41% hypermethylated in non-natives on genic regions.
Project description:Although metals are essential for the molecular machineries of life, systematic methods for discovering metal-small molecule complexes from biological samples are limited. Here, we describe a two-step native electrospray ionization-mass spectrometry method, in which post-column pH adjustment and metal infusion are combined with ion identity molecular networking, a rule-based data analysis workflow. This method enabled the identification of metal-binding compounds in complex samples based on defined mass (m/z) offsets of ion species with the same chromatographic profiles. As this native electrospray metabolomics approach is suited to the use of any liquid chromatography-mass spectrometry system to explore the binding of any metal, this method has the potential to become an essential strategy for elucidating metal-binding molecules in biology.
Project description:Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.
Project description:We tested a number of rRNA removal methods (Illumina RiboZero Plus, NEBNext, NEB Core Depletion Set with custom probes, siTools Panarchaea, siTools RiboPool) on 4 model halophile species: Halobacterium salinarum, Haloferax volcanii, Haloferax meditteranei, Haloarcula hispanica). It was found that methods using custom probes (NEB Core Depletion set with HVO probes, siTools RiboPool with HVO probes) efficiently remove rRNA in species they are targeted to, and that Panarchaea efficiently removes rRNA in all 4 tested species.