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José Pablo Vázquez-Medina, Department of Integrative Biology, University of California, Berkeley

"Prolonged Fasting Increases DNA Methylation in Northern Elephant Seal Pups"

Abstract:

Northern elephant seals (NES) experience extreme physiological conditions, including prolonged food and water deprivation (fasting). NES pups are born during the winter and nurse for a month before they are weaned and fast for two months. During this fasting period, NES pups lose about a quarter of their body mass, while maintaining biochemical homeostasis and supporting muscle development. Metabolic and environmental stress can modify the methylation status of DNA, consequently regulating gene expression. To investigate epigenetic changes associated with fasting in elephant seals, we collected blood samples from early-fasting (1-2 weeks into the fasting period) and late-fasting (7-8 weeks into the fasting period) pups, and from post-fasting pups returning to the beach after their first oceanic foraging trip. We evaluated global DNA methylation levels in NES pups and found that prolonged fasting increases global methylation (p=0.0061). Moreover, methylation levels in post-fasting pups were comparable to early-fasting pups, suggesting a transient decrease in transcription activity during fasting. We then differentiated muscle progenitor cells (myoblasts) derived from early-fasting pups into skeletal muscle fibers (myotubes) in primary cell culture, treated them with three increasing concentrations (0.1, 1.0, and 100 uM) of dexamethasone for 48 hours, and evaluated changes in global methylation. Sustained exposure to dexamethasone decreased global DNA methylation only at the highest concentration (100uM) after 12 (p=0.0023) and 48 (p=0.0059) hours, suggesting a change in gene expression to maintain metabolic functions during glucocorticoid-induced energetic stress. We are currently in the process of quantifying plasma cortisol levels in early-fasting, late-fasting, and post-fasting pups, to further investigate the relationship between DNA methylation and stress hormone levels in NES. To identify methylation changes at single gene levels, we studied Nrf2—a master regulator of the antioxidant response—and the glucocorticoid receptor (GR). Nrf2 showed a consistent pattern of CpG-site methylation in its promoter among the three fasting groups. We are currently identifying methylation patterns in the GR promoter. Our results show that prolonged fasting increases DNA methylation in elephant seal blood cells, but that Nrf2 transcription is not affected. Moreover, our results also suggest that elephant seal muscle cells are extremely tolerant to glucocorticoid-induced changes in DNA methylation.

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Erica Bree Rosenblum, Department of Environmental Science, Policy, and Management, University of California, Berkeley

"An Open-Source Platform for Genotyping Batrachochytrium dendrobatidis, the Chytrid-Inducing Amphibian Fungus"

Abstract:

Amphibian species are declining globally. One major driver of these declines is the worldwide spread of Batrachochytrium dendrobatidis (Bd), a pathogenic fungus known to induce the disease chytridiomycosis in amphibians. Bd can quickly spread across amphibian populations leading to population or even community-level collapses. Such dramatic impact on amphibians necessitates a comprehensive understanding of the emergence and spread of Bd. Molecular data can help reveal pathogen history that cannot be uncovered by field observations alone. Previous work in the Rosenblum lab has developed molecular assays to obtain Bd genetic sequences from amphibian skin swabs, an often overlooked source of pathogen genetic data. Additionally, the Rosenblum lab has generated R scripts to work with genomic sequences for both phylogenetic and population genetic analysis. Such advances in genomic methods have greatly expanded our understanding of Bd both regionally and globally. However, distributing and reproducing methods across the amphibian research community is still a major challenge. While both molecular and data analysis protocols exist, there is no efficient pipeline for broad distribution. Therefore, I developed an open-source platform that combines wet lab protocols and data analysis code developed in the Rosenblum lab. Using both GitHub and RMarkdown, I wrote and compiled R scripts into an interactive, online workspace. This workspace creates a detailed workflow, starting with genomic sequences and resulting in data outputs necessary for phylogenetic and population genetic analysis. It includes example input sequences to walk users through data cleaning, building concatenated amplicon alignments for phylogenetic trees, and calling sequence variants for population genetics. Once established on GitHub, the platform empowers collaborative science by allowing researchers to provide workflow feedback, improve code, and expand the breadth of analyses through novel pipelines. The unique potential of this platform is to enable the amphibian research community to more feasibly apply the method and truly provide open-access science.

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George Brooks, Department of Integrative Biology, University of California Berkeley

"Novel Mass Spectrometric Method of Measuring Fractional Gluconeogenic Rates Using Non-radioactive Isotopic Tracers"

Abstract:

To quantify fractional gluconeogenesis (fGNG), two mass spectrometric methods were utilized and compared. Both relied on deuterium as a non-radioactive isotopic tracer to allow calculation of hydrogen incorporation onto key metabolic intermediates, and subsequently allow calculation of rate of formation of such intermediates, a proxy for flux. The first method, which has been most conventionally established in the literature, relied on measuring ingested heavy water incorporation onto an acetone analyte as a measure of total body water enrichment and subsequently calculated fractional gluconeogenesis via measurement of average enrichment on a glucose penta-acetate derivative. Though proven effective, this method requires larger volumes of biological samples to measure two separate analytes, and disallows the opportunity to re-run samples once the analyte has been derivatized. This project sought to assess the validity of a second method that avoids such constraints. This method measures deuterium incorporation on two separate fragments of the same glucose penta-acetate derivative to isolate enrichment on Carbon 2, which is proportional to overall body water enrichment, bypassing the need for acetone measurements. Blood samples were collected from 24 Sprague-Dawley rats during a Traumatic Brain Injury and feeding study at three different timepoints. Each sample was run in duplicate using each of the two mass spectrometric methods, and statistical testing was performed for comparison of the two methods.

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Lisa Barcellos, University Of California, Berkeley, School of Public Health

"Reproducibility of methylation measurements at CpG sites with Illumina BeadChips"

Abstract:

The Illumina HumanMethylation450 (450K) and MethylationEPIC (EPIC) BeadChip arrays are widely used cost-effective tools for a variety of epigenome-wide association studies (EWAS). The Illumina array measures DNA methylation levels at CpG sites genome-wide in CpG Islands, shores, shelves, and open sea. The original 450K BeadChip covered over 450,000 CpG sites while its newest successor, the EPIC BeadChip, expanded the sites to over 850,000. Two probe types are utilized by the 450K and EPIC BeadChips, type I and type II, which utilize different chemistries to measure methylation. Methylation measurements can be used to find associations between methylation changes and disease outcomes. The reproducibility of this data is crucial for finding meaningful associations. To assess reproducibility of both BeadChips, we utilized quality control data compiled from all projects using the 450K and EPIC chips. The data included 81 repeated runs of DNA extracted from Jurkat cells (immortalized T lymphocytes) run on every plate of other study samples as well as 170 replicates of other human DNA samples. We quantile normalized and removed background signal from samples and removed poor performing CpG sites across samples from the two different BeadChips. At each CpG site, we calculated the pairwise differences between all repeated Jurkat samples and between each set of replicates. We examined both the average difference at each CpG site as well as the maximum difference between repeated runs at each CpG site.The maximum and average differences were analyzed to find patterns by chromosome, probe type, and CpG site location in relation to CpG islands. Preliminary results show a greater concentration of DNA methylation differences within the sex chromosomes for repeated Jurkat and replicate sample runs on both the 450K and EPIC BeadChips, and an increase in differences for CpG sites located in CpG islands on the EPIC BeadChip. Little overlap of CpG sites across the two platforms was observed. Gene Ontology pathway analysis algorithms were utilized to find overrepresentation of variable CpG sites in different genetic pathways. Increased methylation differences were enriched in pathways for nervous system development, adhesion, and integral component in plasma membrane in the 450K replicates, and sequence-specific DNA binding, nucleic acid binding, nervous system development, and pattern specification process in the EPIC replicates.

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Thanh Le, Moichor in Bonneville Labs

"A Linear, Pixel-Specific Color Normalization Algorithm for Hematology Imaging"

Abstract:

The automated cell recognition of hematology microscope images provides crucial information for the qualitative description of cell morphology and other quantitative applications in analyzing blood pathology. Computer-aided diagnostics and cell segmentation are invaluable tools to help reduce the cost of human labor and time. However, discrepancies in stain protocol and imaging hardware pose challenges to automated cell recognition; noise, blur, lighting contrast, and irregular coloration confound cell differentiation. In this study, we describe a linear pre-processing algorithm that addresses the color variation in hematology images. We qualitatively examine the image outputs and quantitatively assess the efficacy of the proposed algorithm by studying the performance of a cell detection model.

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Michael B. Eisen, Department of Molecular & Cell Biology, University of California, Berkeley

"Investigating Zygotic Gene Expression During Drosophila Embryonic Development Using Single-Nucleus RNA-Sequencing"

Abstract:

Maternally-deposited RNAs direct the earliest stages of embryo development, but at a critical point, known as the maternal-to-zygotic transition (MZT), zygotic transcription begins, and embryos gradually lose reliance on maternally-deposited RNAs. In early Drosophila melanogaster embryos, the MZT begins during the 14th nuclear division, which also marks the beginning of cellularization. While we have a general understanding of broad gene expression in the early Drosophila embryo, we need to employ more advanced technologies to understand expression within a single nucleus. Single-nucleus RNA sequencing (snRNA-seq) allows for the separation of zygotic expression from maternally-deposited RNAs, which is not possible using single embryo RNA-seq. Here, I demonstrate the viability of single-nucleus RNA sequencing as a method to assay zygotic gene expression in early Drosophila embryos, specifically by comparing snRNA-seq data collected from earlier nuclear cycles to data collected from nuclear cycle 14. Comparing the variability in expression of patterning genes and non-patterning genes between the two different points in development reveals that zygotic expression increases between nuclear cycles 9-13 and 14. This new method can be applied in the future to better understand how individual nuclei in early embryos respond to manipulations and how gene expression in single nuclei can change during development.

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Elçin Ünal, Department of Molecular & Cell Biology

"Uncovering Factors Affecting LUTI-based Gene Repression"

Abstract:

The central dogma of biology goes as follows: DNA is transcribed to RNA, which is translated into protein. By that logic, more RNA should give rise to higher protein levels. In contrast to this rule, our lab exposed a novel mode of gene regulation in Saccharomyces cerevisiae that suggests the transcription of an mRNA can directly cause gene repression despite carrying a protein-coding sequence. This mechanism occurs via the activation of an alternative transcript from a gene-distal promoter to produce a Long Undecoded Transcript Isoform (LUTI) mRNA. Transcription from the proximal promoter during mitosis and meiotic divisions leads to expression of a protein-coding transcript, while transcription of an upstream, distal promoter during meiotic prophase drives expression of a noncanonical 5’ extended isoform. The LUTI is not productively translated, leading to decreased protein synthesis. While we know that LUTI based transcriptional and translational interference occurs, we do not fully know how it works. Therefore, this analysis aims to identify genes that affect LUTI-based gene repression as well as mutations that suppress the LUTI phenotype. Using a selection assay in which only cells able to escape repression survive, subsequent sequencing of the genomes, and a filtering pipeline, we aim to identify which modifications are causal of LUTI suppression. This will be largely indicative of factors required for establishing the repressive state induced by LUTI transcription.

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Richard Harland, Department of Molecular & Cell Biology

"In Silico Analysis of the Plekhg Family"

Abstract:

Small GTPases are G-proteins involved in signaling cascades for cellular processes such as cell polarity, actin polymerization, and movement. These enzymes work as molecular switches, hydrolyzing GTP to GDP, becoming inactive. GTPase activation occurs via Guanine Exchange Factors, or GEFs, which are associated with specific morphogenetic movements during embryo development. One such GEF is Plekhg5, which is crucially implicated in apical construction during gastrulation in Xenopus. However, Plekhg5 belongs to a family of five other members, of which function is poorly described. Previous work in the lab suggested different roles for the six members of the family during Xenopus development, and in this work we aimed to provide more data towards a deeper understanding of their roles. Here, we describe conserved domains across the Plekhg family members in both species, Xenopus tropicalis and Xenopus laevis, which have helped us identify putative functions through orthology analysis and protein alignments. Additionally, we have designed loss-of-function constructs based on both literature and orthology data to investigate the functionality of each Plekhg protein member in the context of embryo development. We hypothesize that these results will serve as an important benchmark for further experiments into the role of Plekhg GEFs in embryonic development, and their role of putative targets such as Rho/CDC42/Rac1 GTPases.