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Poster Session F (+Lunch and Office Hours)

Session Information

Jun 10, 2021 02:05 PM - 02:35 PM(America/Detroit)
Venue : Posters
20210610T1405 20210610T1435 America/Detroit Poster Session F (+Lunch and Office Hours) Posters NIH Common Fund's 2021 High-Risk, High-Reward Research Symposium becky.miller2@nih.gov

Presentations

Phosphorothioate Epigenetics in the Human Gut Microbiome

Infectious Diseases and Immunology 02:05 PM - 02:35 PM (America/Detroit) 2021/06/10 18:05:00 UTC - 2021/06/10 18:35:00 UTC
Phosphorothioation (PT) is a DNA backbone modification with sulfur replacing a non-bridging phosphate oxygen. As widespread epigenetic marks in bacteria and archaea, PTs are inserted by dnd and ssp genes. Here we show that an abundance of mouse and human gut microbes contain PTs. The discovery of PTs in the microbiome arose in mass spectrometric assays of PT-containing dinucleotides in a limit nuclease digest of fecal DNA. We found 11 of 16 possible dinucleotides in mice and humans. Mice showed more dinucleotide contexts as well as striking similarity and stability among cage mates, while humans showed fewer dinucleotide contexts and significant inter-individual diversity. Further, humans show time-dependent changes in the spectrum and quantities of PTs. We next mined sequenced genomes of ~9000 bacterial isolates for homologs of dnd and ssp genes, finding ~8% of strains containing PT genes. We then used a technique involving iodine-induced cleavage of PTs followed by poly-T tailing at strand breaks to prepare libraries for NGS. Metagenomic alignment of sequencing data revealed a strain distribution similar to isolate genomes. Further, we observed 10 PT motifs: CpsCA from Bacteroidales, GpsAAC/GpsTTC from Enterobacterales, CpsAG and CpsCTC from Bacteroidales, GpsAGC/GpsCTC and CpsCTG from Clostridiales, and GpsTAC, and GpsATC from Enterobacterales. PTs were found to be evenly distributed among genetic and intergenic regions in metagenome assembled genomes. Sequenced bacterial isolates, fecal DNA metagenomics, and LC-MS PT analyses showed remarkable epigenetic correlations in the gut microbiome. While much more analysis across larger populations will define the determinants of the PT-possessing microbiome, the redox-active and nucleophilic PT sulfur is known to affect the bacterial fitness during oxidative stress. Future studies will address the effects of inflammatory bowel disease on PT epigenetics in the gut microbiome.
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Presenters
PD
Peter Dedon
Massachusetts Institute Of Technology
Co-Authors
YY
Yifeng Yuan
Massachusetts Institute Of Technology
SB
Shane Byrne
Massachusetts Institute Of Technology
MD
Michael DeMott
Massachusetts Institute Of Technology
XW
Xiaolin Wu
Singapore-MIT Alliance For Research And Technology
SK
Stefanie Kellner
LMU Muenchen
MG
Mathieu Groussin
Massachusetts Institute Of Technology
KM
Katya Moniz
Massachusetts Institute Of Technology
MP
Mathilde Poyet
Massachusetts Institute Of Technology
EA
Eric Alm
Massachusetts Institute Of Technology

Towards whole-brain electrophysiological readouts of the mammalian nervous system: injectable electromagnetic probes for MRI

Neuroscience 02:05 PM - 02:35 PM (America/Detroit) 2021/06/10 18:05:00 UTC - 2021/06/10 18:35:00 UTC
There is currently a surging effort to develop technologies for recording neural activity from the entire volume of the brain in parallel. Whole-brain direct readouts of neural signals will be critical to understanding the elusive cross-regional communication grid underlying brain function and dysfunction. The goal of Dr. Hai's New Innovator Award is the development and application of a new form of brain imaging using electromagnetic circuits that can be deployed throughout the brain and provide parallel volumetric electrophysiological readouts of neural activity. The project relies on advances made by the principal investigator, demonstrating tetherless microelectronic neural interfaces that transduce neurophysiological events wirelessly to detectable magnetic field perturbations, that are monitored by magnetic resonance imaging (MRI). By combining the unique three-dimensional capabilities of MRI to obtain functional readouts from the entire volume of the brain, with electromagnetic probes-that can directly record electrophysiological neural activity in-situ and transmit its response to the MRI hardware-this project is aiming to transform the way we acquire brain signals. Dr Hai's group uses nanofabrication methods to pioneer cell-sized wireless probes, while employing existing state-of-the-art MRI-compatible microelectrode arrays in rodents for rigorous validation of new technologies and for decoupling electrophysiogical readouts from intrinsic fMRI blood-flow signals. The neuroelectronic MRI probes developed under the umbrella of this award will augment current brain recording capabilities, by presenting a different approach whereby minimally invasive devices are powered by the MRI scanner itself without bulky on-board power, and secondly, by interacting with the imaging scanner to transmit electrical neural activity to the detection hardware outside of the brain with no requirement for a tethered connection. The sensors will be used to directly detect electrical neural activity in three-dimensions and tracing the origins of brain physiology.

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Presenters Aviad Hai
University Of Wisconsin-Madison

Selective control of protein synthesis by DDX3

Molecular and Cellular Biology 02:05 PM - 02:35 PM (America/Detroit) 2021/06/10 18:05:00 UTC - 2021/06/10 18:35:00 UTC
DDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Ded1, the yeast ortholog of DDX3, is a global regulator of translation, whereas DDX3 is thought to preferentially affect a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds highly expressed mRNAs, depletion of DDX3 particularly affects the translation of a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosome, placing it immediately adjacent to the mRNA entry channel. Translation changes caused by depleting DDX3 levels or expressing an inactive point mutation are different, consistent with different association of these genetic variant types with disease. Taken together, this work defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 is altered.

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Presenters
SF
Stephen Floor
UCSF

Highly Multiplexed Fluorescence Microscopy Enabled by Ultrabright Pdot Probes for Interrogation of Complex Tissues

Instrumentation and Engineering 02:05 PM - 02:35 PM (America/Detroit) 2021/06/10 18:05:00 UTC - 2021/06/10 18:35:00 UTC
In situ proteomic studies of the brain and other complex tissues have been severely hindered by limited technical capabilities. We are developing an approach for highly-multiplexed interrogation of biological specimens based on novel, ultra-bright polymer dots with tunable spectral properties. Polymer dots are luminescent semiconducting polymers that exhibit ultrahigh brightness and versatile spectral tunability of excitation and emission wavelengths that can enable highly multiplexed fluorescence microscopy. The methodology should be applicable to many types of tissues or small organisms, although the final goal of our project is to demonstrate this method by studying development of the mouse visual cortex. We also report the development of a simple, small-molecule-based fluorescent labeling method, termed FLARE (fluorescent labeling of abundant reactive entities), that can rapidly label general protein and carbohydrate groups on biological specimens. FLARE is compatible with a wide range of tissue processing procedures, reveals a wealth of details for volumetric studies of the basic physiology of cells and tissues, and is highly complementary to multiplexed proteomics or transcriptomics approaches.

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Presenters
JV
Joshua Vaughan
University Of Washington
Co-Authors
DC
Daniel Chiu
University Of Washington

Topological data analysis reveals a unique hub-like transition state at rest in highly sampled individuals

Neuroscience 02:05 PM - 02:35 PM (America/Detroit) 2021/06/10 18:05:00 UTC - 2021/06/10 18:35:00 UTC
Even in the absence of external stimuli, neural activity is both highly dynamic and organized across multiple spatiotemporal scales. The continuous evolution of brain activity patterns during rest is believed to help maintain a rich repertoire of possible functional configurations. Whether these transitions or "explorations" follow some underlying arrangement or instead lack a predictable ordered plan remains to be determined. Although innovative approaches have been previously developed to reveal the temporal structure underlying transitions in the brain at rest, several methodological and data quality issues precluded the development of a clear understanding of brain dynamics at the individual level. Here, we overcame these limitations by examining the dynamic states during intrinsic brain activity using Topological Data Analysis (TDA) on a maximally denoised precision neuroscience dataset – The Midnight Scan Club (MSC). This dataset comprises of ten highly sampled individuals, with > 5 hours of resting state fMRI data per individual and individualized parcellations of each brain. Without temporal averaging or sliding windows, our TDA-based approach mapped whole-brain resting state volumes onto a set of individually defined intrinsic dynamical manifolds or "state spaces". For reliability, all reported results were validated using split-half cross validation and on an independent dataset from the Human Connectome Project. Using our TDA-based approach, we observed a rich topographic landscape in which the transition of activity from one network to the next involved a large shared attractor-like basin, or "transition state", where all networks were represented equally prior to entering distinct network configurations. The intermediate hub-like transition state seemed to provide the underlying structure for the continuous evolution of brain activity patterns at rest. In addition, differences in the manifold architecture were more consistent within than between subjects, providing evidence that this approach contains potential utility for precision medicine approaches.

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Presenters
MS
Manish Saggar
Stanford University

Microfluidic lightsheet microscopy

Instrumentation and Engineering 02:05 PM - 02:35 PM (America/Detroit) 2021/06/10 18:05:00 UTC - 2021/06/10 18:35:00 UTC
Nearly two meters of DNA are packed into every cell nucleus. Within this volume, gene expression is regulated by the binding of transcription factors, cofactors, and RNA polymerase machinery to the promoters of target genes. However, how a transcription factor navigates through roughly 3.2 billion base pairs to find a 100-1000 base pair long promoter is an open question. Nor is it clear how the underlying organization, composition, and dynamics of chromatin within the nucleus regulates transcription factor search dynamics. Answering these questions requires technologies that are capable of directly visualizing transcription factors, which diffuse in milliseconds, over the hours to days required for cellular fate specification.
With support from the NIH New Innovator Program we are developing a new multifunctional lightsheet microscope. This Multimodal Optical microScope with Adaptive Imaging Correction (MOSAIC) instrument permits rapid cellular imaging under several different types of diffraction limited and super-resolution modalities. In parallel, we are developing microfluidic systems that are compatible with the angled orientation of lightsheet microscopy objectives. We fully characterize the optical performance (aberration, transmission, and polarization effects) of these chips and demonstrate that they are compatible with high resolution lightsheet, single-molecule imaging, and structured illumination microscopy. Additionally, we demonstrate that they support imaging of both adherent and non-adherent cells, allow for rapid exchange of reagents while imaging, and maintain cell growth and sample sterility over multiple days of imaging. 
We are applying these advances to visualize the single-molecule dynamics of transcription factor search and directly visualize how they navigate the genome at different stages of cell differentiation. These studies will provide a novel window into how genes are regulated in both normal and disease settings.



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Presenters
WL
Wesley Legant
UNC - Chapel Hill / UNC - NCSU Joint Department Of Biomedical Engineering
Co-Authors
RM
Regan Moore
UNC - Chapel Hill / UNC-NCSU Joint Department Of Biomedical Engineering

Mitochondrial origin of cytosolic protein aggregation and proteostasis.

Molecular and Cellular Biology 02:05 PM - 02:35 PM (America/Detroit) 2021/06/10 18:05:00 UTC - 2021/06/10 18:35:00 UTC
A key process of mitochondrial biogenesis is the import of mitochondrial proteins that are synthesized in the cytosol. These mitochondrial proteins are maintained in unfolded states in the cytosol and cause cytosolic proteostasis stress if not imported into mitochondria. One common consequence of cytosolic proteostasis stress is the formation of protein aggregates that are attached to the mitochondrial outer membrane. It remains unknown why cytosolic protein aggregates are attached to the mitochondria. Here we show that in budding yeast, Tom70, a conserved receptor for mitochondrial import, nucleates the stress-induced aggregation of cytosolic proteins. Anchoring Tom70 or some of its substrates on the vacuole membrane converts this organelle into a primary site for the formation and attachment of cytosolic protein aggregates. This feature is rooted in the misfolding of some, but not all, Tom70 substrates in the cytosol. Cells monitor this weak point of cytosolic proteostasis by adjusting the transcriptional activity of these mitochondrial proteins to match mitochondrial import through a Tom70-mtDNA-FKH1/2 pathway. Tom70's role in transcriptional control of mitochondrial protein is conserved in fruit fly. Our results suggest that Tom70 sits at the crossroad of cytosolic proteostasis and mitochondrial biogenesis by regulating both the synthesis and import of mitochondrial proteins, while nucleating the aggregation of cytosolic proteins and summoning machineries to degrade these misfolded proteins on mitochondrial surface when this balance is disrupted by stresses. The reduction of Tom70 during aging causes age-dependent mitochondrial defects in the biogenesis of mitochondrial proteins and attachment of cytosolic protein aggregates, both can be rescued by overexpressing TOM70. This interdependence between cytosolic proteostasis and mitochondrial biogenesis explains the observations that mitochondrial dysfunction and protein aggregation are two closely related hallmarks of aging.

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Presenters
CZ
Chuankai Zhou
Buck Institute For Research On Agin

Discovering axonal regeneration genes in the Philippine mouse M. castaneus

High-Throughput and Integrative Biology 02:05 PM - 02:35 PM (America/Detroit) 2021/06/10 18:05:00 UTC - 2021/06/10 18:35:00 UTC
Nerve signals are sent from one region of the body to another along axons, each of which can extend for centimeters. It has been a truism in mammals that once axons get laid down in the central nervous system during development, they do not efficiently regenerate after stroke or trauma. However, neurons of the Philippine mouse Mus castaneus, a relative of the laboratory European species M. musculus, can regenerate axons far beyond those of any other known mouse system. We are mapping the genetic basis of this trait with a high-throughput screen in a cell culture model. Our approach is to mutagenize neurons of the M. castaneus x M. musculus hybrid background. In this scheme, a disruptive viral insertion at one parent's allele of a given locus uncovers the function of the wild-type allele of the other parent. In assays of axonal damage and extension in many such mutant cells, we screen for the M. castaneus alleles that confer a pro-regeneration phenotype. To lay the groundwork for this design, we have developed methods for high-throughput viral mutagenesis of M. castaneus x M. musculus hybrid stem cells, and sequencing of viral insertion positions; neuronal differentiation from these stem cells; and axonal extension assays in the resulting neuron cultures. We will show results from these approaches and describe our ramp-up toward a sequencing-based axonal regeneration screen at scale.

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Presenters
RB
Rachel Brem
UC Berkeley
Co-Authors
ND
Noah Denman
University Of Minnesota Stem Cell Institute
NS
Noah Simon
Biology Of Aging Graduate Program, University Of Southern California And The Buck Institute For Research On Aging,
DN
Duane Nguyen
University Of Minnesota Stem Cell Institute
LE
Lisa Ellerby
Buck Institute For Research On Aging
EV
Eric Verdin
Buck Institute For Research On Aging
DB
Diana Bautista
UC Berkeley
JD
James Dutton
University Of Minnesota Stem Cell Institute
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1623097539NoahDenman_Brem-Discoveringaxonalregeneration-1.png
Discovering axonal regeneration genes...
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Submitted by Rachel Brem
1623087650631_Wesley_Legant___Microfluidic_Lightsheet_Microscopy_1.png
Microfluidic lightsheet microscopy
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Submitted by Wesley Legant
1623104577ChuankaiZhou-Mitochondrialoriginofcytosolic.png
Mitochondrial origin of cytosolic pro...
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Submitted by Chuankai Zhou
1623085950882_Manish_Saggar___Topological_Data_Analysis_Reveals_1.png
Topological data analysis reveals a u...
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Submitted by Manish Saggar
1623089928215_Joshua_Vaughan___Semiconducting_Polymer_Dots_1.png
Highly Multiplexed Fluorescence Micro...
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Submitted by Joshua Vaughan
1623087429ShaneByrne-PhosphorothioateEpigenetics-1.png
Phosphorothioate Epigenetics in the H...
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Submitted by Peter Dedon
1623079448Aviadhai-Towardswhole-brain.jpg
Towards whole-brain electrophysiologi...
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Submitted by Aviad Hai
1623087512StephenFloor-Selectivecontrolofprotein-1.png
Selective control of protein synthesi...
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Submitted by Stephen Floor

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