Poster Session L (+Lunch and Office Hours)

This project is engineering strains of bacteria that replicate, evolve, and use DNA built from 6 independently replicable nucleotides, an Artificially Expanded Genetic Information System (AEGIS). These strains are "Second Examples of Genetics Undergoing Evolution" (SEGUE). By creating living cells capable of Darwinian evolution based on an artificial molecular biology, this project develops biology away from its descriptive roots. Such "grand challenge" synthesis forces us to ask "Why not?" and "What if?" questions as we solve unscripted problems, driving discovery and paradigm change in ways that hypothesis-based research cannot.
For technology, a 6-letter DNA alphabet offers 216 codons, allowing substantial expansion of the number of encoded amino acids in proteins. The value of such platforms is adumbrated by the $2.4 billion price paid for Synthorx, which added just one unnatural amino acid using hydrophobic pairs from Floyd Romesberg. SEGUE also avoids biohazards intrinsic in genetically re-coded bacteria, which may have no viruses to control their population. SEGUE will be used to manufacture AEGIS aptamers and aptazymes, to replace antibodies and long-sought catalytic antibodies in medicine. This past year, our deepened understand of DNA based on this work allowed us to release two "best in class" CoVID-19 diagnostics and surveillance platforms, now being used in India, Europe, and the US.
The scientific impact of this work in biomedical chemistry is also significant. Synthesis is a demonstration of understanding; "What I cannot make, I do not understand." To get a functioning SEGUE, we must dissect existing systems biology in living cells, and then make our own. This includes systems behind a metabolism to make AEGIS components, systems to manage and repair genetic information, and systems to regulate the new core molecular biology. In each, we learn volumes about how natural life manages the elements of living.

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Decades of research on protein folding have primarily focused on a subset of small proteins that can reversibly refold from a denatured state. However, these studies have generally not been representative of the complexity of natural proteomes, which consist of many proteins with complex architectures and domain organizations. Here, we introduce an experimental approach to probe protein refolding kinetics for whole proteomes using mass spectrometry-based proteomics. Our study covers the majority of the soluble E. coli proteome expressed during log-phase growth, and among this group, we find that one third of the E. coli proteome is not intrinsically refoldable on physiological timescales, a cohort that is enriched with certain fold-types, domain organizations, and other biophysical features. We also identify several properties and fold-types that correlate with slow refolding on the minute timescale. Hence, these results illuminate when exogenous factors and processes, such as chaperones or co-translational folding, might be required for efficient protein folding. Finally, we show which features of proteins make them more dependent on chaperonins and the cellular proteostasis machinery, thereby highlighting which proteins are more susceptible to misfolding and initiating proteopathies such as Alzheimer's disease.

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A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify sgRNAs capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.

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The goal of this project is to create a class of electronic materials that can measure signals and interface with the nervous system for two-way communication with biological systems. The project is exploring three classes of materials. (1) Semiconducting polymers with properties inspired by biological tissue. The goal of organic bioelectronics is to detect and treat disease by using signal transducers based on organic conductors and semiconductors in wearable and implantable devices. Except for the carbon framework of these otherwise versatile materials, they have essentially no properties in common with biological tissue: electronic polymers are typically stiff and brittle, and do not degrade under physiological conditions. Such properties can be realized in a single-component polymer by incorporating biocompatible subunits. We have synthesized a new type of stretchable, biodegradable polymeric semiconductor whose electronic performance is unaffected by the biodegradable components. Such materials have applications in wearable and implantable sensors. (2) Metallic nanoislands on single-layer graphene for cellular electrophysiology and wearable sensors. We have used these materials to measure the forces produced by the contractions of cardiomyocytes using a piezoresistive mechanism. Separately, we have developed orthogonal methods of stimulating myoblast cells electrically while measuring the contractions optically (a modality we nicknamed as "piezoplasmonic"). We have also used these sensors to measure the swallowing activity of head-and-neck cancer patients who have received radiation therapy and are at risk of dysphagia arising from fibrosis of the swallowing muscles. The combination of strain sensing, surface electromyography, and machine learning can be used to measure the degree of dysphagia. (3) We have developed ionically conductive organogels for haptic feedback. Medical haptic technology has myriad potential applications, from robotic surgery and surgical training, to tactile therapy for premature infants and patients with neurological impairment.

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Bed bugs (Cimex lectularius) are small, hematophagous ectoparasites of mammals commonly found in close proximity to humans. They are a major indoor pest and notoriously difficult to eradicate from homes. Despite their pest status, bed bugs are widely considered to be of limited medical importance because health issues typically relate to either adverse reactions to their bites, or psychological distress during or following an infestation. Recently, bed bugs were found to produce large amounts of histamine in their feces. Because bed bug populations can attain hundreds or thousands of individuals, it is possible that significant amounts of histamine accumulates in infested homes and poses a potential health risk to residents. Our ongoing work is focused on determining how bed bugs produce histamine, how much histamine accumulates in infested homes, and if exposure to bed bugs or bed bug-produced histamine constitutes a health risk for humans. When evaluated together, these efforts have the potential to identify a new environmental contaminant that has gone undetected and unabated for almost two decades, and additionally determine if bed bugs pose a health risk to humans. Furthermore, this project lends itself to collaboration, where there are countless opportunities for entomology to interact and engage with those working in public health, exposure science, immunology, psychology, and others, allowing me to take this line of inquiry further than I could on my own.

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The gut microbiome extends the capabilities of its host and alters its physiology. Together with diet, host genetic landscapes shape microbiome form and function in the animal gut. Despite its importance, the essential functions that drive microbiome assembly and stability in remain largely elusive. To address this challenge, we leverage the nematode Caenorhabditis elegans to explore how microbiomes assemble in different host genetic backgrounds. This system has several advantages, including: (1) a simple microbiome that can rapidly removed (bleaching) and replaced in high-throughput gnotobiotic experiments; (2) highly conserved intestinal physiology, metabolism and innate immunity; and (3) shared microbial functions for host gut persistence.
To examine the natural variation in acquisition of the microbiome in C. elegans, we first established the natural core microbiome and assembled a functionally redundant, model core microbiome of bacteria (BIGbiome). Then C. elegans wild strains (38) were made 'germ-free' and colonized with BIGbiome to assess strain-level microbiome composition (16S) and levels (CFU) longitudinally using a high-throughput pipeline. The strains clustered into three groups: (1) a highly-selective group that differed greatest from the surrounding environment [Ochrobactrum-dominant]; (2) a 'dysbiotic' group [Bacteroidetes-dominant]; and (3) a 'non-selective' group. By GWAS-, RNAi- and RNAseq-based approaches, we identified ~1000 candidate regulators in highly conserved pathways ( > 60%). Insulin signaling pathways specifically regulate Ochrobactrum colonization, as impaired daf-2/IGFR signaling (mutants or RNAi) limits its colonization.
Last, we next sought to examine alterations in microbiome function. To do this, we sequenced and annotated > 100 bacterial genomes. Virtual metagenomes for each C. elegans microbiome indicate broad microbiome functions are shared, but also point to many emergent functions among the three host groups. Our study highlights the potential a robust platform to identify conserved host and microbial determinants that may underlie assembly and stability of the microbiome.

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Fluorescence microscopy is instrumental in the lab for cellular visualization of immune activity and disease progression, but is yet to be deployed inside the body. A critical application is cancer immunotherapy, a game-changing therapeutic harnessing the immune system to attack cancer, but which is effective in < 50% of patients. The real-time intratumoral response to therapy in patients remains unknown, and as a result, many patients remain on ineffective therapies for months, missing the cure window while incurring needless toxicity. To provide unprecedented real-time and detailed monitoring of tumor response monitoring both activating and immune suppressing components, we propose a "wireless biopsy", leveraging innovations in optics and CMOS technology to personalize treatment based on the individual patient response. The prototype sensor is underdevelopment. Our system aims to provide state-of-the-art fluorescence microscopy-cellular-level resolution, multi-color sensing of different cell types, and 3D image reconstruction-on a millimeter-scale platform compatible with long-term implantation in the body. This requires several key technological innovations: integration of the light source and all optical components on chip, wireless data transmission and power delivery, and advanced image processing techniques. Compact, lens-free imaging is achieved using a custom CMOS image sensor with an integrated micro-laser diode that images tissue directly in contact with its surface. To replace conventional optics, we demonstrate a novel planar filter structure that combines thin-film interference filters with micro-collimators to simultaneously deliver excitation rejection, high resolution, and multi-color sensing. Inspired by current medical implants, an ultrasound power and data communication link is utilized to minimize tissue attenuation, maximize power transfer density and improve robustness for backscattering significant amounts of image data. Additional processing for image enhancement and 3D depth estimation is implemented in software using convolutional neural network models.

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There are over 60 million small open reading frames (smORFs), each of which could encode microproteins smaller than 100 amino acids, yet only few have been well characterized. To date, RNA-seq and ribosome profiling have suggested a few hundred smORFs might be translated. The lack of ultra-sensitive methods of detection, the ambiguity in interpreting mass spectrometry spectra, and the inherent low correlation between RNA and protein levels, however, have hindered the discovery of novel peptides. We have implemented new computational and experimental pipelines based on advanced proteophylogenomics and a combination of low molecular weight fractionation methods and Focused Asymmetric Ion Mobility Mass Spectrometry (FAIMS) to quantify the smORF-encoded peptides (SEPs). Our software identified over 600 k candidates conserved across mammals, some comprised within 155 families of smORFs. So far, we have detected 4,577 circulating SEPs with gender- and age-dependent differences in expression when analyzing human plasma and serum from healthy men and women, ranging 20 to 50 years old. The putative microproteins, none of which are redundant to previously annotated genes, show a median size of 51 amino acids, more than half are predicted to be intergenic, and recurrently display ordered secondary structures (alpha helices, beta sheets or both) and an abundance of positive charges. Several smORFs also show GWAS hits that correlate with specific types of cancer, longevity and other traits and diseases. Some candidates were also found in human and mouse primary cell lines and show dynamic regulation under conditions of senescence or iron deficiency, traits also associated with aging. Work is underway to determine the biological functions of the most interesting hits.

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