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SITE-Seq probes Cas9 cleavage sites in vitro and returns a comprehensive list of off-target sites at different Cas9–sgRNA concentrations. RNA-guided CRISPR–Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9–sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world’s first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5′ terminus capping and 3′ terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.

One Hundred Fifty-Seven nm photodissociation of singly protonated peptides generates unusual distributions of fragment ions. When the charge is localized at the C-terminus of the peptide, spectra are dominated by x-, v-, and w-type fragments. When it is sequestered at the N-terminus, a- and d-type ions are overwhelmingly abundant. Evidence is presented suggesting that the fragmentation occurs via photolytic radical cleavage of the peptide backbone at the bond between the α- and carbonyl-carbons followed by radical elimination to form the observed daughter ions.

Several groups have investigated the photodissociation of peptide ions with ultraviolet light. Significant differences have been reported with 157 and 193 nm excitation. Recent studies have shown that the mass analyzer can also influence the observed photofragment distribution. Comparison of experiments using different peptides, wavelengths, and mass analyzers is undesirably complicated. In the present work, several peptides are analyzed with both 157 and 193 nm photodissociation in tandem-TOF and linear ion trap mass spectrometers. The results indicate that the fragment ion distribution can be influenced by both the photodissociation wavelength and the mass analyzer. The two wavelengths generate similar spectra in an ion trap but quite different results in a tandem-TOF instrument.

One hundred fifty-seven nm photodissociation of singly-charged peptide ions induces the cleavage of α-carbon to carbonyl-carbon bonds along the backbone. a n + 1 radical ions are observed as the primary photolysis products of peptides with N-terminal arginines in a linear ion trap mass spectrometer. The radical elimination pathways undertaken by the a n + 1 radical ions to form more stable even-electron species are studied in hydrogen-deuterium (H/D) exchange experiments. Two types of a n ions along with d-type ions are observed as secondary elimination products. The relative abundance of each depends on the C-terminal residue of the radical fragment ion.

This thesis examines computer vision applications for traffic monitoring and safety analysis. The focus is comparing proprietary computer vision technology and open-source computer vision code we developed. Two case studies are completed using proprietary sensors purchased from a company called Numina. The first case study demonstrates unique opportunities for using computer vision to monitor bicycle travel during snow events. The case study reveals that bicyclists do not use separated bike lanes if they are not adequately cleared of snow. The second case study, using proprietary software, further shows how computer vision can create pedestrian and bicyclist demand models. Three case studies were completed using our computer vision code. The code was written in Python and uses a detection model called YOLOv8. The first case study demonstrates how user counts can be obtained from video feeds and provides examples of insights that can be drawn from these counts. The second case study uses computer vision to create visualizations of user movements at intersections. The third case study develops and demonstrates the application of a new surrogate safety measure for pedestrian and bicyclist safety. Advantages and disadvantages of proprietary and open-source systems are discussed. Shortcomings and future opportunities of computer vision are discussed.

Tandem mass spectrometry is widely used to identify proteins based on the fragmentation of proteolytic peptides. The processes typically employed to activate ions in these experiments induce vibrational excitation. As a result, mass spectra are often dominated by the lowest energy fragmentation processes. Typically, incomplete fragment series are observed. The information contained in these spectra is not sufficient to determine peptide sequences directly. Instead, spectra are interpreted by comparing the experimental results with those obtained from experiments performed in silico using database sequences. Only peptide sequences found in the database will yield matches, so organisms without sequenced genomes cannot be investigated. Likewise, mutated or modified peptides are problematic. In this dissertation, a new peptide ion excitation technique using vacuum ultraviolet light is introduced. The resulting fragmentation spectra are significantly different than those observed with any other activation technique. In the presence of a favorable protonation site, the major fragment series correspond to cleavage of the peptide backbone between the α- and carbonyl-carbons with the charge sequestered at the basic site. Sequence coverage of over 90% is observed, with consistent intensities across each spectrum. Typical photodissociation spectra contain sufficient information to directly determine the sequence of a peptide without a database. By addressing the limitations of current techniques, photodissociation could enable more comprehensive peptide identification during proteomics studies.

The world's first national park is constantly changing. How we understand and respond to recent events putting species under stress will determine the future of ecosystems millions of years in the making. Marshaling expertise from over 30 contributors, Yellowstone's Wildlife in Transition examines three primary challenges to the park's ecology.

In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed \"the Warburg effect.\" Aerobic glycolysis is an inefficient way to generate adenosine 5'-triphosphate (ATP), however, and the advantage it confers to cancer cells has been unclear. Here we propose that the metabolism of cancer cells, and indeed all proliferating cells, is adapted to facilitate the uptake and incorporation of nutrients into the biomass (e.g., nucleotides, amino acids, and lipids) needed to produce a new cell. Supporting this idea are recent studies showing that (i) several signaling pathways implicated in cell proliferation also regulate metabolic pathways that incorporate nutrients into biomass; and that (ii) certain cancer-associated mutations enable cancer cells to acquire and metabolize nutrients in a manner conducive to proliferation rather than efficient ATP production. A better understanding of the mechanistic links between cellular metabolism and growth control may ultimately lead to better treatments for human cancer.