Fluorescence Multiplexing with Combination Probes for Biological and Diagnostic Applications

Cancer refers to a group of diseases containing more than 200 different subtypes. Cancer is a heterogeneous disease by nature, meaning that there are differences among tumors of the same type in different patients, and there are differences among cancer cells within a single tumor of one patient. Since cancer is not a single disease, nor does it have a single cause, it proves to be incredibly hard to diagnose and treat. The ability to study cellular markers, cell and tissue spatial arrangement, and gene function are all integral parts of cancer diagnostic and treatment efforts. Here, I first present a review of current techniques for quantitative tissue imaging at cellular resolution. I broadly divide current imaging techniques into three categories: fluorescence-based, mass spectrometry-based, and sequencing-based. In this work, I primarily concentrate on fluorescence-based methods, with the focus being on our recently developed theory Multiplexing using Spectral Imaging and Combinatorics (MuSIC). The basis for MuSIC is to create combinations of fluorescent molecules (whether it be small molecule fluorophores or fluorescent proteins) to create unique spectral signatures. I then present a protocol for labeling antibodies with combinations of small molecule fluorophores, which I refer to as MuSIC probes. I use fluorescent oligonucleotides (oligos) to arrange the fluorophores at specified distances and orientations from one another in order to produce complex fluorescence spectra when the probe is excited. This labeling protocol is demonstrated using a 3-probe experimental setup, bound to Protein A beads, and analyzed via spectral flow cytometry. When translating this method to staining human cells, our staining intensity was not comparable to that of a conventional antibody labeling kit. Therefore, next I present an improved method to label antibodies with MuSIC probes with increased signal intensity. I re-arrange the oligo-fluorophore arrangement of the MuSIC probe to emit an increased fluorescent signal. Then I validate this approach by comparing the staining intensity of MuSIC probe-labeled antibodies to a conventional antibody labeling kit using human peripheral blood mononuclear cells. Lastly, I present simulation theories for the multiplexing capabilities of MuSIC probes for various biological and diagnostic applications. First, I present a theory for high-throughput genetic interaction screening using MuSIC probes generated from 18 currently available fluorescent proteins. Simulation studies based on constraints of current spectral flow cytometry equipment suggest our ability to perform genetic interaction screens at the human genome-scale. Finally, I adapt this simulation protocol to generate MuSIC probes from 30 currently available small-molecule fluorophores. Using the same constraints as before, I predict that I can perform cell-type profiling of 200+ analytes. I hope that the work presented here provides a foundation for the use of combination probes for various biological and disease applications and ultimately help to better diagnose and treat different types of cancer

Using Whole Genome Sequencing to Track Colibacillosis on Saskatchewan Broiler Flocks

Colibacillosis is a systemic infection caused by Escherichia coli resulting in significant morbidity and mortality in broiler flocks worldwide. Little is known about the group of E. coli that cause colibacillosis, collectively termed avian pathogenic E. coli (APEC). My MSc research focused on determining how APEC differ from resident E. coli that live in the chicken gut but do not cause disease. I hypothesized that systemic and cecal E. coli are genetically distinct, and E. coli that cause colibacillosis are virulent outbreak strains. My objectives were to isolate E. coli from Saskatchewan broilers, sequence their genomes using Nanopore and Illumina technology, and screen them for virulence, antimicrobial resistance, and disinfectant resistance. I developed a pipeline to isolate and sequence E. coli from Saskatchewan colibacillosis outbreaks, selecting isolates based on outbreak, disease status, and biofilm profiles. I sequenced 96 E. coli isolates, consisting of 58 from diseased broilers with confirmed colibacillosis (systemic E. coli), and 38 from the cecal contents of healthy broilers in the same flocks (cecal E. coli). Our initial experiments were optimized for whole genome assembly and excluded DNA fragments under 500bp; therefore, we likely missed plasmids present in E. coli isolates. I tested six plasmid kits and two sequencing protocols to develop a methodology to capture missed plasmids in avian E. coli isolates and successfully identified new plasmids in both types of isolates. Systemic E. coli were more drug-resistant than cecal E. coli against a panel of 27 antimicrobial agents and possessed significantly more plasmids than cecal E. coli. plasmids contained multiple virulence and antimicrobial resistance genes that may contribute to disease. Since biofilms can provide protection from antibiotics and disinfectants, I quantified biofilm formation in three different medias. Systemic isolates were significantly more likely to form biofilms in rich media, but there was no correlation between biofilm formation and antimicrobial resistance. My characterization led us to conclude that systemic and cecal E. coli represent two different populations of strains. This will need to be confirmed with the analysis of more isolates. Characterization of avian pathogenic E. coli will help us understand how these isolates cause disease

Generalized Paley graphs and their complete subgraphs of orders three and four

Let $k \geq 2$ be an integer. Let $q$ be a prime power such that $q \equiv 1 \pmod {k}$ if $q$ is even, or, $q \equiv 1 \pmod {2k}$ if $q$ is odd. The generalized Paley graph of order $q$, $G_k(q)$, is the graph with vertex set $\mathbb{F}_q$ where $ab$ is an edge if and only if ${a-b}$ is a $k$-th power residue. We provide a formula, in terms of finite field hypergeometric functions, for the number of complete subgraphs of order four contained in $G_k(q)$, $\mathcal{K}_4(G_k(q))$, which holds for all $k$. This generalizes the results of Evans, Pulham and Sheehan on the original ($k$=2) Paley graph. We also provide a formula, in terms of Jacobi sums, for the number of complete subgraphs of order three contained in $G_k(q)$, $\mathcal{K}_3(G_k(q))$. In both cases we give explicit determinations of these formulae for small $k$. We show that zero values of $\mathcal{K}_4(G_k(q))$ (resp. $\mathcal{K}_3(G_k(q))$) yield lower bounds for the multicolor diagonal Ramsey numbers $R_k(4)=R(4,4,\cdots,4)$ (resp. $R_k(3)$). We state explicitly these lower bounds for small $k$ and compare to known bounds. We also examine the relationship between both $\mathcal{K}_4(G_k(q))$ and $\mathcal{K}_3(G_k(q))$, when $q$ is prime, and Fourier coefficients of modular forms

Primary reading exercises for use with the Durrell Analysis of Reading Difficulty

Thesis (Ed.M.)--Boston Universit

Aboriginal artefacts on the continental shelf reveal ancient drowned cultural landscapes in northwest Australia

This article reports Australia’s first confirmed ancient underwater archaeological sites from the continental shelf, located off the Murujuga coastline in north-western Australia. Details on two underwater sites are reported: Cape Bruguieres, comprising > 260 recorded lithic artefacts at depths down to −2.4 m below sea level, and Flying Foam Passage where the find spot is associated with a submerged freshwater spring at −14 m. The sites were discovered through a purposeful research strategy designed to identify underwater targets, using an iterative process incorporating a variety of aerial and underwater remote sensing techniques and diver investigation within a predictive framework to map the submerged landscape within a depth range of 0–20 m. The condition and context of the lithic artefacts are analysed in order to unravel their depositional and taphonomic history and to corroborate their in situ position on a pre-inundation land surface, taking account of known geomorphological and climatic processes including cyclone activity that could have caused displacement and transportation from adjacent coasts. Geomorphological data and radiometric dates establish the chronological limits of the sites and demonstrate that they cannot be later than 7000 cal BP and 8500 cal BP respectively, based on the dates when they were finally submerged by sea-level rise. Comparison of underwater and onshore lithic assemblages shows differences that are consistent with this chronological interpretation. This article sets a foundation for the research strategies and technologies needed to identify archaeological targets at greater depth on the Australian continental shelf and elsewhere, building on the results presented. Emphasis is also placed on the need for legislation to better protect and manage underwater cultural heritage on the 2 million square kilometres of drowned landscapes that were once available for occupation in Australia, and where a major part of its human history must lie waiting to be discovered

The effect of frailty on survival in patients with COVID-19 (COPE): a multicentre, European, observational cohort study

Background The COVID-19 pandemic has placed unprecedented strain on health-care systems. Frailty is being used in clinical decision making for patients with COVID-19, yet the prevalence and effect of frailty in people with COVID-19 is not known. In the COVID-19 in Older PEople (COPE) study we aimed to establish the prevalence of frailty in patients with COVID-19 who were admitted to hospital and investigate its association with mortality and duration of hospital stay. Methods This was an observational cohort study conducted at ten hospitals in the UK and one in Italy. All adults (≥18 years) admitted to participating hospitals with COVID-19 were included. Patients with incomplete hospital records were excluded. The study analysed routinely generated hospital data for patients with COVID-19. Frailty was assessed by specialist COVID-19 teams using the clinical frailty scale (CFS) and patients were grouped according to their score (1–2=fit; 3–4=vulnerable, but not frail; 5–6=initial signs of frailty but with some degree of independence; and 7–9=severe or very severe frailty). The primary outcome was in-hospital mortality (time from hospital admission to mortality and day-7 mortality). Findings Between Feb 27, and April 28, 2020, we enrolled 1564 patients with COVID-19. The median age was 74 years (IQR 61–83); 903 (57·7%) were men and 661 (42·3%) were women; 425 (27·2%) had died at data cutoff (April 28, 2020). 772 (49·4%) were classed as frail (CFS 5–8) and 27 (1·7%) were classed as terminally ill (CFS 9). Compared with CFS 1–2, the adjusted hazard ratios for time from hospital admission to death were 1·55 (95% CI 1·00–2·41) for CFS 3–4, 1·83 (1·15–2·91) for CFS 5–6, and 2·39 (1·50–3·81) for CFS 7–9, and adjusted odds ratios for day-7 mortality were 1·22 (95% CI 0·63–2·38) for CFS 3–4, 1·62 (0·81–3·26) for CFS 5–6, and 3·12 (1·56–6·24) for CFS 7–9. Interpretation In a large population of patients admitted to hospital with COVID-19, disease outcomes were better predicted by frailty than either age or comorbidity. Our results support the use of CFS to inform decision making about medical care in adult patients admitted to hospital with COVID-19

“We come together as one…and hope for solidarity to live on”: On designing technologies for activism and the commemoration of lost lives

On the International Day to End Violence Against Sex Workers (IDEVASW), sex worker rights advocates and support services commemorate lives lost due to violence. In this paper we describe and reflect on a Feminist Participatory Action Research project that supported the activities of IDEVASW over two years in North East England. Working alongside a charity that provides services to women who are sex workers or have experienced sexual exploitation, we co-organised the first activist march on the day. As researchers and service providers, we present detailed reflections on the use of digital technologies during the public activist march, a private service for commemoration, and the development of a semi-public archive to collect experiences of the day. We develop three implications for the design of digital technologies for activism and the commemoration of lost lives: as catalysts for reflection and opportunities to layer experience

New Introductions, Spread of Existing Matrilines, and High Rates of Pyrethroid Resistance Result in Chronic Infestations of Bed Bugs (Cimex lectularius L.) in Lower-Income Housing

Infestations of the common bed bug (Cimex lectularius L.) have increased substantially in the United States in the past 10-15 years. The housing authority in Harrisonburg, Virginia, conducts heat-treatments after bed bugs are detected in a lower-income housing complex, by treating each infested unit at 60°C for 4-6 hours. However, a high frequency of recurrent infestations called into question the efficacy of this strategy. Genetic analysis using Bayesian clustering of polymorphic microsatellite loci from 123 bed bugs collected from 23 units from May 2012 to April 2013 in one building indicated that (a) 16/21 (73%) infestations were genetically similar, suggesting ineffective heat-treatments or reintroductions from within the building or from a common external source, followed by local spread of existing populations; and (b) up to 5 of the infestations represented new genotypes, indicating that 5 new populations were introduced into this building in one year, assuming they were not missed in earlier screens. There was little to no gene flow among the 8 genetic clusters identified in the building. Bed bugs in the U.S. often possess one or both point mutations in the voltage-gated sodium channel, termed knockdown resistance (kdr), from valine to leucine (V419L) and leucine to isoleucine (L925I) that confer target-site resistance against pyrethroid insecticides. We found that 48/121 (40%) bed bugs were homozygous for both kdr mutations (L419/I925), and a further 59% possessed at least one of the kdr mutations. We conclude that ineffective heat treatments, new introductions, reintroductions and local spread, and an exceptionally high frequency of pyrethroid resistance are responsible for chronic infestations in lower-income housing. Because heat treatments fail to protect from reintroductions, and pesticide use has not decreased the frequency of infestations, preventing new introductions and early detection are the most effective strategies to avoid bed bug infestations in multistory apartment buildings

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Prognostic value of estimated glomerular filtration rate in hospitalised older patients (over 65) with COVID-19: a multicentre, European, observational cohort study

Background: The reduced renal function has prognostic significance in COVID-19 and it has been linked to mortality in the general population. Reduced renal function is prevalent in older age and thus we set out to better understand its effect on mortality. Methods: Patient clinical and demographic data was taken from the COVID-19 in Older People (COPE) study during two periods (February–June 2020 and October 2020–March 2021, respectively). Kidney function on admission was measured using estimated glomerular filtration rate (eGFR). The primary outcomes were time to mortality and 28-day mortality. Secondary outcome was length of hospital stay. Data were analysed with multilevel Cox proportional hazards regression, and multilevel logistic regression and adjusted for individual patient clinical and demographic characteristics. Results: One thousand eight hundred two patients (55.0% male; median [IQR] 80 [73–86] years) were included in the study. 28-day mortality was 42.3% (n = 742). 48% (n = 801) had evidence of renal impairment on admission. Using a time-to-event analysis, reduced renal function was associated with increased in-hospital mortality (compared to eGFR ≥ 60 [Stage 1&2]): eGFR 45–59 [Stage 3a] aHR = 1.26 (95%CI 1.02–1.55); eGFR 30–44 [Stage 3b] aHR = 1.41 (95%CI 1.14–1.73); eGFR 1–29 [Stage 4&5] aHR = 1.42 (95%CI 1.13–1.80). In the co-primary outcome of 28-day mortality, mortality was associated with: Stage 3a adjusted odds ratio (aOR) = 1.18 (95%CI 0.88–1.58), Stage 3b aOR = 1.40 (95%CI 1.03–1.89); and Stage 4&5 aOR = 1.65 (95%CI 1.16–2.35). Conclusion: eGFR on admission is a good independent predictor of mortality in hospitalised older patients with COVID-19 population. We found evidence of a dose-response between reduced renal function and increased mortality