Nano-probes for point of care diagnostics

The COVID-19 pandemic has exposed deep health inequalities between more economically developed and less economically developed countries: both in terms of diagnostics and vaccinations. Robust and low-cost point of care devices are needed to ease these diagnostic inequalities. Current point of care lateral flow immunoassays, utilise proteins, such as antibodies, to sense for analytes. This is epitomised by the malaria rapid diagnostic test and archetypal home pregnancy test. Glycans are emerging as alternative detection units due to their fundamental role in biological signalling and recognition events. Furthermore, the increased robustness, low-cost and synthetic possibilities offered by glycan-based systems, especially glycosylated polymers, make them a promising alternative to antibody-based biosensing and diagnostic systems. Chapter 1 discusses the current use of protein-based lateral flow and flow-through devices; their advantages and disadvantages versus non-point of care techniques, and the potential of glycan-based lateral flow devices. The concepts introduced in Chapter 1 are then applied in Chapters 2 through 5. Chapter 2 demonstrates the use of glycosylated polymer-coated nanoparticles, produced by controlled radical polymerisation techniques for the sensitive, label-free detection of lectins in lateral flow and flow-through. The systems produced use only glycans, not antibodies, to provide recognition – a “lateral flow glyco-assay.” The lessons learned in Chapter 2 are applied in Chapter 3 to probe the glycan-binding of the SARS-COV-2 spike protein in a “flow-through glyco-assay” and target a pseudovirus mimic of the target coronavirus in a lateral flow glyco-assay. Chapter 4 builds on Chapters 2 and 3, applying the fledgling glyco-assay technology to the “real-world” by sensing for the SARS-COV-2 virus in patient samples, alongside exploring the robustness of the devices themselves. Having explored the concept of glycosylated polymer-coated nanoparticles in lateral flow and flow-through setups; Chapter 5 changes focus and explores the use of polymeric anchors for the design of all-polymer (“vegan”) lateral flow and flow-through devices. This work completely removes proteins as either detecting units or anchors from lateral flow for the first time. Chapters 6 and 7 explore more fundamental Chemistry than the previous chapters. Chapter 6 considers the use of the Mannich reaction to produce monosaccharides with amine functionality at C2, ideal for polymer conjugation, while maintaining hydroxyl functionality at C2. Although unsuccessful with the reagents used, the chapter highlights a potential avenue of future chemical exploration in novel glycan synthesis. Chapter 7 pulls together the x-ray photoelectron analysis data and spectra collected across a range of studies, including data collected in previous chapters, and considers if x-ray photoelectron spectroscopy can be used to determine relative grafting density in glycosylated polymer-coated nanoparticle systems. In summary, the key components of the emerging technology of lateral flow glycoassays are introduced, interrogated and investigated. The prototype devices tested against model proteins, viral proteins and patient samples, are found to show specificities and sensitivities that rival lateral flow immunoassay systems. The understanding developed in this thesis could pave the way to the first generation of lateral flow glyco-assays that are low-cost, stable in a wide range of conditions, and able to target a wide range of analytes and diseases

Intra-session and inter-day reliability of the Myon 320 electromyography system during sub-maximal contractions

Electromyography systems are widely used within the field of scientific and clinical practices. The reliability of these systems are paramount when conducting research. The reliability of Myon 320 Surface Electromyography System is yet to be determined. This study aims to determine the intra-session and inter-day reliability of the Myon 320 Surface Electromyography System. Muscle activity from fifteen participants was measured at the anterior deltoid muscle during a bilateral front raise exercise, the vastus lateralis muscle during a squat exercise and the extensor carpi radialis brevis (ECRB) muscle during an isometric handgrip task. Intra-session and inter-day reliability was calculated by intraclass correlation coefficient, standard error of measurement and coefficient of variation (CV). The normalized root mean squared (RMS) surface electromyographic signals produced good intra-session and inter-day testing intraclass correlation coefficient values (range: 0.63-0.97) together with low standard error of measurement (range: 1.49-2.32) and CV (range: 95% Confidence Interval = 0.36-12.71) measures for the dynamic-and-isometric contractions. The findings indicate that the Myon 320 Surface Electromyography System produces good to fair reliability when examining intra-session and inter-day reliability. Findings of the study provide evidence of the reliability of electromyography between trials which is essential during clinical testing.</p

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How to sequence 10,000 bacterial genomes and retain your sanity: an accessible, efficient and global approach

Non-typhoidal Salmonella(NTS)are typically associated with enterocolitis and linked to the industrialisation of food production. In recent years, NTS has been associated with invasive disease (iNTS disease) causing an estimated 77,000 deaths each year worldwide; 80% of mortality occurs in sub-Saharan Africa. New clades of S. Typhimurium and S. Enteritidis have been identified, which are characterised by genomic degradation, altered prophage repertoires and novel multidrug resistant plasmids. To understand how these clades are contributing to the burden and severity of iNTS disease, it is crucial to expand genome-based surveillance to cover more countries, and incorporate historical isolates to generate an evolutionary timeline of the development of iNTS. We developedand validateda robust and inexpensive method for large-scale collection and sequencing of bacterial genomes. The “10,000 Salmonella genomes” project established a worldwide research collaboration to generate information relevant to the epidemiology, drug resistance and virulence factors of Salmonellae using a whole-genome sequencing approach. By streamlining collection of isolates and developing an efficient logistics pipeline, we gathered 10,419 clinical and environmental isolates from collections in low and middle-income countries within six months. Genome sequences are now available for isolates from 51 countries/territories dating from 1949 to 2017, with ~80 % representing African and Latin-American datasets. Our method can be applied to other large sample collections that require maximisation of resources within a limited timeframe. Detailed genome analyses are in progress and it is hoped that the resulting data will contribute to public health control strategies in low and middle-income countries

Plasmonic detection of SARS-CoV-2 spike protein with polymer-stabilized glycosylated gold nanorods

The COVID-19 pandemic has highlighted the need for innovative biosensing, diagnostic, and surveillance platforms. Here we report that glycosylated, polymer-stabilized, gold nanorods can bind the SARS-CoV-2 spike protein and show correlation to the presence of SARS-CoV-2 in primary COVID-19 clinical samples. Telechelic polymers were prepared by reversible addition–fragmentation chain-transfer polymerization, enabling the capture of 2,3-sialyllactose and immobilization onto gold nanorods. Control experiments with a panel of lectins and a galactosamine-terminated polymer confirmed the selective binding. The glycosylated rods were shown to give dose-dependent responses against recombinant truncated SARS-CoV-2 spike protein, and the responses were further correlated using primary patient swab samples. The essentiality of the anisotropic particles for reducing the background interference is demonstrated. This highlights the utility of polymer tethering of glycans for plasmonic biosensors of infection

An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. Following the optimization of a logistics pipeline that involved shipping isolates as thermolysates in ambient conditions, the project assembled a diverse collection of 10,419 isolates from low- and middle-income countries. The genomes were sequenced using the LITE pipeline for library construction, with a total reagent cost of less than USD$10 per genome. Our method can be applied to other large bacterial collections to underpin global collaborations

Joint effects of climate, tree size, and year on annual tree growth derived from tree-ring records of ten globally distributed forests

Tree rings provide an invaluable long-term record for understanding how climate and other drivers shape tree growth and forest productivity. However, conventional tree-ring analysis methods were not designed to simultaneously test effects of climate, tree size, and other drivers on individual growth. This has limited the potential to test ecologically relevant hypotheses on tree growth sensitivity to environmental drivers and their interactions with tree size. Here, we develop and apply a new method to simultaneously model nonlinear effects of primary climate drivers, reconstructed tree diameter at breast height (DBH), and calendar year in generalized least squares models that account for the temporal autocorrelation inherent to each individual tree\u27s growth. We analyze data from 3811 trees representing 40 species at 10 globally distributed sites, showing that precipitation, temperature, DBH, and calendar year have additively, and often interactively, influenced annual growth over the past 120 years. Growth responses were predominantly positive to precipitation (usually over ≥3-month seasonal windows) and negative to temperature (usually maximum temperature, over ≤3-month seasonal windows), with concave-down responses in 63% of relationships. Climate sensitivity commonly varied with DBH (45% of cases tested), with larger trees usually more sensitive. Trends in ring width at small DBH were linked to the light environment under which trees established, but basal area or biomass increments consistently reached maxima at intermediate DBH. Accounting for climate and DBH, growth rate declined over time for 92% of species in secondary or disturbed stands, whereas growth trends were mixed in older forests. These trends were largely attributable to stand dynamics as cohorts and stands age, which remain challenging to disentangle from global change drivers. By providing a parsimonious approach for characterizing multiple interacting drivers of tree growth, our method reveals a more complete picture of the factors influencing growth than has previously been possible

Human-animal elision: a Darwinian universe in George Eliot’s novels

No abstract available

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis