2,027 research outputs found

    Improving rapid pathogen detection: towards a gram-selective lateral flow test

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    The development of rapid detection assays, such as lateral flow tests, have been effective in helping to detect a range of biological targets, most recently in the COVID-19 pandemic. The effective detection and classification of bacteria, such as their Gram status using rapid low-cost lateral flow assay could help diagnose and target treatment of bacterial infections in medical and veterinary applications. This is important as over prescription of broad-spectrum antibiotics is a well-known contributor to the rise in antimicrobial resistance. My work targeted the development of lateral flow assays to achieve the specific detection of Gram-negative bacteria. To make the assay Gram selective, targeting/binding ligands were synthesised based on Polymyxin B, a Gram-negative selective antibiotic. A literature method of selective functionalisation of Polymyxin B was optimised to service material at the gram scale for the development of three Polymyxin conjugates: a Lipoic acid-Polymyxin conjugate and two Biotin-Polymyxin conjugates with and without a spacer. The most effective binding/labelling agent was determined to be a Biotin-Polymyxin conjugate with a six-carbon spacer, which allowed the fluorescently labelling of E. coli by the generation of a Bacteria-Polymyxin-Biotin-Streptavidin-fluorophore sandwich. Most lateral flow tests use gold nanoparticles to label the target analyte as it provides an intense red colour that is easily detectable by eye, and this was replicated here by the attachment of Polymyxin to gold nanoparticles. Through testing a range of methods for the attachment of Polymyxin, a nanoparticle system that could be used to selectively label the surface of bacteria was developed. This Polymyxin-nanoparticle labelling system was characterised via electron microscopy. Nitrocellulose membranes were laser cut to shape and printed with reagents to give a strip assay platform that could analyse the behaviour of the synthesised gold nanoparticles and the Polymyxin targeting agents in flow. 3D printing was used to fabricate an imaging enclosure to enable repeatable imaging that allowed for smartphone capture of the assays and analysis with a custom Python-based image analysis script. This allowed high-throughput testing and optimisation of the assay and flow conditions. Through the optimisation of the lateral flow assay design Gram-negative E. coli were able to be detected in flow, imaged, and analysed. My PhD work thus provides an example of a lateral flow assay functioning without antibodies or aptamers. This work could provide the foundation for further development, including other antibiotics to target different bacteria

    LIPIcs, Volume 251, ITCS 2023, Complete Volume

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    LIPIcs, Volume 251, ITCS 2023, Complete Volum

    Investigation of the metabolism of rare nucleotides in plants

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    Nucleotides are metabolites involved in primary metabolism, and specialized metabolism and have a regulatory role in various biochemical reactions in all forms of life. While in other organisms, the nucleotide metabolome was characterized extensively, comparatively little is known about the cellular concentrations of nucleotides in plants. The aim of this dissertation was to investigate the nucleotide metabolome and enzymes influencing the composition and quantities of nucleotides in plants. For this purpose, a method for the analysis of nucleotides and nucleosides in plants and algae was developed (Chapter 2.1), which comprises efficient quenching of enzymatic activity, liquid-liquid extraction and solid phase extraction employing a weak-anionexchange resin. This method allowed the analysis of the nucleotide metabolome of plants in great depth including the quantification of low abundant deoxyribonucleotides and deoxyribonucleosides. The details of the method were summarized in an article, serving as a laboratory protocol (Chapter 2.2). Furthermore, we contributed a review article (Chapter 2.3) that summarizes the literature about nucleotide analysis and recent technological advances with a focus on plants and factors influencing and hindering the analysis of nucleotides in plants, i.e., a complex metabolic matrix, highly stable phosphatases and physicochemical properties of nucleotides. To analyze the sub-cellular concentrations of metabolites, a protocol for the rapid isolation of highly pure mitochondria utilizing affinity chromatography was developed (Chapter 2.4). The method for the purification of nucleotides furthermore contributed to the comprehensive analysis of the nucleotide metabolome in germinating seeds and in establishing seedlings of A. thaliana, with a focus on genes involved in the synthesis of thymidilates (Chapter 2.5) and the characterization of a novel enzyme of purine nucleotide degradation, the XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Chapter 2.6). Protein homology analysis comparing A. thaliana, S. cerevisiae, and H. sapiens led to the identification and characterization of an enzyme involved in the metabolite damage repair system of plants, the INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Chapter 2.7). It was shown that this enzyme dephosphorylates deaminated purine nucleotide triphosphates and thus prevents their incorporation into nucleic acids. Lossof-function mutants senesce early and have a constitutively increased content of salicylic acid. Also, the source of deaminated purine nucleotides in plants was investigated and it was shown that abiotic factors contribute to nucleotide damage.Nukleotide sind Metaboliten, die am Primärstoffwechsel und an spezialisierten Stoffwechselvorgängen beteiligt sind und eine regulierende Rolle bei verschiedenen biochemischen Reaktionen in allen Lebensformen spielen. Während bei anderen Organismen das Nukleotidmetabolom umfassend charakterisiert wurde, ist in Pflanzen vergleichsweise wenig über die zellulären Konzentrationen von Nukleotiden bekannt. Ziel dieser Dissertation war es, das Nukleotidmetabolom und die Enzyme zu untersuchen, die die Zusammensetzung und Menge der Nukleotide in Pflanzen beeinflussen. Zu diesem Zweck wurde eine Methode zur Analyse von Nukleotiden und Nukleosiden in Pflanzen und Algen entwickelt (Kapitel 2.1), die ein effizientes Stoppen enzymatischer Aktivität, eine Flüssig-Flüssig-Extraktion und eine Festphasenextraktion unter Verwendung eines schwachen Ionenaustauschers umfasst. Mit dieser Methode konnte das Nukleotidmetabolom von Pflanzen eingehend analysiert werden, einschließlich der Quantifizierung von Desoxyribonukleotiden und Desoxyribonukleosiden mit geringer Abundanz. Die Einzelheiten der Methode wurden in einem Artikel zusammengefasst, der als Laborprotokoll dient (Kapitel 2.2). Darüber hinaus wurde ein Übersichtsartikel (Kapitel 2.3) verfasst, der die Literatur über die Analyse von Nukleotiden und die jüngsten technologischen Fortschritte zusammenfasst. Der Schwerpunkt lag hierbei auf Pflanzen und Faktoren, die die Analyse von Nukleotiden in Pflanzen beeinflussen oder behindern, d. h. eine komplexe Matrix, hochstabile Phosphatasen und physikalisch-chemische Eigenschaften von Nukleotiden. Um die subzellulären Konzentrationen von Metaboliten zu analysieren, wurde ein Protokoll für die schnelle Isolierung hochreiner Mitochondrien unter Verwendung einer Affinitätschromatographie entwickelt (Kapitel 2.4). Die Methode zur Analyse von Nukleotiden trug außerdem zu einer umfassenden Analyse des Nukleotidmetaboloms in keimenden Samen und in sich etablierenden Keimlingen von A. thaliana bei, wobei der Schwerpunkt auf Genen lag, die an der Synthese von Thymidilaten beteiligt sind (Kapitel 2.5), sowie zu der Charakterisierung eines neuen Enzyms des Purinnukleotidabbaus, der XANTHOSINE MONOPHOSPHATE PHOSPHATASE (Kapitel 2.6). Eine Proteinhomologieanalyse, die A. thaliana, S. cerevisiae und H. sapiens miteinander verglich führte zur Identifizierung und Charakterisierung eines Enzyms, das an der Reparatur von geschädigten Metaboliten in Pflanzen beteiligt ist, der INOSINE TRIPHOSPHATE PYROPHOSPHATASE (Kapitel 2.7). Es konnte gezeigt werden, dass dieses Enzym desaminierte Purinnukleotidtriphosphate dephosphoryliert und so deren Einbau in Nukleinsäuren verhindert. Funktionsverlustmutanten altern früh und weisen einen konstitutiv erhöhten Gehalt an Salicylsäure auf. Außerdem wurde die Quelle der desaminierten Purinnukleotide in Pflanzen untersucht, und es wurde gezeigt, dass abiotische Faktoren zur Nukleotidschädigung beitragen

    Effects of municipal smoke-free ordinances on secondhand smoke exposure in the Republic of Korea

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    ObjectiveTo reduce premature deaths due to secondhand smoke (SHS) exposure among non-smokers, the Republic of Korea (ROK) adopted changes to the National Health Promotion Act, which allowed local governments to enact municipal ordinances to strengthen their authority to designate smoke-free areas and levy penalty fines. In this study, we examined national trends in SHS exposure after the introduction of these municipal ordinances at the city level in 2010.MethodsWe used interrupted time series analysis to assess whether the trends of SHS exposure in the workplace and at home, and the primary cigarette smoking rate changed following the policy adjustment in the national legislation in ROK. Population-standardized data for selected variables were retrieved from a nationally representative survey dataset and used to study the policy action’s effectiveness.ResultsFollowing the change in the legislation, SHS exposure in the workplace reversed course from an increasing (18% per year) trend prior to the introduction of these smoke-free ordinances to a decreasing (−10% per year) trend after adoption and enforcement of these laws (β2 = 0.18, p-value = 0.07; β3 = −0.10, p-value = 0.02). SHS exposure at home (β2 = 0.10, p-value = 0.09; β3 = −0.03, p-value = 0.14) and the primary cigarette smoking rate (β2 = 0.03, p-value = 0.10; β3 = 0.008, p-value = 0.15) showed no significant changes in the sampled period. Although analyses stratified by sex showed that the allowance of municipal ordinances resulted in reduced SHS exposure in the workplace for both males and females, they did not affect the primary cigarette smoking rate as much, especially among females.ConclusionStrengthening the role of local governments by giving them the authority to enact and enforce penalties on SHS exposure violation helped ROK to reduce SHS exposure in the workplace. However, smoking behaviors and related activities seemed to shift to less restrictive areas such as on the streets and in apartment hallways, negating some of the effects due to these ordinances. Future studies should investigate how smoke-free policies beyond public places can further reduce the SHS exposure in ROK

    Markov field models of molecular kinetics

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    Computer simulations such as molecular dynamics (MD) provide a possible means to understand protein dynamics and mechanisms on an atomistic scale. The resulting simulation data can be analyzed with Markov state models (MSMs), yielding a quantitative kinetic model that, e.g., encodes state populations and transition rates. However, the larger an investigated system, the more data is required to estimate a valid kinetic model. In this work, we show that this scaling problem can be escaped when decomposing a system into smaller ones, leveraging weak couplings between local domains. Our approach, termed independent Markov decomposition (IMD), is a first-order approximation neglecting couplings, i.e., it represents a decomposition of the underlying global dynamics into a set of independent local ones. We demonstrate that for truly independent systems, IMD can reduce the sampling by three orders of magnitude. IMD is applied to two biomolecular systems. First, synaptotagmin-1 is analyzed, a rapid calcium switch from the neurotransmitter release machinery. Within its C2A domain, local conformational switches are identified and modeled with independent MSMs, shedding light on the mechanism of its calcium-mediated activation. Second, the catalytic site of the serine protease TMPRSS2 is analyzed with a local drug-binding model. Equilibrium populations of different drug-binding modes are derived for three inhibitors, mirroring experimentally determined drug efficiencies. IMD is subsequently extended to an end-to-end deep learning framework called iVAMPnets, which learns a domain decomposition from simulation data and simultaneously models the kinetics in the local domains. We finally classify IMD and iVAMPnets as Markov field models (MFM), which we define as a class of models that describe dynamics by decomposing systems into local domains. Overall, this thesis introduces a local approach to Markov modeling that enables to quantitatively assess the kinetics of large macromolecular complexes, opening up possibilities to tackle current and future computational molecular biology questions

    Modern meat: the next generation of meat from cells

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    Modern Meat is the first textbook on cultivated meat, with contributions from over 100 experts within the cultivated meat community. The Sections of Modern Meat comprise 5 broad categories of cultivated meat: Context, Impact, Science, Society, and World. The 19 chapters of Modern Meat, spread across these 5 sections, provide detailed entries on cultivated meat. They extensively tour a range of topics including the impact of cultivated meat on humans and animals, the bioprocess of cultivated meat production, how cultivated meat may become a food option in Space and on Mars, and how cultivated meat may impact the economy, culture, and tradition of Asia

    Workshop Proceedings of the 12th edition of the KONVENS conference

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    The 2014 issue of KONVENS is even more a forum for exchange: its main topic is the interaction between Computational Linguistics and Information Science, and the synergies such interaction, cooperation and integrated views can produce. This topic at the crossroads of different research traditions which deal with natural language as a container of knowledge, and with methods to extract and manage knowledge that is linguistically represented is close to the heart of many researchers at the Institut für Informationswissenschaft und Sprachtechnologie of Universität Hildesheim: it has long been one of the institute’s research topics, and it has received even more attention over the last few years

    Sum-of-squares representations for copositive matrices and independent sets in graphs

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    A polynomial optimization problem asks for minimizing a polynomial function (cost) given a set of constraints (rules) represented by polynomial inequalities and equations. Many hard problems in combinatorial optimization and applications in operations research can be naturally encoded as polynomial optimization problems. A common approach for addressing such computationally hard problems is by considering variations of the original problem that give an approximate solution, and that can be solved efficiently. One such approach for attacking hard combinatorial problems and, more generally, polynomial optimization problems, is given by the so-called sum-of-squares approximations. This thesis focuses on studying whether these approximations find the optimal solution of the original problem.We investigate this question in two main settings: 1) Copositive programs and 2) parameters dealing with independent sets in graphs. Among our main new results, we characterize the matrix sizes for which sum-of-squares approximations are able to capture all copositive matrices. In addition, we show finite convergence of the sums-of-squares approximations for maximum independent sets in graphs based on their continuous copositive reformulations. We also study sum-of-squares approximations for parameters asking for maximum balanced independent sets in bipartite graphs. In particular, we find connections with the Lovász theta number and we design eigenvalue bounds for several related parameters when the graphs satisfy some symmetry properties.<br/

    On Centrality and Population Size Effects in Urban Pollution: A Meta-Analysis of NO2 and Heat Islands and Spatial Analysis of NO2

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