Fingerprinting and characterisation of Escherichia coli isolates using DNA arrays

Two commercially available DNA whole genome Escherichia coli K12 arrays were compared to identify a subset of markers for typing. The arrays were identical in probe composition but different in substrate (membrane and glass slide arrays) and probe preparation (radio- and fluorescent-labelled). Labelled genomic E. coli DNA from five strains of the E. coli reference (ECOR) collection (ATCT35320 - ATCX35324) and E. coli K12 were hybridised against these arrays. A group of 1240 putative markers was identified on the membrane arrays and 649 were found on the glass slide arrays. Only a small proportion of these sequences (8%) was found through both platforms. Variability in the hybridisation signals from duplicate experiments made it difficult to identify useful markers. In order to investigate whether this technology could be used for characterising or typing E. coli strains, an array for the detection of 29 pathogenicity markers in E. coli strains was produced. This array was used with eight reference strains, including different pathotypes, 72 strains from the ECOR collection, and 49 clinical isolates. A wide range of E. coli pathogenicity markers was detected. The pathogenicity markers that were most common include chuA and iucC, which are both involved in iron metabolism. Additionally, the clinical isolates were grouped into clusters different from groupings based on biochemical tests. This demonstrates that the use of pathogenicity array typing can complement diagnostic tests on clinical E. coli isolates. An extended, second-generation, pathogenicity marker array containing 75 probes was made. The extended array successfully distinguished between ten closely related isolates from an outbreak of urinary tract infections, while previous tests were unable to do so. This array has the potential for providing a rapid and novel means of characterising pathogenic isolates

Sistema de leitura ótica para diagnóstico de malária

Dissertação de mestrado integrado em Engenharia de Eletrónica Industrial e ComputadoresMetade da população mundial, em particular em regiões de subdesenvolvimento económico, encontra se em risco de contrair o parasita da malária, pelo que é urgente o desenvolvimento de métodos diagnóstico eficientes, capazes de detetar a doença na sua fase inicial, rápidos, específicos, de baixo custo e de fácil utilização. A presente dissertação integra um projeto de investigação com o principal objetivo de desenvolver sistemas de deteção de malária através da identificação e quantificação ótica de hemozoína. O objetivo desta dissertação passa pelo desenvolvimento de um sistema eletrónico capaz de identificar e quantificar a presença de hemozoína numa amostra por espectrofotometria de absorção ótica, com dimensões reduzidas, baixo consumo energético, autónomo, de utilização simples, reutilizável e com um custo de utilização reduzido. À medida que o parasita da malária se desenvolve nos glóbulos vermelhos humanos, consome hemoglobina e produz hemozoína, que é um cristal com características óticas específicas e distintas do sangue humano. Assim, o dispositivo proposto baseia-se nas diferenças entre os espectros de absorção ótica do sangue total e da hemozoína à medida que a doença da malária progride. Deste modo, desenvolveu-se um dispositivo eletrónico portátil, baseado num sistema de deteção por espectrofotometria de absorbância, recorrendo apenas a quatro comprimentos de onda do espectro de luz (520, 635, 661 e 783 nm). O sistema foi integrado numa plataforma fluídica, e incluiu díodos laser como emissores, fotodíodos como detetores, eletrónica de aquisição e controlo, assim como um algoritmo de atuação e leitura programado num microcontrolador. Desenvolveram-se duas abordagens de encapsulamento para o sistema de leitura, compatíveis com diferentes poços e reservatórios para as amostras. O sistema foi testado com amostras de diferentes concentrações, até um limite de deteção de 1 µg/mL de hemozoína em sangue total. O dispositivo desenvolvido foi capaz de identificar variações na absorbância, permitindo, através dos quatro comprimentos de onda reproduzir as principais oscilações dos espectros de absorção da hemozoína e do sangue total. Os resultados obtidos demonstram, assim, que a utilização de sistemas óticos portáteis poderá ser a resposta para colmatar as limitações atuais dos métodos e tecnologias de diagnóstico da malária.Half of the world's population, particularly in economically underdeveloped regions, is at risk of contracting malaria. So, the development of efficient, fast, specific, low cost and user-friendly diagnostic methods, able to detect the disease in its initial phase, is urgent. This dissertation is part of a research project with the main objective of developing malaria detection systems through the identification and optical quantification of hemozoin. The objective of this dissertation is the development of an electronic system capable of identifying and quantifying the presence of hemozoin in a sample by optical absorption spectrophotometry. Such system must be small, with low energy consumption, autonomous, simple to use, reusable and low cost. As the malaria parasite develops in human red blood cells, it consumes hemoglobin and produces hemozoin, which is a crystal with specific and optical characteristics distinct from human blood. Therefore, the proposed device is based on the differences between the optical absorption spectra of whole blood and hemozoin as the malarial disease progresses. Thus, a portable electronic device was developed, based on a detection system by absorbance spectrophotometry, using only four wavelengths of the light spectrum (520, 635, 661 and 783 nm). The system was integrated in a fluidic platform, and included laser diodes as emitters, photodiodes as detectors, acquisition and control electronics, as well as an actuation and reading algorithm programmed in a microcontroller. Two encapsulation approaches were developed for the reading system, compatible with different wells and reservoirs for the samples. The system was tested with different concentration samples, up to a detection limit of 1 µg/mL of hemozoin in whole blood. The developed device was able to identify variations in absorbance, allowing, through the four wavelengths, to reproduce the main oscillations of the absorption spectra of hemozoin and whole blood. The results obtained enhance that the use of portable optical systems can be the answer to overcome the current limitations of malaria diagnostic methods and technologies.O trabalho desenvolvido teve o apoio do projeto MalariaChip, NORTE-01-0145-FEDER-028178, financiado pelo Programa Operacional Regional do Norte (NORTE 2020), sob o Acordo de Parceria PORTUGAL 2020, através do Fundo Europeu de Desenvolvimento Regional (FEDER) e pela Fundação para a Ciência e Tecnologia (FCT), IP

The characterisation of the peanut agglutinin an evolved plant lectin, with improved specificity to the Thompson Freidenriech antigen

Includes abstract.Includes bibliographical references.Peanut agglutinin (PNA), a carbohydrate binding protein, is able to recognise and bind a number of distinct carbohydrate structures that have been implicated in a number of disease pathologies in humans. In vitro studies of PNA have previously been shown to have some specificity for the Thomson Freidenriech antigen (T-antigen), found on malignant human cells, and this specificity has made PNA an important target for protein engineering experiments aimed at improving its specificity and affinity. A number of tumour cells are characterised by altered states and patterns of glycosylation on cell surfaces and suitably engineered lectins may be able to recognise tumour specific carbohydrate structures. This study was aimed at carrying out the biophysical characterisation of a set of PNA mutants which showed apparent improvement in specificity for the T-Antigen. Previous studies have aimed to engineer this lectin in order to direct its recognition properties towards the T-antigen and away from lactose, the preliminary binding affinities of these mutants being determined using Surface Plasmon Resonance (SPR). Here a set of PNA mutants were characterised, proteins expressed and purified to determine binding activities to the T-antigen, N-Acetyl-Dlactosamine (LacNAc) and lactose through the use of Protein Micro Array technology as well as Enzyme linked immunosorbant assays (ELISA)

Novel Analytical Methods in Food Analysis

This reprint provides information on the novel analytical methods used to address challenges occurring at academic, regulatory, and commercial level. All topics covered include information on the basic principles, procedures, advantages, limitations, and applications. Integration of biological reagents, (nano)materials, technologies, and physical principles (spectroscopy and spectrometry) are discussed. This reprint is ideal for professionals of the food industry, regulatory bodies, as well as researchers

The red cell storage lesion and therapeutic blood transfusion in the critically ill patient

1.1 BACKGROUND Anaemia is a common finding in critically ill patients. Currently, the transfusion of stored blood is the only treatment available to most patients. Despite this reliance on blood transfusion there is a marked lack of data about both the efficacy of red cell transfusion products and the clinical situations in which they are likely to be effective. It has recently been suggested that red blood cell (RBC) transfusions may have detrimental effects in critically ill patients and that these effects may be related to the transfusion of stored RBCs in particular. It is well recognised that RBCs undergo many metabolic and structural changes during refrigerated storage, these changes are termed the red cell storage lesion. The clinical implications of the red cell storage lesion are not known.1.2 AIM To assess the implications of the red cell storage lesion of the current UK RBC product, namely leucodepleted RBCs stored in saline-adenine-glucose-mannitol additive solution, using a combination of in vitro and in vivo studies.1.3 Methods 1. The quality of the current RBC product was assessed using in-vitro assays of RBC oxygenation/de-oxygenation, namely P50 and the 2,3 diphosphoglycerate concentration, and RBC deformability. || 2. Radiolabel studies were performed to determine the 24 and 48-hour recovery of stored allogeneic blood in critically ill patients. || 3. The in-vivo regeneration of red cell 2,3 diphophoglycerate (2,3 DPG) in stored blood transfused to critically ill patients was investigated. || 4. Antigenic differences between donor and recipient were used to track allogeneic RBCs following therapeutic transfusions to determine RBC survival using a nonradioisotopic technique.1.4 RESULTS 1. In-vitro tests showed that current collection processing and storage procedures: (a) Result in a very rapid reduction in red cell 2,3 DPG concentration. Approximately 50% of 2,3 DPG had been lost by day 2 of storage and it was barely detectable by day 14. The in-vitro Ps0 also decreased rapidly during storage; the time-frame of the decrease matched that of the decrease in 2,3 DPG. || (b) Result in a slight reduction in red cell deformability. 2. The current red cell product, stored for between 10 to 29 days, had a mean 24-hour recovery of 91% in critically ill patients. || 3. Following transfusion to critically ill patients stored blood rapidly regenerated 2,3 DPG. || 4. Red cell antigens were used to track allogeneic red cells for up to 12 weeks post¬ transfusion. The estimated median red cell lifespan was 104 days (range 86 to 124 days).1.5 CONCLUSIONS Current red cell storage methods fail to maintain red cell 2,3 DPG and result in a loss of red cell deformability. Although 2,3 DPG regeneration was found to occur rapidly it still took 24 to 72 hours for levels to approach normal; whether or not this is clinically significant is not known.The current UK red blood cell product has good short-term and long-term survival characteristics following therapeutic transfusion

Biofouling of dental handpieces

Dental handpieces (HP’s) are used during semi-critical and critical dental procedures that imply the HP must be sterile at the point of use. The aim of this study was to undertake a quantitative and qualitative analysis of dental HP contamination to inform the development of HP cleaning. Preliminary validation work on protein desorbtion methods and protein detection assays resulted in boiling in 1% sodium dodecylsulphate (SDS) and the o-phthaldialedhyde (OPA) assay (sensitivity 5 μg/ml) selected for further use in this study. A quantitative and qualitative analysis of HP microbial and protein contamination was then undertaken. Before decontamination, bacteria were isolated from high speed HP’s (n=40) (median 200 cfu, range 0-1.9x104 CFU/instrument), low speed HP’s(n-40) (median 400 cfu, range 0-1x104 CFU/instrument) and surgical HP’s (n=20) (median 1x103, range 0-3.7x104 CFU/instrument). A range of oral bacteria were identified in addition to Staphylococcus aureus and Propionibacterium acnes. Protein was detected from high speed HP’s (median 1.3, range 0- 210g), low speed HP’s (median 15.41 μg, range 0 - 448 μg) and surgical HP’s (median 350 μg, range 127.5– 1,936 μg) before decontamination. Serum albumin and salivary mucin were identified on surgical HP’s before decontamination. Calcium based deposits and contaminants trapped in lubricating oil were also detected using scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDX). The efficacy of detergents and a HP cleaning solution at cleaning HP contaminants was assessed in vitro with a standard test soil and disruption of biofilms with a range of cleaning efficacies noted from each cleaning solution tested. Alkaline detergents caused a significant biomass disruption of P. acnes biofilms compared to ROH2O alone. HP cleaning solution resulted in fixation of the biofilm and blood to the surface. The efficacy of novel HP cleaning machines was also assessed using a test soil based on the data generated in this study. Efficacy varied between devices tested with one demonstrating efficient protein removal in all but 1 HP location. The data presented describes a quantitative and qualitative assessment of common contaminants of HP’s, mainly bacteria, salivary mucin and serum albumin. In-vivo biofouling levels of HP’s are several fold lower than standard test soil formulations and consideration should be given to use of HP test soil based on in-vivo data to validate HP cleaning processes. The data generated in this thesis should aid in designing dental HP test soils and cleaning regimens

Cell surface changes induced by vaccinia virus

Virus-specified antigens induced in the membranes of infected cells have been implicated as primary targets in the recognition and destruction of virus-infected cells by the immune system of mammalian hosts. Antigenic changes induced in the plasma membranes of vaccinia-infected HeLa cells 2 h post-infection were detected by immunofluorescence, immune haemadsorption, and to a lesser extent by complement-fixation and complement-mediated cytolysis. Cytopathic effects (cell rounding) were seen prior to viral DM replication and also when this was blocked. Other changes occurred at the cell surface later in infection when turkey erythrocytes and concanavalin A were bound by cells. There was no evidence for an overall increase in mucopolysaccharide synthesis in the form of sialic acid. Quantitative spectrophotometric assays for haemadsorption, immune haemadsorption and concanavalin A binding were developed and the effect of varying growth or reaction conditions on the expression of vaccinia haemagglutinin at the surface of infected cells was investigated. Vaccinia cell surface haemagglutinin (VHA), detected by the binding of turkey erythrocytes, was synthesised around 10 h post-infection, even under conditions of low multiplicity of infection, and was closely coincident with the appearance of infectious virus. Of seven different cell lines tested, haemadsorption was greatest in HeLa cells and least in L-929 cells. Other red cell species were bound to infected cells to a lesser degree than turkey cells. Haemadsorption occurred within the physiological and alkaline pH ranges and at temperatures between 2

Development of a Cobinamide-Based Optical Sensor for Hydrogen Cyanide and Hydrogen Sulfide

In an occupational or military environment, a personal air-purifying respirator must be provided when breathing-air is contaminated by harmful dust, fumes, gases, aerosols, or vapors. Too often, a respirator user does not have enough information to know when to change his/her cartridge/canister and thus is potentially exposed to toxic gases. Currently, there is no definitive way to determine when respirators\u27 carbon beds have begun to fail. When an end-of-service-life indicator (ESLI) is incorporated into the carbon bed, it informs one, in real-time, when imminent breakthrough is occurring and to replace the cartridge/canister. ESLIs are a more reliable and safer way to determine respirator end-of-service-life. To date, there are no commercially available active ESLIs for inorganic gases.;The objective of this research is to develop an inexpensive, optical sensor for the detection of hydrogen cyanide (HCN) and hydrogen sulfide (H 2S) gas, which can be used to determine the end-of-service-life of a respirator carbon bed. The sensor relies on diffuse reflectance from a paper substrate fixed with cobinamide, a Vitamin B12 derivative. Cobinamide undergoes a metal-ligand binding interaction with HCN, whereas both a binding and reduction reaction may occur with exposure to H2S. Characteristic and different spectral shifts rapidly occurred after exposure to HCN and H 2S, implying a dual ESLI could be developed to simultaneously detect both gases. Upon increasing the relative humidity from 25 to 85%, the sensitivity was found to increase 7x for cobinamide-immobilized cellulose fiber filter paper and 50x for glass fiber filter paper upon exposure to 5.0 parts-per-million (ppm) HCN---the NIOSH recommended exposure limit for HCN. The cobinamide-immobilized paper sensor successfully detected low concentrations of HCN and H2S upon imminent breakthrough of respirator canisters and cartridges (respectively). The breakthrough curves of the cobinamide paper sensor correlated well with commercial electrochemical detectors, implying that cobinamide may be used to detect both gases at a certain location in the respirator carbon bed and inform the user to replace his or her cartridge/canister.;Additionally, a low-power, inexpensive 3-color (RGB) sensor was prototyped to actively monitor the total change in color of the cobinamide complex on paper upon HCN exposure. The photodiodes detected, in real-time, a rapid change in the red, green, and blue values of the cobinamide compound upon exposure to HCN at various concentrations and relative humidity levels. Total change in color from initial cobinamide on paper increased as a function of HCN concentration, where faster reaction kinetics were observed at higher relative humidity. Response times at all relative humidity levels were within 20 seconds for 5.0 ppm HCN exposure. The color sensor offers an economical and more quantitative approach to determining color change compared to current, subjective end-of-service-life indicators