Intravenous lipid emulsions are used ubiquitously through the medical field as a source of parenteral nutrition. Development of new formulations requires an understanding of the metabolism of the product. Current methods of rate determination and of metabolism analysis have several drawbacks. Radioactive labelled assays have lower biological relevance, are time consuming due to separation steps and require long substrate preparation. Existing fluorescence assays based around triglyceride hydrolysis are impractical in emulsion systems due to high signal-noise ratio as well as the use of non-specific fluorescent dyes. Colorimetric methods such as the non-esterified fatty acids (NEFA) assay is expensive, requires multiple steps and specialised machinery. Due to the limitations of these techniques we developed a novel fluorescent assay using a lipoprotein lipase specific substrate incorporated into lipid emulsions. The lipoprotein lipase substrate, EnzChek® Lipase Substrate, green fluorescent, 505/515, is based on a triglyceride structure with a fluorescent dye at the Sn1 position and a dark quencher at the Sn2 position. LPL cleaves preferentially at the Sn1 position of triglycerides, which separates the dye from the quencher creating a fluorescent signal. The signal can then be detected using a fluorescent plate reader. Both lipid emulsion particles and EnzChek are substrates for LPL, so the hydrolysis of EnzChek is analogous for native emulsions particles. Over time, in the presence of LPL, fluorescent signal increases as more EnzChek is hydrolysed in tandem with emulsion particles. We have designed a range of emulsions with varied oil and surfactant composition. Using the EnzChek emulsion assay detailed above we are able to follow the rate of metabolism in real-time. This assay has been tested and found to be robust and reproducible. It is able to investigate differences in metabolism between Soybean oil, medium-chain triglyceride and fish oil emulsions. As well as changes in surfactant type and concentration.

University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

This work had the objective to achieve the knowledge of the dosimetric quantities related to chest and abdomen computed tomography (CT) examinations of pediatric patients, in Belo Horizonte city. The reason of this work is based on the fact that the probability of health detriment in children, which it may be caused by radiation, is higher than in adults. Besides, although in many countries the knowledge and control of patient doses is a normal procedure, this safety culture does not exist in Brazil. Another objective of this work was to compare the dosimetric quantity values with the Diagnostic Reference Levels (DRLs); when it was needed, an optimization process was applied and the quality of the diagnostic image obtained with the optimized technical parameters was analyzed. This study was carried out in five hospitals, where the weighted air kerma index (Cw), the volumetric air kerma index (Cvol), the air kerma length product (PKL,CT), the Effective Dose (E) and the Normalized Effective Dose (En) were determined; three methods were adopted for measurements: the ionization chamber inside a chest pediatric phantom, radiochromic films and the CT-EXPO software. The optimization process was applied to a single hospital through variations in the current (mA) and voltage (kV) of the x-ray tube for the protocols used for abdomen CT examinations. The analysis of the quality of the diagnostic image was done by Normal Distribution and ROC analysis; spatial resolution analysis was used through MTF determination and the noise level was judged in terms quantitative and qualitative. Results of the dosimetric quantities showed that they significantly differed between single-slice and multi-slice tomography units, but their values were always below the recommended DRLs. The optimized values of the dosimetric quantities obtained after the optimization process showed that it was possible to reduce the radiation exposure of pediatric patient without losing the image quality, despite the low dose values found. Dose values very lower than DRLs might induce to an unjustified practice of increasing the dose for improving the image diagnostic quality; the results showed that if it was done there was not a relevant change in the quality. Based on the results of this work, it was possible to state that the dosimetric quantity values in the studied pediatric tomographic procedures were lower than DRLs, but there was still room of optimization for patient protection and to maintain an adequate image diagnostic quality in computed tomography. / Este trabalho teve como um dos objetivos o conhecimento das grandezas dosimétricas em exames de tomografia computadorizada pediátrica do tórax e do abdômen na cidade de Belo Horizonte. A justificativa para este objetivo está no fato da probabilidade de efeitos danosos à saúde, causados pela radiação, ser maior em crianças que em adultos e, diferentemente de outros países onde o conhecimento e controle dos valores das doses é fato normal, esta cultura de segurança ainda não existe no Brasil. Outro objetivo deste trabalho foi confrontar os valores das grandezas dosimétricas com os Níveis de Referência em Diagnóstico (NRD), aplicando, quando necessário, o processo de otimização e fazendo uma análise da qualidade diagnóstica das imagens obtidas com os parâmetros técnicos otimizados. O estudo foi realizado em cinco hospitais, sendo as grandezas Índice Ponderado de Kerma no Ar (Cw), Índice Volumétrico de Kerma no Ar (Cvol), Produto Kerma-Comprimento (PKL,CT), Dose Efetiva (E) e Dose Efetiva Normalizada (En) obtidas por meio de três métodos distintos: com uma câmara de ionização e um objeto simulador de tronco pediátrico, com filmes radiocrômicos e com o software CT-EXPO. O processo de otimização, aplicado em apenas um hospital, consistiu de sugestões para variações na corrente (mA) e da tensão (kV) do tubo no protocolos para a obtenção de exames de tomografia do abdômen. A metodologia para a análise da qualidade diagnóstica foi feita a partir da Distribuição Normal e Análise ROC, ambas de cunho inédito, pela análise da resolução espacial através da obtenção da MTF e julgamento quantitativo e qualitativo do ruído. Os resultados obtidos para as grandezas dosimétricas mostraram uma diferença significativa entre tomógrafos singleslice e multi-slice, mas com valores abaixo do NRD recomendado. Os valores obtidos com o processo de otimização indicaram que é possível reduzir a exposição do paciente pediátrico à radiação, mesmo quando as doses são consideradas baixas, não acarretando na perda em qualidade diagnóstica. A obtenção de doses com valores muito inferiores aos NRD pode induzir ao aumento da exposição à radiação, na tentativa de ganho na qualidade diagnóstica da imagem; porém, os resultados das análises mostraram que não houve ganho significativo na qualidade da imagem, tornando o procedimento uma prática não justificada. Com base nos resultados, foi possível afirmar que as grandezas dosimétricas envolvidas em procedimentos tomográficos pediátricos estão abaixo dos NRD recomendados, porém que há espaço para o processo de otimização, preservando ainda mais a integridade física do paciente e mantendo a adequada qualidade diagnóstica da imagem tomográfica.

A esquistossomose mansoni é uma das principais doenças parasitárias no mundo e é conhecida por ser endêmica em aproximadamente 54 países nos continentes americano e africano. No Brasil o número de pessoas infectadas é estimado em 7 milhões. Em suas diversas formas clínicas, a esquistossomose mansoni assemelha-se a muitas outras doenças; portanto, essa inespecificidade e a variação nos sintomas dificultam o seu diagnóstico apenas pelo exame clínico do paciente. A doença é diagnosticada por métodos parasitológicos de fezes. A técnica de Kato/Katz é o procedimento internacionalmente recomendado pela Organização Mundial da Saúde (OMS) para o diagnóstico do Schistosoma mansoni, porque associa baixo custo à facilidade de execução. Atualmente, estão disponíveis Kits comerciais, a fim de minimizar as dificuldades de operacionalização e melhorar a sensibilidade do diagnóstico parasitológico, principalmente em áreas de baixa endemicidade. Este estudo avaliou comparativamente a efetividade de quatro técnicas parasitológicas de fezes em diagnosticar casos de esquistossomose mansoni. As amostras fecais foram examinadas pelas técnicas Kato/Katz, Sedimentação espontânea e pelos Kits Midi Parasep® e Paratest®. Foi coletada 1 amostra de cada indivíduo e para cada amostra foram preparadas 2 lâminas das quatro metodologias. Um total de 442 exames coproparasitológicos foram realizados em migrantes de áreas endêmicas para a esquistossomose, de três municípios sob jurisdição da Superintendência Regional de Saúde de Alfenas (SRS). A taxa de positividade da esquistossomose mansoni detectada pela técnica Kato/Katz foi de 1,8%. Pela técnica de Sedimentação espontânea e pelo Kit Paratest® foi detectada uma positividade de 0,45%. Pelo Kit Midi Parasep® a positividade foi de 0,23%. A menor efetividade da técnica de sedimentação espontânea e dos Kits Midi Parasep® e Paratest® pode ser comprovada pelo baixo grau de concordância diagnóstica com o exame referência (técnica Kato/Katz). As técnicas Kato/Katz e sedimentação espontânea apresentam os menores custos para realizar o exame de fezes, cerca de R$ 0,82/exame e R$ 0,48/exame, respectivamente. Entretanto, são as que apresentam o maior grau de exposição do analista à amostra biológica. Os Kits Midi Parasep® e Paratest® apresentam o menor grau de exposição à amostra fecal, mas são relativamente mais caros para realização do exame de fezes, cerca de R$ 3,10/exame e R$ 1,65/exame, respectivamente. Com relação as parasitoses intestinais, pelo “Padrão Ouro”, foram encontrados 97 (21,9%) indivíduos parasitados. As taxas de positividade das parasitoses intestinais detectadas pelas técnicas Kato/Katz, sedimentação espontânea, Midi Parasep® e Paratest®, foram respectivamente, 5,2%, 19,5%, 17,6% e 18,3%. Os resultados mostraram que a técnica de Kato/Katz apresentou melhor desempenho dos quatro testes no diagnóstico da esquistossomose mansoni. Assim, a vigilância epidemiológica através do exame censitário de todos os migrantes pela técnica Kato/Katz, deve ser implementada com o propósito de impedir o estabelecimento da transmissão da esquistossomose. / Schistosomiasis mansoni is a major parasitic disease in the world and is known to be endemic in about 54 countries in the American and African continents. In Brazil the number of infected is estimated at 7 million. In different clinical forms, schistosomiasis mansoni resembles many other diseases. So this lack of specificity and symptoms variety hinder its diagnosis only by clinical examination. The disease is diagnosed using stool examinations. The Kato/Katz technique is the procedure recommended by the World Health Organization (WHO) for the diagnosis of Schistosoma mansoni, because it associates low cost and easy execution. Currently, there are commercial Kits available, in order to minimize the operational difficulties and improve the sensitivity of parasitological diagnosis, especially in areas of low endemicity. This study comparatively evaluated the effectivity of four stool parasitological techniques in diagnosing cases of schistosomiasis. Stool samples were examined by the Kato/katz and spontaneous sedimentation techniques, Midi Parasep® and Paratest® kits. One sample was collected from each individual and for each sample were prepared two slides of the four methods. A total of 442 coproparasitological exams were accomplished with migrants from endemic areas for schistosomiasis, of three cities under Health Superintendence Office of Alfenas (HSO) jurisdiction. The positivity rate of Kato/Katz technique for schistosomiasis was 1,8%. The positivity rate schistosomiasis by the spontaneous sedimentation technique and Paratest® was of 0,45%. By Midi Parasep® Kit the positivity rate was of 0,23%. The lower effectivity of spontaneous sedimentation and Midi Parasep® and Paratest® can be demonstrated by low agreement diagnostic with the reference examination (Kato/Katz). Kato/Katz and spontaneous sedimentation techniques showed low cost for the exam stool, about R$ 0,82 per exam and R$ 0,48 per exam, respectively. However, are those with greatest degree of exposure of the expert to biological samples. Midi Parasep® and Paratest® Kits showed the lowest degree of exposure to fecal sample, but are relatively more expensive for perform the fecal exam, about R$ 3,10 per exam and R$ 1,65 per exam, respectively. Regarding intestinal parasites, by the "gold standard" were found 97 (21,9%) individuals parasitized. The positivity rates of intestinal parasitosis detected by the Kato/Katz, spontaneous sedimentation, Midi Parasep® and Paratest®, were respectively, 5,2%, 19,5%, 17,6% e 18,3%. The results showed that the Kato/Katz technique presented best of performance diagnosising of schistosomiasis mansoni. Thus, epidemiological surveillance should implement the Kato/Katz technique in order to prevent the establishment of transmission of schistosomiasis. / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq

The modular polyketide synthase responsible for biosynthesis of the antibiotic mupirocin occupies 75 kb of Pseudomonas fluorescens NCIMB 10586, while a hybrid of PKS/NRPS is responsible for biosynthesis of the antibiotic thiomarinol located on a 97 kb plasmid pTML1 in Pseudoalteromonas spp SANK 73390. Biosynthesis of the acyl side chains in mupirocin and thiomarinol are thought to be either through esterification of the fully synthesised fatty acid (C\(_9\) or C\(_8\)) or through extension of the PK derived ester starter unit which is predicted to be carried out on MmpB and TmpB. mupU/O/V/C/F and macpE are proposed to be sufficient for the conversion of pseudomonic acid B to pseudomonic acid A. Mupirocin is regulated via quorum sensing, while regulation of thiomarinol was not identified. Production of thiomarinol was determined to occur after 8 hours of growth, while acidic conditions and use of acetone with ethyl acetate improved the extraction. TmlU, the thiomarinol amide ligase, did not complement a mupU mutant in mupirocin, and was found to block the biosynthesis of 9-hydroxynonanoic acid, causing truncation of 9-HN. This suggests that MupU, prevents MmpB from being an iterative PKS. KS-B2/ACP-B2 was shown to be involved in the removal of C8-OH from thiomarinol. Genetically manipulated mupU increased the production of mupirocin to 3 to 4 fold without abolishing PA-B production. Fused mupU-macpE complemented the NCIMB10586ΔmupUΔmacpE double mutant. However, insertion of this fusion into MmpB blocked the biosynthesis of mupirocin, while insertion after MmpA did not changed the pathway. Attempts to mobilise pTML1 revealed that a hybrid plasmid of RK2-R6K γ-ori was integrated into pTML1 but recovery of this cointegrate has not yet been recovered in E. coli.

Haemorrhage remains a leading cause of death in civilian and military environments. Recent research into emergency treatments for severe haemorrhaging injuries has resulted in production of a number of advanced haemostatic products. In certain scenarios, concomitant release of toxic materials may exacerbate trauma and ultimately reduce survival from such injuries. In particular, systemic absorption of the nerve agent VX (S-[2-(diisopropylamino)ethyl]-O-ethyl methylphosphonothioate) via wounds may rapidly cause muscle paralysis and death. This research explored the hypothesis that a haemostatic product could perform an additional function as a wound decontaminant. […] In summary, this project has successfully demonstrated that haemostatic products, based on an adsorptive mechanism of action, offer a highly effective means of countering the rapidly fatal effects of the chemical warfare agent VX present in wounded skin tissue. Further development of the concept of a haemostatic decontaminant has life-saving implications.

An analysis of over 34,000 free-text messages assigned by pharmacists to prescription orders over a 12-month period showed a sub-optimal exchange of information with the physician. Focus groups and observational research were conducted to provide a more in-depth understanding of the factors involved. The use of CPOE did not reduce opportunities for personal interaction. The capability to communicate electronically facilitated a non-interruptive workflow, beneficial for staff time and for limiting distractions. It also improved clinical documentation, which helped coordinate care of patients between members of the pharmacy team. However, the research identified several barriers to the effectiveness of communication via the CPOE system, including: the increased frequency of messages sent; poor display characteristics of the message; poor access to information to inform decision-making; one-way communication; and no assigned responsibility to respond. These factors need to be considered in the design of systems and supported by interprofessional training to optimise communication between the professionals.

There is a clear and well-established need for new antibiotics. The biosynthesis of the antibiotic mupirocin is an important model trans-AT polyketide synthase pathway. A key tailoring step is the removal of a hydroxyl from the intermediate pseudomonic acid B (PA-B), which generates active pseudomonic acid A (PA-A). This is proposed to occur on discrete proteins after release from enzyme MmpB. In this work, a systematic screen for genes required for this key step was developed, which implicated ten genes. On a multi-gene expression plasmid, these ten genes encoded all functions necessary for conversion. This re-identified seven genes from the existing proposed model. A novel requirement for MupM, MupN, and the DNA sequence but not protein function ofMupL was uncovered. This represents the first identification of all genes required for this surprisingly convoluted pathway. With the aim of re-engineering the location of these tailoring steps, domains from thiomarinol enzyme TmpB were inserted into MmpB. Low-level conversion to PA-A was achieved using thiomarinol tailoring enzymes. The TmpB KS0 domain was used to load the normally free-standing MacpE, and high-level conversion was achieved on the hybrid enzyme. This success opens the future path to further MmpB modifications to generate mupirocin derivatives.

Polyketide synthases (PKS) are enzyme complexes that synthesise many natural products of medicinal interest, notably a large number of antibiotics. The present work investigated the mupirocin biosynthesis system, comparing it with similar pathways such as thiomarinol and kalimantacin. The focus was on the structural modelling of the protein complexes involved in antibiotic synthesis, via molecular simulation and the analysis of structural and sequence data. Structural docking of acyl carrier proteins (ACP) cognate for an HMG-CoA synthase orthologue responsible for β-methylation (MupH) identified key residues involved in the recognitions specificity of the interacting partners, further supported by mutagenesis experiments, which thus allows prediction of β-methylation sites in PKS. Moreover, complementation and mutagenesis experiments performed on MupH homologs from kalimantacin and thiomarinol systems suggests specificity between the ACP:HCS proteins in the β-branching suggesting the possibility of engineering multiple specific β-branching modifications into the same pathway. Molecular dynamics simulations of ACPs from the mupirocin cluster revealed that the PKS ACPs form a cavity upon the attachment of the phosphopantetheine and acyl chains similar to what is seen in the fatty acid synthase ACPs and provide a better understanding of the structure function relationship in these small proteins. Molecular docking of the putative cognate substrate with the ketosynthase (KS) homo dimer of module 5 of the MmpA in the mupirocin pathway revealed a loop that may control specificity for the α-hydroxylated substrate and mutagenesis experiments support this proposition.