Eppin: A Novel Protein Possessing Antimicrobial Properties Against Oral Pathogens

Background: Epididymal protease inhibitor (eppin) is a dual motif protein belonging to the whey acidic protein (WAP) family. Although expressed in numerous different tissues, to date, its functional characterisation is limited. It has been shown to exhibit antibacterial activity against Gram-negative bacteria (Escherichia coli) and antiprotease activity against some proteases of the serine protease family. We are interested in determining the role of eppin in innate immune defence. Objectives: This study aims to determine eppin's potential function in the innate immune response in the oral cavity by investigating the antimicrobial activity of eppin against relevant oral pathogens. Methods: Eppin was recombinantly expressed in E. coli cells and purified by immobilised metal affinity chromatography (IMAC). The antimicrobial effects of the protein were then assessed against two oral pathogens, Fusobacterium nucleatum and Candida albicans, using a double layer radial diffusion assay. Results: Eppin displayed antimicrobial activities against both oral pathogens tested and these activities were shown to be comparable to the well characterised antimicrobial peptide, LL-37. The antifungal effects of eppin were shown to be more potent than those of the human cathelicidin, LL-37. Conclusions: Eppin has been shown to possess both antibacterial and antifungal properties against oral pathogens, suggesting an important role for this protein in the innate immune response in the oral cavity. This study furthers our knowledge of the physiological role exerted by eppin and its possible role in the modulation of chronic diseases such as periodontitis and oral candidiasis.

Identification of human neutrophil defensins (HNPs1-3) in biopsies of squamous cell carcinomas of the human tongue

It has previously been reported that the a-defensins, found in the granules of polymorphonuclear leukocytes (neutrophils/ PMNs), are cytolytic for human tumour cells in vitro. Objective: To identify and quantify the a- defensins, HNP-1, HNP-2 and HNP-3 in healthy and tumour tissue from patients with oral squamous cell carcinoma using HPLC, mass spectrometry and amino acid sequencing. Methods: All patients (n=5) were diagnosed with oral squamous cell carcinoma of the tongue.Biopsy tissue from the site of the tumour (n=5) and a non-affected region of the tongue (n=5) was snap frozen and subsequently stored at -70 ºC until analysed. Peptides were extracted from the 10 tissue biopsies using acidified ethanol. Peptide extracts were separated by reverse-phase HPLC . All tumour and control tissue samples were individually analysed under identical conditions with a flow rate of l ml/min, ambient column temperature and absorbance detection at 214 and 280 nm. Fractions (1ml) were collected automatically. HPLC fractions were analysed by MALDI-MS using a linear time-of-flight Voyager DE-mass spectrometer (PerSeptive Biosystems, UK). Using this system the detection limit was 10 fmol. Peptides with molecular masses corresponding to those reported for the a-defensins were deemed of interest and were further subject to complete structural analysis by automated Edman degradation using an Applied Biosystems 491 Procise microsequencer. Results: MALDI-MS revealed a triad of peptides of molecular masses 3442 Da, 3371 Da and 3486 Da in both healthy and tumour tissue. Full length sequence data were obtained for the three a-defensins, unequivocally identifying their presence in both tumour and healthy tissue. Analysis of the MALDI-MS and sequence data indicated that the a-defensins were overexpressed (up to 12 fold) in tumour tissue. Conclusion: This study demonstrates the feasibility of screening tumour tissue for novel peptides/proteins using HPLC and MALDI-MS.The role of a-defensins in oral squamous cell carcinoma of the tongue requires further investigation.

Justice, Truth and Oral History: Legislating the Past 'From Below' in Northern Ireland

Drawing on the ‘from below’ perspective which has emerged in transitional justice scholarship and practice
over the past two decades, this article critically examines the dealing with the past debate in Northern
Ireland. The paper begins by offering an outline of the from below perspective in the context of post-conflict
or post-authoritarian societies which are struggling to come to terms with past violence and human rights
abuses. Having provided some of the legal and political background to the most recent efforts to deal with
the past in Northern Ireland, it then critically examines the relevant past-related provisions of the Stormont
House Agreement, namely the institutions which are designed to facilitate ‘justice’, truth recovery and the
establishment of an Oral History Archive. Drawing from the political science and social movement
literature on lobbying and the ways in which interests groups may seek to influence policy, the paper then
explores the efforts of the authors and others to contribute to the broader public debate, including through
drafting and circulating a ‘Model Bill’ on dealing with the past (reproduced elsewhere in this issue) as a
counterweight to the legislation which is required from the British government to implement the Stormont
House Agreement. The authors argue that the combination of technical capacity, grass-roots
credibility and ‘international-savvy’ local solutions offers a framework for praxis from below in other
contexts where activists are struggling to extend ownership of transitional justice beyond political elites.
Keywords: transitional justice; from below; dealing with the past; legislation; truth
recovery; prosecutions; oral history

Influence of speciation in the bioavailability of cadmium to an isopod

Devido às actividades antropogénicas várias substâncias químicas têm sido introduzidas no meio ambiente em concentrações que de outro modo não ocorreriam de forma tão elevada naturalmente. Assim, o conhecimento acerca das características de um químico, tais como, o potencial para se acumular em diferentes níveis tróficos, a sua mobilidade dentro do ecossistema, a toxicidade específica e a bioacumulação, é fundamental para compreender os seus efeitos nos ecossistemas. Esta tese investiga a influência de especiação, na biodisponibilidade do cádmio (Cd) para o isópode Porcellio dilatatus, incluindo os efeitos de especiação do metal: (i) na assimilação do Cd, (ii) no modo como o Cd se distribui internamente no organismo, e (iii) como a sobrevivência e a reprodução são afectadas em isópodes terrestres. Num primeiro ensaio laboratorial avaliou-se a importância da transferência trófica na assimilação do Cd em P. dilatatus. Para tal analisou-se a eficiência de assimilação (EA) do Cd em isópodes, adicionado superficialmente ao alimento (alface) na forma de Cd(NO3)2 e contaminando o meio de crescimento da alface. A hipótese era de que a alface contaminada biologicamente através do cultivo em meio hidropónico contaminado teria uma maior proporção de complexos com proteína ou conjugado na forma de Cd (ex. Cd cisteína). A EA de Cd foi maior entre os isópodes que foram alimentados com o sal (71%, SE = 7%), do que entre os isópodes que se alimentaram de alface contaminada biologicamente (52%, SE = 5%), demonstrando-se assim num teste laboratorial que é provável que a especiação do Cd influencie a taxa de assimilação e acumulação do Cd. Na experiência alimentar que se seguiu, estudou-se em detalhe a especiação do metal comparando as EA do Cd conjugado com cisteína (Cd(Cys)2) e na forma de Cd(NO3)2, com os quais se contaminou gelatina com alface. A utilização de Cd-cisteína, proporcionou uma forma experimental para explorar a biodisponibilidade do Cd complexado dentro do tecido biológico. Como esperado, a EA de Cd em isópodes alimentados com nitrato de Cd (64%, SE = 5%) foi maior do que no caso de isópodes alimentados com o conjugado de cisteína (20%, SE = 3%). De seguida estudou-se a distribuição subcelular das espécies de Cd assimilado através de um processo de fraccionamento. Supunha-se que as diferenças de especiação de Cd reflectiria diferentes estratégias de compartimentalização, com consequências ao nível da detoxificação, armazenamento celular e distribuição subcelular do metal. O “sequestro” na forma de metal biologicamente detoxificado (BDM = proteínas estáveis ao calor - HSP e grânulos ricos em metal - RMG) foi maior nos isópodes alimentados com Cd(NO3)2, sugerindo que são mais eficientes na detoxificação de Cd (22%) do que quando alimentados com Cd(Cys)2 (15%). Foi também demonstrado que os isópodes alimentados com Cd(Cys)2 possuíam níveis de armazenamento de Cd superior nas fracções sensíveis ao metal (MSF = organelos e proteínas desnaturadas pelo calor - HDP) consideradas fracções potencialmente vulneráveis e afectando os isópodes em termos de toxicidade. As diferentes distribuições internas que se seguiram à assimilação e detoxificação das diferentes espécies de Cd foram finalmente avaliadas em termos da sobrevivência e reprodução dos isópodes. O tratamento com Cd(Cys)2 teve maior mortalidade, provavelmente devido à maior disponibilidade de Cd ingerido com implicações ao nível dos processos fisiológicos. Os isópodes alimentados com Cd(NO3)2 armazenaram o Cd nos MRG, como estratégia de detoxificação, sendo mais eficientes a detoxificar o Cd ainda que aumentando a concentração total do metal que se tornou menos tóxico para o isópode. Desta forma, o Cd nos grânulos não estava disponível para os processos fisiológicos e deixou de ser tóxico. Isso poderia estar relacionado com a resistência e tolerância aos metais devido à capacidade dos isópodes compartimentalizarem o Cd no hepatopâncreas, que actua como um mecanismo de detoxificação e contribui para a tolerância a altos níveis de cádmio. Em termos de parâmetros reprodutivos, observou-se uma redução de gestações e duração da gestação na presença de ambas as espécies de metal, mas no caso do Cd(Cys)2 as gravidezes não se concluíram. O número de jovens produzido por fêmeas alimentadas com Cd(NO3)2 foi menor do que no controlo, mas os pesos dos juvenis foram superiores. Finalmente sugere-se assim que esta abordagem seja considerada em estudos do movimento trófico de metais nas cadeias alimentares dado que se espera que a especiação de metais implique diferentes fluxos, dentro de uma dada cadeia trófica.

Characterizing Human Cysticercosis In Portugal

Abstract text Introduction: Cysticercosis results from the ingestion Taenia solium eggs directly by faecal-oral route or contaminated food or water. While, still considered a leading cause of acquired epilepsy in developed countries, this zoonosis has been controlled or eradicated in industrialized countries due to significant improvements in sanitation, pig rearing and slaughterhouse control systems. Objectives: the health burden of human cysticercosis in Portugal. Material and Metodes: We developed a retrospective study on human neurocysticercosis (NCC) hospitalisations based on the national database resulting from National Health Service (NHS) hospital episodes except those of Madeira and Azores Islands. Results: Between 2006 and 2013 there were 357 hospitalized NCC cases in Portugal. Annual frequency of cases between 2006-2013 kept stable (mean 45). NCC was most frequent in those aged 25-34 years (59; 16,5%) and those >75 years (65; 18,2%). Overall, mean age was 47,3 years (median age 45, standard deviation 41,1, mode 28) and 176 cases were in males (49,3%); no significant differences were observed between age and gender (t-student, p>0,05). In Norte Region cases tended to be older than in Lisboa and Vale do Tejo Region. Conclusions: The Directorate-General of Health established the National Observatory of Cysticercosis and Teniiasis which will define criteria for NCC cases monitoring and surveillance (hospitalized and non-hospitalized cases).

Reduction of trimethylamine N-oxide to trimethylamine by the human gut microbiota: supporting evidence for ‘metabolic retroversion’

Dietary sources of methylamines such as choline, trimethylamine (TMA), trimethylamine N-oxide (TMAO), phosphatidylcholine (PC) and carnitine are present in a number of foodstuffs, including meat, fish, nuts and eggs. It is recognized that the gut microbiota is able to convert choline to TMA in a fermentation-like process. Similarly, PC and carnitine are converted to TMA by the gut microbiota. It has been suggested that TMAO is subject to ‘metabolic retroversion’ in the gut (i.e. it is reduced to TMA by the gut microbiota, with this TMA being oxidized to produce TMAO in the liver). Sixty-six strains of human faecal and caecal bacteria were screened on solid and liquid media for their ability to utilize trimethylamine N-oxide (TMAO), with metabolites in spent media profiled by Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy. Enterobacteriaceae produced mostly TMA from TMAO, with caecal/small intestinal isolates of Escherichia coli producing more TMA than their faecal counterparts. Lactic acid bacteria (enterococci, streptococci, bifidobacteria) produced increased amounts of lactate when grown in the presence of TMAO, but did not produce large amounts of TMA from TMAO. The presence of TMAO in media increased the growth rate of Enterobacteriaceae; while it did not affect the growth rate of lactic acid bacteria, TMAO increased the biomass of these bacteria. The positive influence of TMAO on Enterobacteriaceae was confirmed in anaerobic, stirred, pH-controlled batch culture fermentation systems inoculated with human faeces, where this was the only bacterial population whose growth was significantly stimulated by the presence of TMAO in the medium. We hypothesize that dietary TMAO is used as an alternative electron acceptor by the gut microbiota in the small intestine/proximal colon, and contributes to microbial population dynamics upon its utilization and retroversion to TMA, prior to absorption and secondary conversion to TMAO by hepatic flavin-containing monooxygenases. Our findings support the idea that oral TMAO supplementation is a physiologically-stable microbiota-mediated strategy to deliver TMA at the gut barrier.

Precious transition metals: the importance of Zn2+, Mn2+ and Cu2+ in the human pathogen Enterococcus faecalis

Dissertation presented to obtain the Ph.D degree in Biology

Understanding Human Carboxylesterase 2

Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences, Biotechnology

Exploring the bioavailability of (poly)phenols from berries and their potential activities in humans

(Poly)phenols are the most widely distributed secondary metabolites, in plants, and, therefore, are regular constituents of human food products. The regular ingestion of (poly)phenol-containing foods has been associated with a reduced risk of acquiring chronic diseases and many studies are currently trying to corroborate this theory. However, the precise contribution of (poly)phenols to disease prevention is still unknown.(...)

Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions.

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 microgram of VLP given at weekly intervals to anesthetized mice induced high (>10(4)) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-microgram VLP systemic priming followed by two 5-microgram VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Rapid high-performance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry.

Furosemide (FD: Lasix) is a loop diuretic which strongly increases both urine flow and electrolyte urinary excretion. Healthy volunteers were administered 40 mg orally (dissolved in water) and concentrations of FD were determined in serum and urine for up to 6 h for eight subjects, who absorbed water at a rate of 400 ml/h. Quantification was performed by HPLC with fluorescence detection (excitation at 233 nm, emission at 389 nm) with a limit of detection of 5 ng/ml for a 300-microliters sample. The elution of FD was completed within 4 min using a gradient of acetonitrile concentration rising from 30 to 50% in 0.08 M phosphoric acid. The delay to the peak serum concentration ranged from 60 to 120 min. FD was still easily measurable in the sera from all subjects 6 h after administration. In urine, the excretion rates reached their maximum between 1 and 3 h. The total amount of FD excreted in the urine averaged 11.2 mg (range 7.6-14.0 mg), with a mean urine volume of 3024 ml (range 2620-3596 ml). Moreover, the urine density was lower than 1.010 (recommended as an upper limit in doping analysis to screen diuretics) only for 2 h. An additional volunteer was administered 40 mg of FD and his urine was collected over a longer period. FD was still detectable 48 h after intake. Gas chromatography-mass spectrometry with different types of ionization was used to confirm the occurrence of FD after permethylation of the extract. Negative-ion chemical ionization, with ammonia as reactant gas, was found to be the most sensitive method of detection.

The effects of R(+)-lipoic acid supplementation on regulation of human skeletal muscle pyruvate dehydrogenase

This thesis investigated whole body glucose disposal and the adaptive changes in skeletal muscle carbohydrate metabolism following 28 d of supplementation with 1000 mg R(+)-lipoic acid in young sedentary males (age, 22.1 ± 0.67 yr, body mass, 78.7 ± 10.3 kg, n=9). In certain individuals, lipoic acid decreased the 180-min area under the glucose concentration and insulin concentration curve during an oral glucose tolerance test (OGTT) (n=4). In the same individuals, lipoic acid supplementation decreased pyruvate dehydrogenase kinase activity (PDK) (0.09 ± 0.024 min"^ vs. 0.137 ± 0.023 min'\ n=4). The fasting levels of the activated form of pyruvate dehydrogenase (PDHa) were decreased following lipoic acid (0.42 ± 0.13 mmol-min'kg'^ vs. 0.82 ± 0.32 mmolrnin'^kg"\ n=4), yet increased to a greater extent during the OGTT (1.21 ± 0.34 mmol-min'kg"' vs. 0.81 ±0.13 mmolmin"'kg'\ n=4) following hpoic acid supplementation. No changes were demonstrated in the remaining subjects (n=5). It was concluded that improved glucose clearance during an OGTT following lipoic acid supplementation is assisted by increased muscle glucose oxidation through increased PDHa activation and decreased PDK activity in certain individuals.

Who is to protect whom from what? The human security dilemmas of Iraq, North Korea and Darfur

Brian Job, Professor, Department of Political Science, University of British Columbia, Director, Center of International Relations. Présentation dans le cadre du cycle de conférences organisé par le CRDP intitulé « Le droit à la sécurité ... la sécurité par le droit ».

Moderate Champagne consumption promotes an acute improvement in acute endothelial-independent vascular function in healthy human volunteers

Epidemiological studies have suggested an inverse correlation between red wine consumption and the incidence of CVD. However, Champagne wine has not been fully investigated for its cardioprotective potential. In order to assess whether acute and moderate Champagne wine consumption is capable of modulating vascular function, we performed a randomised, placebo-controlled, cross-over intervention trial. We show that consumption of Champagne wine, but not a control matched for alcohol, carbohydrate and fruit-derived acid content, induced an acute change in endothelium-independent vasodilatation at 4 and 8 h post-consumption. Although both Champagne wine and the control also induced an increase in endothelium-dependent vascular reactivity at 4 h, there was no significant difference between the vascular effects induced by Champagne or the control at any time point. These effects were accompanied by an acute decrease in the concentration of matrix metalloproteinase (MMP-9), a significant decrease in plasma levels of oxidising species and an increase in urinary excretion of a number of phenolic metabolites. In particular, the mean total excretion of hippuric acid, protocatechuic acid and isoferulic acid were all significantly greater following the Champagne wine intervention compared with the control intervention. Our data suggest that a daily moderate consumption of Champagne wine may improve vascular performance via the delivery of phenolic constituents capable of improving NO bioavailability and reducing matrix metalloproteinase activity.