Development of a LC-MS method for analysis of thiol ratios as an indicator of oxidative stress

Reactive oxygen species are free radicals capable of damaging the cellular components in a process called oxidative stress. Among the different biomarkers that are used to determine level of oxidative stress is the ratio between reduced and oxidized thiols, such as glutathione and oxidized glutathione. The use of glutathione ratio as a biomarker of oxidative stress is possible because the thiols are responsible for reducing the oxidizing species in a process that oxidizes the thiols into their disulfides. Under normal conditions, the cells can regenerate the reduced thiols by the action of reductases, which keeps the ratio constant. However, under oxidative stress, the cell cannot regenerate the reduced thiols rapidly enough. This in turn increases the concentration of the disulfide, and the ratio decreases. The ratio can also be inadvertently altered during sample manipulation because thiols can autoxidize. Therefore, for their accurate determination, thiols should be derivatized prior to analysis. The existing protocols using liquid chromatography-mass spectrometry (LC-MS) for thiol analysis largely focus on urine or plasma analysis, and do not consider exposure to oxidation during sample handling, while the few studies on intracellular thiol concentrations employ derivatization after cell lysis. The main objective of this thesis was to develop a LC-MS method to accurately measure individual thiols and disulfides, and their ratios in Jurkat cells. To achieve this goal, the selectivity and efficiency of two different derivatizing agents that are able to permeate the cell membrane were first compared in detail: N-ethyl maleimide (NEM) and N-phenyl ethyl maleimide (NPEM). They were compared in terms of their derivatization efficiency, electrospray ionization enhancement, stability and selectivity/side product formation with focus on four abundant intracellular thiols: cysteine (CYS), homocysteine (HCY), N-acetyl cysteine (NAC), glutathione (GSH) and their corresponding disulfides. While NPEM provided greater ionization efficiency than NEM (NPEM/NEM varies from 2.1x for GSH to 5.7x for CYS), it was also more unstable, forming more side-products. The instability of its maleimide ring led to reaction with amines, as well as double derivatization and cyclization reactions, which corresponded to about 10% of the signal of CYS. NEM showed only minor contribution of side reactions (about 1.5% of the signal of CYS), so it was chosen as the derivatizing reagent for the protocol. The derivatizing conditions with NEM were further optimized to minimize side product formation, and pH 7.0 was selected for further assay development while being compatible with cell handling. In the next step, a full cell extraction protocol was developed to quantify the thiol ratios in Jurkat T cells. Briefly, the optimized protocol required 1 × 106 cells and combined NEM derivatization prior to cell lysis, cell lysis and extraction using 20% methanol (v/v) and protein precipitation by methanol. The thiols were then chromatographically separated using a biphenyl, reversed-phase, separation in combination with Quadrupole Time of Flight Mass Spectrometry (QToF-MS) analysis. Protocol optimization included evaluation of different lysis solvents, recovery, matrix effects, and evaluation of the number of washes required to ensure as complete removal of extracellular metabolites as possible without compromising cellular integrity. The final method was tested for its capacity to evaluate oxidative stress in cells stimulated by hydrogen peroxide, a known inducer of oxidative damage. The results show that the method was capable of differentiating between the control, mild and intense oxidative stress conditions. To the best of my knowledge, this is the first cellular protocol that combines NEM derivatization prior to cell lysis with LC-MS determination of individual thiol ratios. An innovative aspect of this procedure is the protection of reduced thiols prior to lysis, which minimizes changes in the ratio caused by sample manipulation, as opposed to the typical procedure which has the derivatization after extraction. This work is also the first systematic comparison of NEM versus NPEM derivatization for LC-MS analysis and shows clearly the propensity of NPEM for side-product formation under conditions commonly used for maleimide derivatization. In summary, this research contributes towards more accurate measurement of thiol ratios as readouts of oxidative stress

Investigating the critical variables of azithromycin oral absorption using <i>in vitro </i>tests and PBPK Modeling

Azithromycin is an antibiotic listed in the essential list of medicines for adults and pediatrics. Conflicting evidence has been found regarding azithromycin classification according to the Biopharmaceutics classification system (BCS). The purpose of this study was to identify the critical variables that influence the oral absorption of azithromycin in adults and pediatrics.Azithromycin solubility and dissolution studies (oral suspension) were performed in buffers and biorelevant media simulating the fasted and fed gastrointestinal tract. A PBPK model was developed for azithromycin for healthy adult volunteers and pediatrics (Simcyp® v18.2) informed by in vitro solubility and dissolution studies to predict drug performance after administration of azithromycin as an oral suspension.The developed PBPK model predicted azithromycin plasma concentrations-time profiles after administration of an oral suspension to adults and pediatrics. Sensitivity analysis of solubility vs dose suggests that absorption is independent of solubility within the therapeutic dose range in both adults and pediatrics. The developed PBPK model for adults and pediatrics was consistent with the mechanism of permeation through the intestinal membrane (passive and active processes) being the rate-limiting step of azithromycin's absorption.The physiologically based approach proposed was shown to be useful to determine the factors controlling drug absorption in adults and pediatrics

ARTE E ABORDAGENS CLÍNICAS DO TRABALHO UMA REVISÃO BIBLIOGRÁFICA: INTRODUÇÃO AO PARADOXO DA ATIVIDADE MUSICAL

O presente trabalho é um esforço de interlocução entre as Abordagens Clínicas do Trabalho, em especial a Clínica da Atividade proposta por Yves Clot e um campo de trabalho ainda relativamente pouco explorado por teóricos da área: a música. O ártico consta inicialmente de uma breve revisão bibliográfica que buscou por trabalhos que envolvessem a temática Psicologia do trabalho/Abordagens Clíncias do Trabalho (ACT) arte/música e alienação. A escassez de referencial teórico encontrado nos conduziu a reflexões iniciais que articulasse estes campos fazendo algumas proposições: primeiro a operacionalidade do conceito de “atividade” no mundo da música suas implicações; segundo os atravessamentos da indústria fonográfica nas produções musicais da atualidade; e terceiro o paradoxo que envolve a criação musical submetida a uma lógica mercadológica

Adaptation and survival of Burkholderia cepacia and B. contaminans during long-term Incubation in saline solutions containing benzalkonium chloride

TheBurkholderia cepaciacomplex (Bcc) is a group of opportunistic pathogenic bacteria with a remarkable metabolic capacity and broad genotypic/phenotypic plasticity, allowing their adaptation to hostile conditions, including nutrient depleted solutions containing antimicrobial agents. Bcc bacteria are feared contaminants in pharmaceutical industries and cause nosocomial outbreaks, posing health threats to immunocompromised individuals and cystic fibrosis (CF) patients. In this study, the adaptation and survival ofB. cepaciaandB. contaminansisolates was investigated after long-term incubation in nutrient depleted saline solutions supplemented with increasing concentrations of the biocidal preservative benzalkonium chloride (BZK), recreating the storage conditions of pharmaceutical products. These epidemiologically related isolates were recovered from intrinsically contaminated saline solutions for nasal application and from two CF patients. Long-term incubation in saline solutions containing BZK led to the development of bacterial sub-populations that survived for at least 16 months, despite an initial 2-3 log decrease in viability, displaying a progressive dose-dependent decrease of colony and cell size, including the appearance of small colony variants (SCVs). Bacterial colonies lost pigmentation, changed the morphotype from rough to smooth and produced more spherical cells during extended incubation with BZK. The development of macroscopically visible cellular aggregates, rich in polysaccharide and harboring viable cells in their interior was triggered by BZK. The existence of a metabolic pathway for BZK degradation was confirmed through genome analysis. This study reveals mechanisms underlying the prevalence of Bcc bacteria as contaminants of pharmaceutical products containing BZK, which often lead to false-negative results during quality control and routine testing

Ab initio study of magnetism at the TiO2/LaAlO3 interface

In this paper we study the possible relation between the electronic and magnetic structure of the TiO2/LaAlO3 interface and the unexpected magnetism found in undoped TiO2 films grown on LaAlO$_3$. We concentrate on the role played by structural relaxation and interfacial oxygen vacancies. LaAlO3 has a layered structure along the (001) direction with alternating LaO and AlO2 planes, with nominal charges of +1 and -1, respectively. As a consequence of that, an oxygen deficient TiO2 film with anatase structure will grow preferently on the AlO2 surface layer. We have therefore performed ab-initio calculations for superlattices with TiO2/AlO2 interfaces with interfacial oxygen vacancies. Our main results are that vacancies lead to a change in the valence state of neighbour Ti atoms but not necessarily to a magnetic solution and that the appearance of magnetism depends also on structural details, such as second neighbor positions. These results are obtained using both the LSDA and LSDA+U approximations.Comment: Accepted for publication in Journal of Materials Scienc

Microglial Sirtuin 2 shapes long-term potentiation in hippocampal slices

Copyright © 2020 Sa de Almeida, Vargas, Fonseca-Gomes, Tanqueiro, Belo, Miranda-Lourenço, Sebastião, Diógenes and Pais. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Microglial cells have emerged as crucial players in synaptic plasticity during development and adulthood, and also in neurodegenerative and neuroinflammatory conditions. Here we found that decreased levels of Sirtuin 2 (Sirt2) deacetylase in microglia affects hippocampal synaptic plasticity under inflammatory conditions. The results show that long-term potentiation (LTP) magnitude recorded from hippocampal slices of wild type mice does not differ between those exposed to lipopolysaccharide (LPS), a pro-inflammatory stimulus, or BSA. However, LTP recorded from hippocampal slices of microglial-specific Sirt2 deficient (Sirt2-) mice was significantly impaired by LPS. Importantly, LTP values were restored by memantine, an antagonist of N-methyl-D-aspartate (NMDA) receptors. These results indicate that microglial Sirt2 prevents NMDA-mediated excitotoxicity in hippocampal slices in response to an inflammatory signal such as LPS. Overall, our data suggest a key-protective role for microglial Sirt2 in mnesic deficits associated with neuroinflammation.This study was supported by Santa Casa da Misericórdia de Lisboa (MB37-2017), GAPIC Research Program of the University of Lisbon Medical School (n° 2014002 and n° 2015028) and the following doctoral grants: PD/BD/128091/2016, SFRH/BD/118238/2016, PD/BD/114337/2016, and PD/BD/1144- 41/2016.info:eu-repo/semantics/publishedVersio

Fluoromycobacteriophages for rapid, specific, and sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis

Rapid antibiotic susceptibility testing of Mycobacterium tuberculosis is of paramount importance as multiple- and extensively- drug resistant strains of M. tuberculosis emerge and spread. We describe here a virus-based assay in which fluoromycobacteriophages are used to deliver a GFP or ZsYellow fluorescent marker gene to M. tuberculosis, which can then be monitored by fluorescent detection approaches including fluorescent microscopy and flow cytometry. Pre-clinical evaluations show that addition of either Rifampicin or Streptomycin at the time of phage addition obliterates fluorescence in susceptible cells but not in isogenic resistant bacteria enabling drug sensitivity determination in less than 24 hours. Detection requires no substrate addition, fewer than 100 cells can be identified, and resistant bacteria can be detected within mixed populations. Fluorescence withstands fixation by paraformaldehyde providing enhanced biosafety for testing MDR-TB and XDR-TB infections. © 2009 Piuri et al

Efficacy of novel recombinant fowlpox vaccine against recent Mexican H7N3 highly pathogenic avian influenza virus

Since 2012, H7N3 highly pathogenic avian influenza (HPAI) has produced negative economic and animal welfare impacts on poultry in central Mexico. In the present study, chickens were vaccinated with two different recombinant fowlpox virus vaccines (rFPV-H7/3002 with 2015 H7 hemagglutinin [HA] gene insert, and rFPV-H7/2155 with 2002 H7 HA gene insert), and were then challenged three weeks later with H7N3 HPAI virus (A/chicken/Jalisco/CPA-37905/2015). The rFPV-H7/3002 vaccine conferred 100% protection against mortality and morbidity, and significantly reduced virus shed titers from the respiratory and gastrointestinal tracts. In contrast, 100% of sham and rFPV-H7/2155 vaccinated birds shed virus at higher titers and died within 4 days. Pre- (15/20) and post- (20/20) challenge serum of birds vaccinated with rFPV-H7/3002 had antibodies detectable by hemagglutination inhibition (HI) assay using challenge virus antigen. However, only a few birds (3/20) in the rFPV-H7/2155 vaccinated group had antibodies that reacted against the challenge strain but all birds had antibodies that reacted against the homologous vaccine antigen (A/turkey/Virginia/SEP-66/2002) (20/20). One possible explanation for differences in vaccines efficacy is the antigenic drift between circulating viruses and vaccines. Molecular analysis demonstrated that the Mexican H7N3 strains have continued to rapidly evolve since 2012. In addition, we identified in silico three potential new N-glycosylation sites on the globular head of the H7 HA of A/chicken/Jalisco/CPA-37905/2015 challenge virus, which were absent in 2012 H7N3 outbreak virus. Our results suggested that mutations in the HA antigenic sites including increased glycosylation sites, accumulated in the new circulating Mexican H7 HPAIV strains, altered the recognition of neutralizing antibodies from the older vaccine strain rFPV-H7/2155. Therefore, the protective efficacy of novel rFPV-H7/3002 against recent outbreak Mexican H7N3 HPAIV confirms the importance of frequent updating of vaccines seed strains for long-term effective control of H7 HPAI virus.info:eu-repo/semantics/publishedVersio

Short-Term Evaluation in Growing Rats of Diet Containing Bacillus thuringiensis Cry1Ia12 Entomotoxin: Nutritional Responses and Some Safety Aspects

The Cry1Ia12 entomotoxin from a Brazilian Bacillus thuringiensis strain is currently being expressed in cotton cultivars to confer resistance to insect-pests. The present study aimed to assess the effects of a diet containing Cry1Ia12 protein on growing rats. A test diet containing egg white and Cry1Ia12 (0.1% of total protein) as a protein source was offered to rats for ten days. In addition, an acute toxicity bioassay was performed in rats with a single oral dose of the entomotoxin (12 mg/animal). No adverse effects were observed in the animals receiving the test diet when compared to those receiving a control diet (egg white). The analysed parameters included relative dry weight of internal organs, duodenum histology, blood biochemistry, and nutritional parameters. The results of the acute toxicity test showed no mortality or behaviour alteration. Thus, Cry1Ia12 toxin at the tested concentration does not cause deleterious effects on growing rats when incorporated in the diet for 10 days