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

Noise in an Intensive Care Nursery/Newborn Unit

info:eu-repo/semantics/publishedVersio

MAGNETIC FORCES IN ORTHODONTICS

BACKGROUND: Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity. RESULTS: A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements. CONCLUSIONS: NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies

Desempenho silvicultural de híbridos de Eucalyptus em função do preparo de solo, na Chapada do Araripe, PE.

Resumo

Efeito do espaçamento sobre o desenvolvimento inicial de híbridos de Eucalyptus na Chapada do Araripe, Pernambuco.

Seção: Silvicultura e Produção de Biomassa

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

Influência do preparo de solo no crescimento de híbridos de Eucalyptus na Chapada do Araripe, Pernambuco.

This study aimed to evaluate the effect of two soil management approaches: plowing and harrowing (AG) and plowing and harrowing with sub-soiling (AGS) on wood productivity of two Eucalyptus hybrids, in the Chapada do Araripe, Pernambuco. Sub-soiling was done down to 40 cm deep in the row for two hybrids: Eucalyptus grandis x E. camaldulensis (EGC) and E. brassiana x E. urophylla (Ebu), planted in with a spacing of 3.0 x 3.0m. The experimental design was a randomized block with four treatments and eight replications. At six years old, the height, diameter at breast height, survival (number of plants in relation to the initial stand), and timber production per plant were measured. The survival of hybrids in different soil preparations varied from 95 to 99%, and provided no significant difference between treatments. The hybrid Ebu planted in soil under plowing and harrowing showed significantly lower values for height (10.6 cm), timber volume (99 m3.ha-1), and IMA (16.5 m3.ha-1.year-1), compared to the other treatments. Additional parameters were obtained: mean annual increments AG-EGC = 27.0 m3.ha-1.year-1, AG-Ebu = 16.5 m3.ha-1.year-1, AGS-EGC = 24.6 m3.ha-1.year-1, AGS-Ebu = 23.7 m3.ha-1.year-1. The hybrid EGC performed better for both treatments analyzed in relation to the hybrid Ebu