High-performance liquid chromatographic assay with fluorescence detection for the routine monitoring of the antidepressant mirtazapine and its demethyl metabolite in human plasma

A validated HPLC method for the simultaneous quantitative analysis of the antidepressant mirtazapine and its demethyl metabolite in human plasma is described. The active constituents including internal standard were extracted from 1 ml of plasma with hexane and separated on a μBondapak Phenyl column with fluorescence detection. The lower limit of quantification was 0.5 ng/ml, without significant interferences with endogenous or exogenous components. Inter- and intra-assay accuracy determined at quality control levels of 2, 10 and 80 ng/ml were, respectively, 104.6–113.7% and 105.1–117.7% for mirtazapine, and 91.7–99.3% and 89.9–103.7% for demethylmirtazapine. In all cases the precision was below 6.8%.     

Functional characteristics of human peripheral blood alpha/betaTCR+, CD4- and CD8- double-negative (DN) T cells

The function of human peripheral blood alpha/betaTCR positive, CD4- and CD8- double-negative T lymphocytes (DN cells) in vivo is not completely understood. The response of immunomagnetically isolated DN cells to PHA and anti-CD3 was compared to the response of single-positive (SP) CD4 and CD8 subsets. Proliferation of DN cells in response to PHA was largely independent of APC. This suggests activation requirements for DN cells that are different from SP cells. Upon activation, HLA-DR was found to be upregulated early on DN cells, and IL-4 and IL-10 were detected in the supernatants of DN cells. These observations in vitro could correspond with an immunoregulatory role of human DN cells in vivo.     

Post-pandemic seroprevalence of pandemic influenza A (H1N1) 2009 infection (swine flu) among children <18 years in Germany

Background

We determined antibodies to the pandemic influenza A (H1N1) 2009 virus in children to assess: the incidence of (H1N1) 2009 infections in the 2009/2010 season in Germany, the proportion of subclinical infections and to compare titers in vaccinated and infected children.

Methodology/Principal Findings

Eight pediatric hospitals distributed over Germany prospectively provided sera from in- or outpatients aged 1 to 17 years from April 1^st to July 31^st 2010. Vaccination history, recall of infections and sociodemographic factors were ascertained. Antibody titers were measured with a sensitive and specific in-house hemagglutination inhibition test (HIT) and compared to age-matched sera collected during 6 months before the onset of the pandemic in Germany. We analyzed 1420 post-pandemic and 300 pre-pandemic sera. Among unvaccinated children aged 1–4 and 5–17 years the prevalence of HI titers (≥1∶10) was 27.1% (95% CI: 23.5–31.3) and 53.5% (95% CI: 50.9–56.2) compared to 1.7% and 5.5%, respectively, for pre-pandemic sera, accounting for a serologically determined incidence of influenza A (H1N1) 2009 during the season 2009/2010 of 25,4% (95% CI : 19.3–30.5) in children aged 1–4 years and 48.0% (95% CI: 42.6–52.0) in 5–17 year old children. Of children with HI titers ≥1∶10, 25.5% (95% CI: 22.5–28.8) reported no history of any infectious disease since June 2009. Among vaccinated children, 92% (95%-CI: 87.0–96.6) of the 5–17 year old but only 47.8% (95%-CI: 33.5–66.5) of the 1–4 year old children exhibited HI titers against influenza A virus (H1N1) 2009.

Conclusion

Serologically determined incidence of influenza A (H1N1) 2009 infections in children indicates high infection rates with older children (5–17 years) infected twice as often as younger children. In about a quarter of the children with HI titers after the season 2009/2010 subclinical infections must be assumed. Low HI titers in young children after vaccination with the AS03_B-adjuvanted split virion vaccine need further scrutiny.     

Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine, and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein‐engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N‐acetyl‐D‐glucosamine units at their subsite (?2), which is impossible for the wildtype enzyme.     

Antiadhesive Properties of Abelmoschus esculentus (Okra) Immature Fruit Extract against Helicobacter pylori Adhesion

Background

Traditional Asian and African medicine use immature okra fruits (Abelmoschus esculentus) as mucilaginous food to combat gastritis. Its effectiveness is due to polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach tissue. The present study investigates the antiadhesive effect in mechanistic detail.

Methodology

A standardized aqueous fresh extract (Okra FE) from immature okra fruits was used for a quantitative in vitro adhesion assay with FITC-labled H. pylori J99, 2 clinical isolates, AGS cells, and fluorescence-activated cell sorting. Bacterial adhesins affected by FE were pinpointed using a dot-blot overlay assay with immobilized Lewis^b, sialyl-Lewis^a, H-1, laminin, and fibronectin. ¹²⁵I-radiolabeled Okra FE polymer served for binding studies to different H. pylori strains and interaction experiments with BabA and SabA. Iron nanoparticles with different coatings were used to investigate the influence of the charge-dependence of an interaction on the H. pylori surface.

Principal findings

Okra FE dose-dependently (0.2 to 2 mg/mL) inhibited H. pylori binding to AGS cells. FE inhibited the adhesive binding of membrane proteins BabA, SabA, and HpA to its specific ligands. Radiolabeled compounds from FE bound non-specifically to different strains of H. pylori, as well as to BabA/SabA deficient mutants, indicating an interaction with a still-unknown membrane structure in the vicinity of the adhesins. The binding depended on the charge of the inhibitors. Okra FE did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors.

Conclusion

Non-specific interactions between high molecular compounds from okra fruits and the H. pylori surface lead to strong antiadhesive effects.     

The Metabolic Status Drives Acclimation of Iron Deficiency Responses in Chlamydomonas reinhardtii as Revealed by Proteomics Based Hierarchical Clustering and Reverse Genetics

Iron is a crucial cofactor in numerous redox-active proteins operating in bioenergetic pathways including respiration and photosynthesis. Cellular iron management is essential to sustain sufficient energy production and minimize oxidative stress. To produce energy for cell growth, the green alga Chlamydomonas reinhardtii possesses the metabolic flexibility to use light and/or carbon sources such as acetate. To investigate the interplay between the iron-deficiency response and growth requirements under distinct trophic conditions, we took a quantitative proteomics approach coupled to innovative hierarchical clustering using different “distance-linkage combinations” and random noise injection. Protein co-expression analyses of the combined data sets revealed insights into cellular responses governing acclimation to iron deprivation and regulation associated with photosynthesis dependent growth. Photoautotrophic growth requirements as well as the iron deficiency induced specific metabolic enzymes and stress related proteins, and yet differences in the set of induced enzymes, proteases, and redox-related polypeptides were evident, implying the establishment of distinct response networks under the different conditions. Moreover, our data clearly support the notion that the iron deficiency response includes a hierarchy for iron allocation within organelles in C. reinhardtii. Importantly, deletion of a bifunctional alcohol and acetaldehyde dehydrogenase (ADH1), which is induced under low iron based on the proteomic data, attenuates the remodeling of the photosynthetic machinery in response to iron deficiency, and at the same time stimulates expression of stress-related proteins such as NDA2, LHCSR3, and PGRL1. This finding provides evidence that the coordinated regulation of bioenergetics pathways and iron deficiency response is sensitive to the cellular and chloroplast metabolic and/or redox status, consistent with systems approach data.The green alga Chlamydomonas reinhardtii has an enormous metabolic versatility (1) and possesses the flexibility to grow in the presence of different carbon sources. It may use carbon dioxide (CO₂) for photoautotrophic, acetate for heterotrophic, and both carbon sources for mixotrophic growth. In this alga CO₂ is fixed via the Calvin Benson Bassham cycle (2), while acetate can be taken up, converted to acetyl-CoA, and enter the glyoxylate cycle where it may be incorporated into C4 acids (3). In addition to the use of acetate as a source of energy and carbon backbone for biosynthetic processes, acetate can control respiration and photosynthesis in conjunction with the light intensity and CO₂ availability (4–6). Moreover, acclimation responses to iron- and copper-deficiencies significantly vary in photoautotrophic versus heterotrophic conditions (7–10), indicating that the metabolic status of the cells influence overall cellular acclimation responses.Transition metals like copper, manganese, and iron possess the ability to donate and accept electrons, making these metals suitable cofactors in enzymes that catalyze redox reactions. In particular, iron is used as a cofactor in numerous biochemical pathways and is therefore an essential nutrient. Cells require relatively high levels of iron because it is present in heme-, iron-sulfur and other proteins that function in respiratory and photosynthetic energy transducing. Correspondingly, in eukaryotic cells, the mitochondrion is a major iron-utilizing compartment. It is well established that iron is transported into mitochondria for heme synthesis and iron-sulfur cluster assembly. This is required for the formation of a functional respiratory electron transport machinery (11). Therefore, mitochondrial metabolism in mammals, fungi and plants is significantly affected under iron deficiency, as demonstrated by a number of studies (12–14). In plants, the chloroplasts are a primary target of iron deficiency. Changes in chloroplast structure, photosynthetic capacity and the composition of thylakoid membranes have been described for plants deprived of iron (15–21).Plants have devised various strategies for acquiring iron (22). Generally, iron deficiency leads to the activation of the iron uptake systems in photosynthetic organisms. For example, the accumulation of the ferroxidase, a component of the high affinity iron uptake system in C. reinhardtii, is very rapidly enhanced when iron becomes limiting (23). Inactivation of IRT1, the most prevalent Fe²⁺ transporter in Arabidopsis thaliana leads to a dramatic iron deficiency that is reflected by chlorosis (24–26). Despite the evolution of elaborate iron-uptake mechanisms in plants, iron deficiency-induced chlorosis remains a major agricultural problem (27, 28).The global impact of iron deficiency on photosynthetic productivity has been also shown in vast ocean regions, which are severely limited for iron (29, 30). Generally, one can conclude that photosynthesis in the oceans and on land can occur in environment where iron availability is restricted.Photosystem I (PSI) is a prime target of iron deficiency as it contains 12 atoms of iron per core complex. In algae, the degradation of PSI is also linked to remodeling of PSI-associated light-harvesting antenna (LHCI) (31–33). Cyanobacteria respond to iron deficiency by degradation of light harvesting phycobilisomes (34) and induction of the “iron-stress-induced” gene isiA. The ISIA protein, which has significant sequence similarity with CP43, a chlorophyll a-binding protein of photosystem II (PSII; (35, 36), forms a ring of 18 molecules around a PSI trimeric reaction center, as shown by electron microscopy (37, 38). The overall reorganization of the PSI complex from 900 kDa into 1.7 MDa complex highlights the large adaptive nature of the cellular response to iron deficiency, which helps to optimize the architecture of the photosynthetic apparatus to conditions in which iron is a limiting factor.The marine diatom Thalassiosira oceanica shows a remarkable retrenchment of cellular metabolism and remodeling of bioenergetic pathways in response to iron availability (39). Low iron triggers a reduction in the level of iron-rich photosynthetic proteins while iron-rich mitochondrial proteins are preserved. Furthermore, iron deprivation causes a remodeling of the photosynthetic machinery resulting in the adjustment of light energy use to an overall decline in the level of photosynthetic electron transport complexes (39). These responses, reported for green algae such as C. reinhardtii (31, 40, 41), are important for minimizing photo-oxidative stress and optimizing photosynthetic function. As observed for T. oceanica, under conditions of low iron availability (in the presence of organic carbon) a hierarchy of iron allocation responses in C. reinhardtii result in the down-regulation of iron-rich photosynthetic complexes while iron-rich mitochondrial complexes remain stable (41). Notably, under photoautotrophic and mixotrophic conditions C. reinhardtii displays distinct iron deprivation responses, suggesting that the cell''s response to iron deficiency is also dependent on trophic conditions (7–9). Thus bioenergetics pathways are remodeled in response to iron availability as well as to the type of carbon source available. Moreover, recent data has indicated that the regulation of iron-induced remodeling of the photosynthetic apparatus is linked to energy metabolism. Depletion of Proton Gradient Regulation Like1 protein (PGRL1) in C. reinhardtii has revealed a decreased efficiency of cyclic electron transfer under low iron conditions resulting in higher vulnerability toward iron deprivation (42).It was our aim to generate a more comprehensive picture of how the proteome of C. reinhardtii varies in response to low iron under distinct trophic conditions and how these changes compare with differences observed for cells grown under photoautotrophic and photoheterotrophic iron replete conditions. Quantitative proteomics in conjunction with a novel hierarchical clustering approach revealed information about the responses of C. reinhardtii to low iron conditions and the iron requirements of photoautotrophic growth. These analyses provide novel insights into the relationships between protein networks required for photosynthesis and iron deprivation-elicited stress responses; these studies are providing the knowledge required for modulating the level of available iron to improve the photosynthetic performance of plants (43, 44).     

Effect of age on homeostasis of lymphocytes in an interleukin-7-deficient mouse model

Interleukin (IL)-7 is thought to be a non-redundant cytokine for lymphopoiesis as there is a reduction of T and B cells in peripheral blood (PB) and a loss of TCRγδ+ cells in PB and bone marrow (BM) in IL-7?/? mice. To investigate whether the absence of IL-7 influences the organ-dependent distribution of the lymphocytes, we analyzed single cell suspensions of several organs (BM, lung, liver, small intestine, and spleen) at different ages (three and 12 months) of IL-7+/+ and IL-7?/? mice using flow cytometry; immunohistochemical staining was performed on frozen sections of various organs. We observed lymphocytopenia in almost all organs of IL-7?/? mice, but normal counts in the liver and the lung of three-month-old IL-7?/? mice. CD4+ and CD8+ cell numbers were decreased in the spleen and the BM. With aging, we found a greater increase in CD4+ and CD8+ cells in the BM of IL-7?/? than in IL-7+/+ mice, particularly of memory cells. The spleen of IL-7?/? mice was characterized by lymphocytopenia. We challenge the view that IL-7 is a non-redundant cytokine for lymphocyte development. Some of the changes observed, e.g. partial absence of TCRγδ+ T cells in the PB, BM and small intestine and complete loss in liver, lung and spleen, may be due to the altered organ distribution instead of a defect in γδ+ T cell lymphopoiesis. In this model, aging leads to a significantly altered composition of lymphocyte subsets, and the lack of IL-7 seems to accelerate this process.     

The Interplay of Light and Oxygen in the Reactive Oxygen Stress Response of Chlamydomonas reinhardtii Dissected by Quantitative Mass Spectrometry

Light and oxygen are factors that are very much entangled in the reactive oxygen species (ROS) stress response network in plants, algae and cyanobacteria. The first obligatory step in understanding the ROS network is to separate these responses. In this study, a LC-MS/MS based quantitative proteomic approach was used to dissect the responses of Chlamydomonas reinhardtii to ROS, light and oxygen employing an interlinked experimental setup. Application of novel bioinformatics tools allow high quality retention time alignment to be performed on all LC-MS/MS runs increasing confidence in protein quantification, overall sequence coverage and coverage of all treatments measured. Finally advanced hierarchical clustering yielded 30 communities of co-regulated proteins permitting separation of ROS related effects from pure light effects (induction and repression). A community termed redox^II was identified that shows additive effects of light and oxygen with light as the first obligatory step. Another community termed 4-down was identified that shows repression as an effect of light but only in the absence of oxygen indicating ROS regulation, for example, possibly via product feedback inhibition because no ROS damage is occurring. In summary the data demonstrate the importance of separating light, O₂ and ROS responses to define marker genes for ROS responses. As revealed in this study, an excellent candidate is DHAR with strong ROS dependent induction profiles.Life originated in an environment in which the atmosphere was reducing. More than 2.2 Gyr ago, photosynthetic bacteria managed to extract electrons from water, thereby releasing oxygen (O₂) as a side product (1). Although molecular O₂ is a triplet state (³O₂), and is thus kinetically inhibited, its related reactive oxygen species (ROS)¹, i.e., superoxide (O₂^•−), peroxides (ROOR), singlet oxygen (¹O₂), and hydroxyl radicals (HO^•) are not. Nevertheless, molecular O₂ itself oxidizes biomolecules, for example, thiol groups, albeit at a much slower rate. The fundamental change in environment and the appearance of O₂ and ROS triggered the biggest mass extinction ever seen on Earth (2, 3). Soon after, the much more efficient O₂ based metabolism (compared with fermentation) lead to an evolutionary explosion (4). Today, cells obtain energy from reduced organic molecules through O₂ based respiration.In the past ROS were associated with cellular stress but strong evidence points toward a cellular ROS network that keeps ROS production and ROS scavenging in tight balance to ensure the maintenance of the cellular redox homeostasis and protection against ROS stress (5, 6). An imbalance in this network has been associated with a wide array of human diseases such as cancer (7), neurodegeneration (8), Keshan disease (9), and many others (see also review (6)), although arguments have been brought forward that the origin of some diseases is not directly linked to ROS and that ROS are more likely to be the result of deteriorating cells (10). In any case, the cellular ROS network response to ROS stress is implicated in the progress of these diseases and understanding the network dynamics will have a significant impact in medicine.Equally important, reduced ROS capacity or imbalance in the ROS network results in decreased crop yields and simple attempts to increase production yields by increasing ROS scavenging capacities in plants failed because those plants lost their ability to mount a defense against pathogens efficiently by the hypersensitive reaction (11), which implicates intended localized high yield ROS production. On the other hand Chang et al. could show that the knock-out of glutathione peroxidase 7 (gpx7), i.e., reducing ROS scavenging capacity, leads to an increased pathogen resistance but, unfortunately, to an increased photosensitivity as well (12), thus resulting in reduced crop production. The quintessence is that plants require the ability to produce sufficient amounts of ROS as part of their defense mechanism yet require some ROS scavenging capacity because photosynthetic growth inevitably produces damaging ROS. In order to effectively mount a hypersensitivity defense reaction, the ROS scavenging capacities have to be suppressed. Thus understanding the ROS network is an important global issue in the light of hunger in some parts of the world and the need for biofuels. Elucidating the key players of the ROS network will allow high production crop plants to be designed.It seems clear that the ROS network, its dynamics and homeostasis are poorly understood. Understanding how to evaluate the ROS balance and how to restore ROS balance within a cell would have a strong impact on a medical and agricultural level. To put it in the words of Barry Halliwell: “the likely clinical value of ‘antioxidant therapy’ will depend on how well the exact role of reactive oxygen species,” i.e., the ROS network, “is known” (13).ROS can be divided into two classes, i.e., H₂O₂ and ¹O₂ based ones. Especially in plants, algae, and cyanobacteria, it is now widely accepted that the signaling pathways of H₂O₂ (14) and ¹O₂ (15) are complex and entangled (16, 17) simply through the nature of their production, i.e., via an active photosynthetic electron transport chain. However, there have been reports that clearly show the independence of H₂O₂ and ¹O₂ mediated responses (see e.g. (18, 19)). In Arabidopsis thaliana the ROS network, in particular the ¹O₂ aspect has been widely studied, but comprehensive proteomic studies are still required. The A. thaliana flu mutant was used to reveal ¹O₂ related retrograde signaling. The flu mutant accumulates protochlorophyllide when grown in the dark, and seedlings bleach and die whereas mature plants stop growing when transferred into light (20). ¹O₂ production yielded an induction of distinct genes and these differed significantly from genes induced by H₂O₂ (15). Apel and co-workers identified the chloroplast localized EXECUTER1/2 proteins as key players in ¹O₂ retrograde signaling (18, 21), highlighting that specific ¹O₂ induced signals trigger programmed cell death (PCD) rather than ROS induced damage. A flu-like gene (flp) was identified in Chlamydomonas reinhardtii, and its gene product FLP in its two splicing variants was shown to be involved in the chlorophyll biosynthesis (22). Regulation of FLPs were suggested to occur via light and retrograde plastid signals (22). The specific ¹O₂ signaling mechanism in A. thaliana was further extended by Ramel et al. (23). The authors could show that ¹O₂ induced damage to β-carotene, a major component in a ROS defense strategy, yields β-cyclocitral, which when produced and applied exogenously triggers a selective ¹O₂ response, similar to the one reported by Apel and co-workers when describing the effects of the flu mutant (15, 18, 21). However, the signaling pathways involving EXECUTER and β-cyclocitral show more and more independent features (see e.g. Lundquist et al. (24)).ROS production is an inevitable part of the oxygenic photosynthesis and thus can be controlled noninvasively by light intensities. This is why plants, algae, and cyanobacteria offer a unique opportunity to investigate the ROS network. However, in plants the majority of ROS is produced in the chloroplast requiring O₂ as educt and the presence of light. Therefore, careful separation of the light, O₂, and ROS responses is required. As a consequence, simple high light/low light comparisons are overshadowed by additional ROS production, and vice versa. A classical example is HSP70A in C. reinhardtii, which was originally reported to be light regulated (25) and later proven to be regulated by ROS (19), via two promoters that react specifically on H₂O₂ and ¹O₂, to be precise.We have devised an experimental setup, which allows the ROS, high light/low light (HL/LL) and aerobic/anaerobic (AR/AN) responses to be dissected on a proteome level using metabolic labeling and quantitative proteomics. We used an interlinked experimental setup that connects all four possible treatments in such a way that each treatment is compared with two other treatments. This offers a strong internal control because the changes in protein levels comparing two not directly connected treatments can be measured by two independent estimates. MS data was analyzed employing high quality retention time alignment to increase overall confidence in protein quantification, increase protein sequence coverage and increase coverage of all conditions. PyGCluster, a novel hierarchical clustering approach (26) was used to identify communities of proteins that are coregulated. Five communities/expression profiles are discussed: a) light and O₂ dependent induction, i.e., potential ROS related regulations, b) a novel regulation type, which shows induction of protein expression influenced additively by light and O₂, but with light as the obligatory first step, c) light related induction (O₂ independent), d) light dependent repression (O₂ independent), and e) light dependent repression in the absence of O₂, which might be a regulation linked to feedback inhibition by for example, molecules that are normally damaged by ROS.