Metabolite targeting: development of a comprehensive targeted metabolomics platform for the assessment of diabetes and its complications

Biomarker studies for metabolic disorders like diabetes mellitus (DM) are an important approach towards a better understanding of the underlying pathophysiological mechanisms of diseases (Roberts and Gerszten in Cell Metab 18:43–50, 2013; Wilson et al. in Proteome Res 4:591–598, 2005). Furthermore, screening of potential metabolic biomarkers opens the opportunity of early diagnosis as well as therapy and drug monitoring of metabolic disorders (Rhee et al. in J Clin Invest 10:1–10, 2011; Wang et al. in Nat Med 17:448–458, 2011; Wenk in Nat Rev Drug Discov 4:594–610, 2005). The aim of the present study was to develop methods for the quantitative determination of 74 potential metabolite biomarkers for DM and diabetic nephropathy (DN) in serum. Several studies have shown that the concentrations of many polar metabolites like amino or organic acids are changed in subjects suffering from diabetes (Wang et al. in Nat Med 17:448–458, 2011; Yuan et al. in J Chromatogr B 813:53–58, 2007). Analyzing polar analytes presents a challenge in liquid chromatography (LC) coupled with ESI–MS/MS (Gika et al. in J Sep Sci 31:1598–1608, 2008; Spagou et al. in J Sep Sci 33:716–727, 2010). Considering those reasons we decided to develop a specific HILIC–ESI–QqQ–MS/MS-method for quantitative determination of these polar metabolites. A subsequent method validation was carried out for both HILIC and RP chromatography with respect to the guidelines of the Food and Drug Administration (FDA in Food and Drug Administration: Guidance for industry, bioanalytical method validation, 2001). The HILIC and RP LC–MS methods were successfully validated. Furthermore, the HILIC method presented here was applied to serum samples of GIPR^dn transgenic mice, a diabetic strain developing DN, and non transgenic littermate controls. Significant, diabetes-associated changes were observed for the concentrations of 21 out of 62 metabolites. The new methods described here accurately quantify 74 metabolites known to be regulated in diabetes, allowing for direct comparison between studies and laboratories. Thus, these methods may be highly adoptable in clinical research, providing a starting point for early diagnosis and metabolic screening.     

Possible origins of CTnBST, a conjugative transposon found recently in a human colonic Bacteroides strain

A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species.     

Enhancement of the microbial community biomass and diversity during air sparging bioremediation of a soil highly contaminated with kerosene and BTEX

In order to obtain insights in complexity shifts taking place in natural microbial communities under strong selective pressure, soils from a former air force base in the Czech Republic, highly contaminated with jet fuel and at different stages of a bioremediation air sparging treatment, were analyzed. By tracking phospholipid fatty acids and 16S rRNA genes, a detailed monitoring of the changes in quantities and composition of the microbial communities developed at different stages of the bioventing treatment progress was performed. Depending on the length of the air sparging treatment that led to a significant reduction in the contamination level, we observed a clear shift in the soil microbial community being dominated by Pseudomonads under the harsh conditions of high aromatic contamination to a status of low aromatic concentrations, increased biomass content, and a complex composition with diverse bacterial taxonomical branches. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The online version of an erratum to this article can be found at http://dx.doi.org/. An erratum to this article can be found at     

Antigen delivery to plasmacytoid dendritic cells via BST2 induces protective T cell-mediated immunity

Plasmacytoid dendritic cells (PDCs) are capable of presenting Ags to T cells in a tolerogenic or immunogenic manner depending on the formulation of the Ag and the mode of stimulation. It has not been investigated whether effective adaptive immune responses useful for vaccination can be induced by Ab-mediated Ag targeting to PDCs in vivo. In this study, we show that Ag delivered to murine PDCs via bone marrow stromal cell Ag 2 (BST2)/CD317 in combination with TLR agonists as adjuvants is specifically presented by PDCs in vivo and elicits strong cellular and humoral immune responses. These include IFN-γ production by CD4(+) T cells and high Ab titers with a broad range of IgG isotypes. In addition, BST2-mediated Ag delivery in the presence of polyinosinic-polycytidylic acid as adjuvant induces cytotoxic T lymphocytes that are functional in vivo. A single immunization with Ag-fused anti-BST2 Ab together with polyinosinic-polycytidylic acid as adjuvant is sufficient to trigger protective immunity against subsequent viral infection and tumor growth. We conclude that despite the potential tolerogenic properties of PDCs, Ag targeting to PDCs in combination with TLR agonists as adjuvants is an effective vaccination strategy.     

Suppression of hyperglycemia in NOD mice after inoculation with recombinant vaccinia viruses

In autoimmune (type 1) diabetes, autoreactive lymphocytes destroy pancreatic β-cells responsible for insulin synthesis. To assess the feasibility of gene therapy for type 1 diabetes, recombinant vaccinia virus (rVV) vectors were constructed expressing pancreatic islet autoantigens proinsulin (INS) and a 55-kDa immunogenic peptide from glutamic acid decarboxylase (GAD), and the immunomodulatory cytokine interleukin (IL)-10. To augment the beneficial effects of recombinant virus therapy, the INS and GAD genes were fused to the C terminus of the cholera toxin B subunit (CTB). Five-week-old non-obese diabetic (NOD) mice were injected once with rVV. Humoral antibody immune responses and hyperglycemia in the infected mice were analyzed. Only 20% of the mice inoculated with rVV expressing the CTB::INS fusion protein developed hyperglycemia, in comparison to 70% of the mice in the uninoculated animal group. Islets from pancreatic tissues isolated from euglycemic mice from this animal group showed no sign of inflammatory lymphocyte invasion. Inoculation with rVV producing CTB::GAD or IL-10 was somewhat less effective in reducing diabetes. Humoral antibody isotypes of hyperglycemic and euglycemic mice from all treated groups possessed similar IgG1/IgG2c antibody titer ratios from 19 to 32 wk after virus inoculation. In comparison with uninoculated mice, 11-wk-old NOD mice injected with virus expressing CTB::INS were delayed in diabetes onset by more than 4 wk. The experimental results demonstrate the feasibility of using rVV expressing CTB::INS fusion protein to generate significant protection and therapy against type 1 diabetes onset and progression.     

Differential Impact of Lipoxygenase 2 and Jasmonates on Natural and Stress-Induced Senescence in Arabidopsis

Jasmonic acid and related oxylipins are controversially discussed to be involved in regulating the initiation and progression of leaf senescence. To this end, we analyzed profiles of free and esterified oxylipins during natural senescence and upon induction of senescence-like phenotypes by dark treatment and flotation on sorbitol in Arabidopsis (Arabidopsis thaliana). Jasmonic acid and free 12-oxo-phytodienoic acid increased during all three processes, with the strongest increase of jasmonic acid after dark treatment. Arabidopside content only increased considerably in response to sorbitol treatment. Monogalactosyldiacylglycerols and digalactosyldiacylglycerols decreased during these treatments and aging. Lipoxygenase 2-RNA interference (RNAi) plants were generated, which constitutively produce jasmonic acid and 12-oxo-phytodienoic acid but do not exhibit accumulation during natural senescence or upon stress treatment. Chlorophyll loss during aging and upon dark incubation was not altered, suggesting that these oxylipins are not involved in these processes. In contrast, lipoxygenase 2-RNAi lines and the allene oxid synthase-deficient mutant dde2 were less sensitive to sorbitol than the wild type, indicating that oxylipins contribute to the response to sorbitol stress.Senescence is an important, highly regulated process at the end of development. Senescence is characterized by breakdown of organelles and molecules, export and transport of these nutrients to other organs/parts of the organism, and finally programmed cell death of the senescing organ.The process of senescence has been intensively studied in leaves, and morphological as well as molecular changes in senescing leaves have been described. Yellowing as a consequence of chlorophyll and chloroplast degradation is the most obvious process during natural leaf senescence. In addition, gene expression changes dramatically during senescence. Some senescence-associated genes (SAG, SEN) have been reported that are induced during this process, and several of the encoded proteins function in macromolecule degradation, detoxification and defense metabolism, or signal transduction (Gepstein et al., 2003). Based on the degradation of chloroplasts and macromolecules, leaf metabolism changes from carbon assimilation to catabolism (Lim et al., 2007).The initiation and progression of senescence is regulated by endogenous as well as exogenous factors. Among the endogenous factors, the developmental status of the organ and of the whole plant (e.g. age and progress in flowering and seed production) has a great impact on the process of senescence. Different stress factors such as pathogen attack, drought, osmotic stress, heat, cold, ozone, UV light, and shading can induce or accelerate senescence (Quirino et al., 2000). Phytohormones are very important regulators that integrate information about the developmental status and the environmental factors. Cytokinins are antagonistic signals and delay senescence. Endogenous levels of cytokinins decrease during senescence, and exogenous application and transgenic approaches, enhancing endogenous levels of these compounds, lead to delayed senescence (Gan and Amasino, 1995). In contrast, the gaseous phytohormone ethylene is known to induce and accelerate senescence (John et al., 1995). There are also several indications that abscisic acid modulates senescence (van der Graaff et al., 2006). The roles of other phytohormones/signaling compounds such as auxin, salicylic acid, and jasmonates are less clear (Lim et al., 2007).Jasmonates are oxylipin signaling molecules derived from linolenic acid. The term jasmonates comprises 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and derivatives such as the methyl ester and amino acid conjugates of JA. One of the first biological activities described for these compounds was the promotion of senescence in oat (Avena sativa) leaves by methyl jasmonate (MeJa) isolated from Artemisia absinthium (Ueda and Kato, 1980). Later on, the induction of senescence-like phenotypes by exogenous application of MeJa was also found in other plant species (Ueda and Kato, 1980; Weidhase et al., 1987a; He et al., 2002). On the molecular level, this senescence-promoting effect of MeJa is accompanied by chlorophyll loss and decreases in Rubisco and photosynthesis (Weidhase et al., 1987a, 1987b). In addition, expression of some senescence-up-regulated genes is also responsive to JA; examples are SEN1, SEN4, SEN5, SAG12, SAG14, and SAG15 (Park et al., 1998; Schenk et al., 2000; He et al., 2002). Due to the results described above, jasmonates have been described for decades as compounds with senescence-promoting activities, while the function of these compounds in natural senescence in planta was critically discussed (Parthier, 1990; Sembdner and Parthier, 1993; Creelman and Mullet, 1997; Wasternack, 2007; Balbi and Devoto, 2008; Reinbothe et al., 2009). Additional indications for a role of jasmonates in regulating senescence are the transient up-regulation of expression of some enzymes involved in JA biosynthesis, such as allene oxide synthase (AOS) and OPDA reductase 3 (OPR3), and the increase in JA levels during natural senescence (He et al., 2002; van der Graaff et al., 2006). Furthermore, alterations in natural and induced senescence have been reported for some mutants with defects in the JA pathway. The mutant coi1, which is impaired in JA signaling, exhibited delayed chlorophyll loss upon dark incubation of detached leaves (Castillo and Leon, 2008). Plants with reduced expression of the 3-ketoacyl-CoA-thiolase KAT2, which is involved in β-oxidation and JA production, showed delayed yellowing during natural senescence and upon dark incubation of detached leaves (Castillo and Leon, 2008).However, there are also several reports that cast doubt on an important function of JA in senescence. For most mutants in JA biosynthesis or signaling, no differences in natural senescence are apparent (He et al., 2002; Schommer et al., 2008). In addition, mutants defective in the expression of AOS or OPR3 do not show altered senescence-like phenotypes upon dark treatment (Schommer et al., 2008; Kunz et al., 2009). It has to be taken into consideration that the knockout in these mutants has pleiotrophic effects during whole plant development. For example, the leaves of plants with reduced expression of the lipase DGL or of OPR3 are larger (Hyun et al., 2008). In addition, several knockout mutants defective in JA biosynthesis or signaling do not produce fertile flowers (Feys et al., 1994; McConn and Browse, 1996; Sanders et al., 2000; Stintzi and Browse, 2000; Ishiguro et al., 2001; von Malek et al., 2002). These changes in development might affect other developmental processes such as senescence.To investigate the function of jasmonates in senescence in more detail, we compared the oxylipin profile of wild-type leaves during natural senescence and upon stress induction of senescence-like phenotypes. The analysis of lipoxygenase 2 (LOX2)-RNA interference (RNAi) plants, which produce low basal levels of oxylipins but are impaired in the accumulation of OPDA and JA during senescence or in response to stress, indicates that 13-LOX products are not necessary for natural senescence or dark-induced chlorophyll loss but are involved in the response to sorbitol.     

Mitochondrial DNA repair of oxidative damage in mammalian cells

Nuclear and mitochondrial DNA are constantly being exposed to damaging agents, from endogenous and exogenous sources. In particular, reactive oxygen species (ROS) are formed at high levels as by-products of the normal metabolism. Upon oxidative attack of DNA many DNA lesions are formed and oxidized bases are generated with high frequency. Mitochondrial DNA has been shown to accumulate high levels of 8-hydroxy-2'-deoxyguanosine, the product of hydroxylation of guanine at carbon 8, which is a mutagenic lesion. Most of these small base modifications are repaired by the base excision repair (BER) pathway. Despite the initial concept that mitochondria lack DNA repair, experimental evidences now show that mitochondria are very proficient in BER of oxidative DNA damage, and proteins necessary for this pathway have been isolated from mammalian mitochondria. Here, we examine the BER pathway with an emphasis on mtDNA repair. The molecular mechanisms involved in the formation and removal of oxidative damage from mitochondria are discussed. The pivotal role of the OGG1 glycosylase in removal of oxidized guanines from mtDNA will also be examined. Lastly, changes in mtDNA repair during the aging process and possible biological implications are discussed.     

Light-induced fluorescence changes in<Emphasis Type="Italic"> Phycomyces</Emphasis>: evidence for blue light-receptor associated flavo-semiquinones

Light-induced fluorescence changes (LIFCs) were detected in sporangiophores of the blue-light-sensitive fungus Phycomyces blakesleeanus (Burgeff). The LIFCs can be utilized as a spectrophotometric assay for blue-light photoreceptors and for the in vivo characterization of their photochemical primary reactions. Blue-light irradiation of sporangiophores elicited a transient decrease and subsequent regeneration of flavin-like fluorescence emission at 525 nm. The signals recovered in darkness in about 120 min. In contrast to blue light, near-UV (370 nm) caused an increase in the fluorescence emission at 525 nm. Because the LIFCs were altered in a light-insensitive madC mutant with a defective photoreceptor, the fluorescence changes must be associated with early photochemical events of the transduction chain. Action spectra for the fluorescence changes at 525 nm showed major peaks near 470 and 600 nm. Double-pulse experiments involving two consecutive pulses of either blue and near-UV, blue and red, or near-UV and red showed that the responses depended on the sequence in which the different wavelengths were applied. The results indicate a blue-light receptor with intermediates in the near-UV, blue and red spectral regions. We explain the results in the framework of a general model, in which the three redox states of the flavin photoreceptor, the oxidized flavin (Fl), the flavo-semiquinone (FlH^·), and the flavo-hydroquinone (FlH₂) are each acting as chromophores with their own characteristic photochemical primary reactions. These consist of the photoreduction of the oxidized flavin generating semiquinone, the photoreduction of the semiquinone generating hydroquinone, and the photooxidation of the flavo-hydroquinone regenerating the pool of oxidized flavins. The proposed mechanism represents a photocycle in which two antagonistic photoreceptor forms, Fl and FlH₂, determine the pool size of the biological effector molecule, the flavo-semiquinone. The redox changes that are associated with the photocycle are maintained by redox partners, pterins, that function in the near-UV as secondary chromophores.Abbreviations FAD flavin adenine dinucleotide - Fl oxidized flavin - FlH flavo-semiquinone radical - FlH₂ flavo-hydroquinone - LIAC light-induced absorbance change - LIFC light-induced fluorescence change - Pt oxidized pterin - PtH₂ dihydro-pterin - PtH₄ tetrahydro-pterin     

Detection of Borrelia burgdorferi Sensu Stricto ospC Alleles Associated with Human Lyme Borreliosis Worldwide in Non-Human-Biting Tick Ixodes affinis and Rodent Hosts in Southeastern United States

Comparative analysis of ospC genes from 127 Borrelia burgdorferi sensu stricto strains collected in European and North American regions where Lyme disease is endemic and where it is not endemic revealed a close relatedness of geographically distinct populations. ospC alleles A, B, and L were detected on both continents in vectors and hosts, including humans. Six ospC alleles, A, B, L, Q, R, and V, were prevalent in Europe; 4 of them were detected in samples of human origin. Ten ospC alleles, A, B, D, E3, F, G, H, H3, I3, and M, were identified in the far-western United States. Four ospC alleles, B, G, H, and L, were abundant in the southeastern United States. Here we present the first expanded analysis of ospC alleles of B. burgdorferi strains from the southeastern United States with respect to their relatedness to strains from other North American and European localities. We demonstrate that ospC genotypes commonly associated with human Lyme disease in European and North American regions where the disease is endemic were detected in B. burgdorferi strains isolated from the non-human-biting tick Ixodes affinis and rodent hosts in the southeastern United States. We discovered that some ospC alleles previously known only from Europe are widely distributed in the southeastern United States, a finding that confirms the hypothesis of transoceanic migration of Borrelia species.     

Development of an in vitro integration assay for the Bacteroides conjugative transposon CTnDOT

Integrated self-transmissible elements called conjugative transposons (CTns) are responsible for the transfer of antibiotic resistance genes in many different species of bacteria. One of the best characterized of these newly recognized elements is the Bacteroides CTn, CTnDOT. CTnDOT is thought to have a circular transfer intermediate that transfers to and integrates into the genome of the recipient cell. Previous investigations of the mechanism of CTnDOT integration have been hindered by the lack of an in vitro system for checking this model of integration and determining whether the CTnDOT integrase alone was sufficient to catalyze the integration reaction or whether host factors might be involved. We report here the development of an in vitro system in which a plasmid containing the joined ends of CTnDOT integrates into a plasmid carrying a CTnDOT target site. To develop this in vitro system, a His-tagged version of the integrase gene of CTnDOT was cloned and shown to be active in vivo. The protein produced by this construct was partially purified and then added to a reaction mixture that contained the joined ends of the circular form of CTnDOT and a plasmid carrying one of the CTnDOT target sites. Integration was demonstrated by using a fairly simple mixture of components, but integration was stimulated by a Bacteroides extract or by purified Escherichia coli integration host factor. The results of this study demonstrate both that the circular form of CTnDOT is the form that integrates into the target site and that host factors are involved in the integration process.