UPLC-ESI-TOFMS-based metabolomics and gene expression dynamics inspector self-organizing metabolomic maps as tools for understanding the cellular response to ionizing radiation

Global transcriptomic and proteomic profiling platforms have yielded important insights into the complex response to ionizing radiation (IR). Nonetheless, little is known about the ways in which small cellular metabolite concentrations change in response to IR. Here, a metabolomics approach using ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry was used to profile, over time, the hydrophilic metabolome of TK6 cells exposed to IR doses ranging from 0.5 to 8.0 Gy. Multivariate data analysis of the positive ions revealed dose- and time-dependent clustering of the irradiated cells and identified certain constituents of the water-soluble metabolome as being significantly depleted as early as 1 h after IR. Tandem mass spectrometry was used to confirm metabolite identity. Many of the depleted metabolites are associated with oxidative stress and DNA repair pathways. Included are reduced glutathione, adenosine monophosphate, nicotinamide adenine dinucleotide, and spermine. Similar measurements were performed with a transformed fibroblast cell line, BJ, and it was found that a subset of the identified TK6 metabolites were effective in IR dose discrimination. The GEDI (Gene Expression Dynamics Inspector) algorithm, which is based on self-organizing maps, was used to visualize dynamic global changes in the TK6 metabolome that resulted from IR. It revealed dose-dependent clustering of ions sharing the same trends in concentration change across radiation doses. "Radiation metabolomics," the application of metabolomic analysis to the field of radiobiology, promises to increase our understanding of cellular responses to stressors such as radiation.

Transient dominance in a central African rain forest

The large-crowned emergent tree Microberlinia bisulcata dominates rain forest groves at Korup National Park, Cameroon, along with two codominants, Tetraberlinia bifoliolata and T. korupensis. M. bisulcata has a pronounced modal size frequency distribution around 110 cm stem diameter: its recruitment potential is very poor. It is a long-lived light-demanding species, one of many found in African forests. Tetraberlinia species lack modality, are more shade tolerant, and recruit better. All three species are ectomycorrhizal. M. bisulcata dominates grove basal area, even though it has similar numbers of trees (≥50 cm stem diameter) as each of the other two species. This situation presented a conundrum that prompted a long-term study of grove dynamics. Enumerations of two plots (82.5 and 56.25 ha) between 1990 and 2010 showed mortality and recruitment of M. bisulcata to be very low (both rates 0.2% per year) compared with Tetraberlinia (2.4% and 0.8% per year), and M. bisulcata grows twice as fast as the Tetraberlinia. Ordinations indicated that these three species determined community structure by their strong negative associations while other species showed almost none. Ranked species abundance curves fitted the Zipf-Mandelbrot model well and allowed “overdominance” of M. bisulcata to be estimated. Spatial analysis indicated strong repulsion by clusters of large (50 to <100 cm) and very large (≥100 cm) M. bisulcata of their own medium-sized (10 to <50 cm) trees and all sizes of Tetraberlinia. This was interpreted as competition by M. bisulcata increasing its dominance, but also inhibition of its own replacement potential. Stem coring showed a modal age of 200 years for M. bisulcata, but with large size variation (50–150 cm). Fifty-year model projections suggested little change in medium, decreases in large, and increases in very large trees of M. bisulcata, accompanied by overall decreases in medium and large trees of Tetraberlinia species. Realistically increasing very-large-tree mortality led to grove collapse without short-term replacement. M. bisulcata most likely depends on climatic events to rebuild its stands: the ratio of disturbance interval to median species' longevity is important. A new theory of transient dominance explains how M. bisulcata may be cycling in abundance over time and displaying nonequilibrium dynamics.

Fetuin-A and cystatin C are endogenous inhibitors of human meprin metalloproteases

Meprin and , zinc metalloproteinases, play significant roles in inflammation, including inflammatory bowel disease (IBD), possibly by activating cytokines, like interleukin 1 , interleukin 18, or tumor growth factor . Although a number of potential activators for meprins are known, no endogenous inhibitors have been identified. In this work, we analyzed the inhibitory potential of human plasma and identified bovine fetuin-A as an endogenous meprin inhibitor with a K(i) (inhibition constant) of 4.2 × 10(-5) M for meprin and a K(i) of 1.1 × 10(-6) M meprin . This correlated with data obtained for a fetuin-A homologue from carp (nephrosin inhibitor) that revealed a potent meprin and inhibition (residual activities of 27 and 22%, respectively) at a carp fetuin concentration of 1.5 × 10(-6) M. Human fetuin-A is a negative acute phase protein involved in inflammatory diseases, thus being a potential physiological regulator of meprin activity. We report kinetic studies of fetuin-A with the proteolytic enzymes astacin, LAST, LAST_MAM, trypsin, and chymotrypsin, indeed demonstrating that fetuin-A is a broad-range protease inhibitor. Fetuin-A inhibition of meprin activity was 40 times weaker than that of meprin activity. Therefore, we tested cystatin C, a protein structurally closely related to fetuin-A. Indeed, cystatin C was an inhibitor for human meprin (K(i) = 8.5 × 10(-6) M) but, interestingly, not for meprin . Thus, the identification of fetuin-A and cystatin C as endogenous proteolytic regulators of meprin activity broadens our understanding of the proteolytic network in plasma.

Xenobiotic metabolism: a view through the metabolometer

The combination of advanced ultraperformance liquid chromatography coupled with mass spectrometry, chemometrics, and genetically modified mice provide an attractive raft of technologies with which to examine the metabolism of xenobiotics. Here, a reexamination of the metabolism of the food mutagen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), the suspect carcinogen areca alkaloids (arecoline, arecaidine, and arecoline 1-oxide), the hormone supplement melatonin, and the metabolism of the experimental cancer therapeutic agent aminoflavone is presented. In all cases, the metabolic maps of the xenobiotics were considerably enlarged, providing new insights into their toxicology. The inclusion of transgenic mice permitted unequivocal attribution of individual and often novel metabolic pathways to particular enzymes. Last, a future perspective for xenobiotic metabolomics is discussed and its impact on the metabolome is described. The studies reviewed here are not specific to the mouse and can be adapted to study xenobiotic metabolism in any animal species, including humans. The view through the metabolometer is unique and visualizes a metabolic space that contains both established and unknown metabolites of a xenobiotic, thereby enhancing knowledge of their modes of toxic action.

Metallic copper as an antimicrobial surface

Bacteria, yeasts, and viruses are rapidly killed on metallic copper surfaces, and the term "contact killing" has been coined for this process. While the phenomenon was already known in ancient times, it is currently receiving renewed attention. This is due to the potential use of copper as an antibacterial material in health care settings. Contact killing was observed to take place at a rate of at least 7 to 8 logs per hour, and no live microorganisms were generally recovered from copper surfaces after prolonged incubation. The antimicrobial activity of copper and copper alloys is now well established, and copper has recently been registered at the U.S. Environmental Protection Agency as the first solid antimicrobial material. In several clinical studies, copper has been evaluated for use on touch surfaces, such as door handles, bathroom fixtures, or bed rails, in attempts to curb nosocomial infections. In connection to these new applications of copper, it is important to understand the mechanism of contact killing since it may bear on central issues, such as the possibility of the emergence and spread of resistant organisms, cleaning procedures, and questions of material and object engineering. Recent work has shed light on mechanistic aspects of contact killing. These findings will be reviewed here and juxtaposed with the toxicity mechanisms of ionic copper. The merit of copper as a hygienic material in hospitals and related settings will also be discussed.

High-throughput procedure for tick surveys of tick-borne encephalitis virus and its application in a national surveillance study in Switzerland

Tick-borne encephalitis (TBE), a viral infection of the central nervous system, is endemic in many Eurasian countries. In Switzerland, TBE risk areas have been characterized by geographic mapping of clinical cases. Since mass vaccination should significantly decrease the number of TBE cases, alternative methods for exposure risk assessment are required. We established a new PCR-based test for the detection of TBE virus (TBEV) in ticks. The protocol involves an automated, high-throughput nucleic acid extraction method (QIAsymphony SP system) and a one-step duplex real-time reverse transcription-PCR (RT-PCR) assay for the detection of European subtype TBEV, including an internal process control. High usability, reproducibility, and equivalent performance for virus concentrations down to 5 x 10(3) viral genome equivalents/microl favor the automated protocol compared to the modified guanidinium thiocyanate-phenol-chloroform extraction procedure. The real-time RT-PCR allows fast, sensitive (limit of detection, 10 RNA copies/microl), and specific (no false-positive test results for other TBEV subtypes, other flaviviruses, or other tick-transmitted pathogens) detection of European subtype TBEV. The new detection method was applied in a national surveillance study, in which 62,343 Ixodes ricinus ticks were screened for the presence of TBE virus. A total of 38 foci of endemicity could be identified, with a mean virus prevalence of 0.46%. The foci do not fully agree with those defined by disease mapping. Therefore, the proposed molecular test procedure constitutes a prerequisite for an appropriate TBE surveillance. Our data are a unique complement of human TBE disease case mapping in Switzerland.

Effect of Acute Psychological Stress on Prefrontal GABA Concentration Determined by Proton Magnetic Resonance Spectroscopy

Objective Impaired function of the central gamma-aminobutyric acid (GABA) system, which provides the brain’s major inhibitory pathways, is thought to play an important role in the pathophysiology of anxiety disorders. The effect of acute psychological stress on the human GABA-ergic system is still unknown, however. The purpose of this study was to determine the effect of acute stress on prefrontal GABA levels. Method A recently developed noninvasive magnetic resonance spectroscopy method was used to measure changes in the GABA concentration of the prefrontal cortex in 10 healthy human subjects during a threat-of-shock condition and during a safe condition (two sessions on different days). The main outcome measure was the mean GABA concentration within a 3×3×2-cm3 voxel selected from the medial prefrontal cortex. Results Prefrontal GABA decreased by approximately 18% in the threat-of-shock condition relative to the safe condition. This reduction was specific to GABA, since the concentrations of N-acetyl-aspartate, choline-containing compounds, and glutamate/glutamine levels obtained in the same spectra did not change significantly. Conclusions This result appeared compatible with evidence from preclinical studies in rodents, which showed rapid presynaptic down-regulation of GABA-ergic neurotransmission in response to acute psychological stress. The molecular mechanism and functional significance of this reduced inhibitory effect of acute psychological stress in relation to impaired GABA-ergic function in anxiety disorders merit further investigation.

Reduced metabotropic glutamate receptor 5 density in major depression determined by [(11)C]ABP688 PET and postmortem study

Clinical and preclinical evidence suggests a hyperactive glutamatergic system in clinical depression. Recently, the metabotropic glutamate receptor 5 (mGluR5) has been proposed as an attractive target for novel therapeutic approaches to depression. The goal of this study was to compare mGluR5 binding (in a positron emission tomography [PET] study) and mGluR5 protein expression (in a postmortem study) between individuals with major depressive disorder and psychiatrically healthy comparison subjects.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Ruminant rhombencephalitis-associated Listeria monocytogenes alleles linked to a multilocus variable-number tandem-repeat analysis complex

Listeria monocytogenes is among the most important food-borne pathogens and is well adapted to persist in the environment. To gain insight into the genetic relatedness and potential virulence of L. monocytogenes strains causing central nervous system (CNS) infections, we used multilocus variable-number tandem-repeat analysis (MLVA) to subtype 183 L. monocytogenes isolates, most from ruminant rhombencephalitis and some from human patients, food, and the environment. Allelic-profile-based comparisons grouped L. monocytogenes strains mainly into three clonal complexes and linked single-locus variants (SLVs). Clonal complex A essentially consisted of isolates from human and ruminant brain samples. All but one rhombencephalitis isolate from cattle were located in clonal complex A. In contrast, food and environmental isolates mainly clustered into clonal complex C, and none was classified as clonal complex A. Isolates of the two main clonal complexes (A and C) obtained by MLVA were analyzed by PCR for the presence of 11 virulence-associated genes (prfA, actA, inlA, inlB, inlC, inlD, inlE, inlF, inlG, inlJ, and inlC2H). Virulence gene analysis revealed significant differences in the actA, inlF, inlG, and inlJ allelic profiles between clinical isolates (complex A) and nonclinical isolates (complex C). The association of particular alleles of actA, inlF, and newly described alleles of inlJ with isolates from CNS infections (particularly rhombencephalitis) suggests that these virulence genes participate in neurovirulence of L. monocytogenes. The overall absence of inlG in clinical complex A and its presence in complex C isolates suggests that the InlG protein is more relevant for the survival of L. monocytogenes in the environment.

Role of ADAMTS13 in the pathogenesis, diagnosis, and treatment of thrombotic thrombocytopenic purpura

The regulation of VWF multimer size is essential in preventing spontaneous microvascular platelet clumping, a central pathophysiologic finding in thrombotic thrombocytopenic purpura (TTP). In the majority of TTP patients, ADAMTS13, the principal regulator of VWF size, is severely deficient. Today, 2 forms of severe ADAMTS13 deficiency are recognized. The acquired form is caused by circulating autoantibodies inhibiting ADAMTS13 activity or increasing ADAMTS13 clearance. Pathogenic anti-ADAMTS13 Abs are mainly of the IgG class, predominantly of subclass IgG4, and inhibitory Abs recognize a defined epitope in the ADAMTS13 spacer domain. The reasons underlying the failure to maintain immunologic tolerance to ADAMTS13, however, are still poorly understood. Constitutional ADAMTS13 deficiency leading to hereditary TTP, also known as Upshaw-Schulman syndrome, is the result of homozygous or compound heterozygous ADAMTS13 gene mutations.